Part 8 — Thompson & Thompson Genetics and Genomics in Medicine

Chapter 14: The Treatment of Genetic Disease

Ch14 · Pt1 chapter 14 The Treatment of Genetic Disease Ronald Doron Cohn ▶

Ada Hamosh The understanding of genetic disease at a molecular level, as presented in Chapters 11, 12, and 13, is the foundation of rational therapy. In the coming decades, increasing annotation of th...

Ch14 · Pt2 294 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE and other -omic technologies. However, even when the gene is known, knowledge of the pathophysiologic mechanism is often inadequate and can lag well behind gene discovery. In phenylketonuria (PKU), for example, despite decades of study, the mechanisms by which the elevation in phenylalanine impairs brain development and function are still poorly understood (see Chapter 13). Prediagnostic fetal damage. Some variants act early in development or cause irreversible pathologic changes before they are diagnosed. These problems can sometimes be anticipated if there is a family history of the genetic disease or if carrier screening identifies couples at risk. In some cases, prenatal treatment is possible (e.g., maternal dexamethasone [a cortisol analog] to prevent virilization in female fetuses known to have congenital adrenal hyperplasia). Severe phenotypes are less amenable to intervention. The initial cases of a disease to be recognized are usually the most severely affected, but they are often less amenable to treatment. In such individuals, the variant frequently leads to the absence of the encoded protein or to a severely compromised mutant protein with no residual activity. In contrast, when the variant is less disruptive, the mutant protein may retain some residual function, and it may be possible to increase the small amount of function sufficiently to have a therapeutic effect, as described later. Level of intervention Treatment strategy Mutant gene Modification of the somatic genotype i) transplantation e.g., bone marrow transplantation in -thalassemia ii) gene therapy e.g., transfer of the c cytokine receptor submit gene of the interleukin receptor in X-linked SCID Pharmacological modulation of gene expression RNA interference to degrade mutant mRNA e.g., RNAi for transthyretin amyloidosis Protein replacement e.g., glucocerebrosidase administration in Gaucher disease, factor VIII in hemophilia A Enhancement of residual function Disease-specific compensation e.g., pyridoxine in classic homocystinuria e.g., migalastat in Fabry disease i) dietary ii) pharmacologic e.g., low-phenylalanine diet in PKU e.g., phenylbutryate or sodium phenylacetate and sodium benzoate in urea cycle defects Medical intervention Surgical intervention Genetic counseling Carrier screening Presymptomatic diagnosis e.g., transfusion in thalassemia e.g., correction of congenital heart disease e.g., after child born with trisomy 21 e.g., for Tay-Sachs disease e.g., Huntington disease Mutant protein Metabolic or other biochemical dysfunction Clinical phenotype The family iii) genome editing e.g., CRISPR/Cas 9 editing of the BCL11 enhancer to promote fetal hemoglobin expression Mutant mRNA Figure 14.1 The various levels of treatment that are relevant to genetic disease, with the corresponding strategies used at each level. For each level, a disease discussed in the book is given as an example. All the therapies listed are used clinically in many centers, unless indicated otherwise. Hb F, Fetal hemoglobin; mRNA, messenger RNA; PKU, phenylketonuria; RNAi, RNA interference; SCID, severe combined immunodeficiency. (Modified from Valle D: Genetic disease: an overview of current therapy, Hosp Pract 22:167–182, 1987.) ▶

294 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE and other -omic technologies. However, even when the gene is known, knowledge of the pathophysiologic mechanism is often inadequate and can...

Ch14 · Pt3 CHAPTER 14 — The Treatment of Genetic Disease 295 The challenge of dominant negative alleles. For some dominant disorders, the mutant protein interferes with the function of the normal allele. The challenge is to decrease the expression or impact of the variant allele or its encoded altered protein specifically, without disrupting expression or function of the normal allele or its normal protein. SPECIAL CONSIDERATIONS IN TREATING GENETIC DISEASE Long-Term Assessment of Treatment Is Critical For treating monogenic diseases, long-term evaluation of cohorts of treated individuals, often over decades, is critical for several reasons. First, treatment initially judged as successful may eventually be revealed to be imperfect; for example, although well-managed children with PKU have escaped severely impaired intellectual development and have normal or nearly normal IQs (see later), they may manifest subtle learning disorders and behavioral disturbances that impair their academic performance in later years. Second, successful treatment of the pathologic changes in one organ may be followed by unexpected problems in tissues not previously observed to be clinically involved because the patients typically did not survive long enough for the new phenotype to become evident. Galactosemia, a well-known inborn error of carbohydrate metabolism, illustrates this point. This disorder results from an inability to metabolize galactose, a component of lactose (milk sugar), because of the autosomal recessive deficiency of galactose-1-phosphate uridyltransferase (GALT). Affected infants are usually normal at birth but develop gastrointestinal problems, cirrhosis of the liver, and cataracts in the weeks after they are given lactosecontaining milk. The pathogenesis is thought to be due to the negative impact of galactose-1-phosphate accumulation on other critical enzymes. If not recognized, galactosemia causes severe intellectual disability and is often fatal. Complete removal of milk from the diet, however, can protect against most of the harmful consequences, although learning disabilities are now recognized to be common, even in well-treated patients. Moreover, despite conscientious treatment, most females with galactosemia have ovarian failure that appears to result from endogenously produced galactose toxicity. Another example is provided by hereditary retinoblastoma (Case 39) due to germline variants in the retinoblastoma (RB1) gene (see Chapter 16). Patients successfully treated for the eye tumor in the first years of life are unfortunately at increased risk for development of other independent malignant neoplasms, particularly osteosarcoma, after the first decade of life. Ironically, therefore, treatment that successfully prolongs life provides an opportunity for the manifestation of a previously unrecognized phenotype. Figure 14.2 Timeline of major developments in the treatment and diagnosis of inborn errors of metabolism (IEM) from 1955 to present. ADA-SCID, Adenosine deaminase-severe combined immunodeficiency, LC-FAO, long chain fatty acid oxidation defects; Mo CDa, molybdenum cofactor deficiency type A; NAGS, N-acetylglutamate synthetase; UCD, urea cycle defects; levocarnitine and medical foods are used to treat many IEMs. (Adapted from Vernon HJ, Manoli I: Milestones in treatments for inborn errors of metabolism: reflections on where chemistry and mediine meet, Am J Med Genet 185a:3350–3358, 2021.) ▶

CHAPTER 14 — The Treatment of Genetic Disease 295 The challenge of dominant negative alleles. For some dominant disorders, the mutant protein interferes with the function of the normal allele. The cha...

Ch14 · Pt4 296 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE In addition, therapy that is free of side effects in the short term may be associated with serious problems in the long term. For example, clotting factor infusion in hemophilia (Case 21) sometimes results in the formation of antibodies to the infused protein, and blood transfusion in thalassemia (Case 44) invariably produces iron overload, which must then be managed by the administration of iron-chelating agents, such as deferoxamine. Genetic Heterogeneity and Treatment The optimal treatment of single-gene defects requires an unusual degree of diagnostic precision; one must often define not only the biochemical abnormality but also the specific gene that is affected. For example, as we saw in Chapter 13, hyperphenylalaninemia can result from variants in either the phenylalanine hydroxylase (PAH) gene or in one of the genes that encodes the enzymes required for the synthesis of tetrahydrobiopterin (BH4), the cofactor of the PAH enzyme (see Fig. 13.2). The treatment of these two different causes of hyperphenylalaninemia is entirely different, as shown in Table 13.1. Allelic heterogeneity (see Chapter 7) may also have critical implications for therapy. Some alleles may produce a protein that is decreased in abundance but has some residual function, so strategies to increase the expression, function, or stability of such a partially functional mutant protein may correct the biochemical defect. This situation is again illustrated by some patients with hyperphenylalaninemia due to variants in the PAH gene; the variants in some patients lead to the formation of a mutant PAH enzyme whose activity can be increased by the administration of high doses of the BH4 cofactor (see Chapter 13). Of course, if a patient carries two alleles with no residual function, nothing will be gained by increasing the abundance of the mutant protein. One of the most striking examples of the importance of knowing the specific mutant allele in a patient with a genetic disease is exemplified by cystic fibrosis (CF); the drug ivacaftor (Kalydeco) was approved for treating CF patients carrying any one of only nine of the many hundreds of CFTR missense alleles. Further work has resulted in identification of a three-drug regimen that can treat over 90% of CF patients (see later). TREATMENT BY THE MANIPULATION OF METABOLISM Presently, the most successful disease-specific approach to the treatment of genetic disease is directed at the metabolic abnormality in inborn errors of metabolism. The principal strategies used to manipulate metabolism in the treatment of this group of diseases are listed in Table 14.1. The necessity for patients with pharmacogenetic diseases, such as glucose-6-phosphate dehydrogenase deficiency (Case 19), to avoid certain drugs and chemicals is described in Chapter 19. Substrate Reduction As illustrated by the damaging effects of hyperphenylalaninemia in PKU, enzyme deficiencies may lead to substrate accumulation, with pathophysiologic consequences (see Chapter 13). Strategies to prevent the accumulation of the offending substrate have been one of the most effective methods of treating genetic disease. The most common approach is to reduce the dietary intake of the substrate or of a precursor of it, and presently several dozen disorders – most involving amino acid catabolic pathways – are managed in this way. The drawback is that severe lifelong restriction of dietary protein intake is often necessary, requiring strict adherence to an artificial diet that is onerous for TABLE 14.1 Treatment of Genetic Disease by Metabolic Manipulation Type of Metabolic Intervention Substance or Technique Disease Avoidance Antimalarial drugs G6PD deficiency Isoniazid Slow acetylators Dietary restriction Phenylalanine PKU Galactose Galactosemia Replacement Thyroxine Biotin Monogenic forms of congenital hypothyroidism Biotinidase deficiency Diversion Sodium benzoate/ sodium phenylacetate Urea cycle disorders Drugs that sequester bile acids in the intestine (e.g., colesevelam) Familial hypercholesterolemia heterozygotes Enzyme inhibition Statins PCSK9 inhibitors Familial hypercholesterolemia heterozygotes Substrate reduction Miglustat and eliglustat for Gaucher disease: competitive inhibitors of the first step of glycosylation of ceramide FDA approved, oral agents, can be instead of enzyme replacement therapy Receptor antagonism Losartan Marfan syndrome Depletion LDL apheresis (direct removal of LDL from plasma) Familial hypercholesterolemia homozygotes FDA, US Food and Drug Administration; G6PD, glucose-6-phosphate dehydrogenase; LDL, low-density lipoprotein; PKU, phenylketonuria. Updated from Rosenberg LE: Treating genetic diseases: lessons from three children, Pediatr Res 27:S10–S16, 1990. ▶

296 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE In addition, therapy that is free of side effects in the short term may be associated with serious problems in the long term. For example, c...

Ch14 · Pt5 CHAPTER 14 — The Treatment of Genetic Disease 297 the patient as well as for the family. Nutrients such as 20 essential amino acids cannot be withheld entirely, however; their intake must be sufficient for anabolic needs such as protein synthesis. A diet restricted in phenylalanine largely circumvents the neurologic damage in classic PKU (see Chapter 13). Children with PKU are normal at birth because the maternal enzyme protects them during prenatal life. Treatment is most effective if begun promptly after diagnosis by newborn screening. Without treatment, irreversible neurologic damage occurs, with the degree of intellectual deficit being directly related to the delay in commencing the low-phenylalanine diet and adherence to it. It is now recommended that patients with PKU remain on a low-phenylalanine diet for life because neurologic and psychiatric (including attention- deficit/hyperactivity disorder, anxiety, and depression) problems develop in many (although perhaps not all) patients if the diet is stopped. However, even PKU patients who have been effectively treated throughout life may have neuropsychologic deficits (e.g., impaired conceptual, visual-spatial, and language skills), despite their having normal intelligence as measured by IQ tests. Nonetheless, treatment produces results vastly superior to the severe intellectual disability that occurs without treatment. As discussed in Chapter 13, continued and tightly controlled phenylalanine restriction is particularly important in women with PKU during pregnancy to prevent damage to the fetus, even though the fetus is highly unlikely to be affected by PKU. Substrate Augmentation Substrate augmentation can either drive an enzyme reaction or stabilize the mutant protein (discussed later). An example of substrate augmentation to drive limited enzyme activity is giving pharmacologic doses of mannose to treat MPI-CDG (CDG1b) (see Chapter 13). High-dose galactose and high-dose manganese are being studied to treat SLC35A2-CDG (CDGIIf) and SLC39A8-CDG (CDGIIn), respectively (Fig. 14.3). Replacement The provision of essential metabolites, cofactors, or hormones whose deficiency is due to a genetic disease is simple in concept and often simple in application. Some of the most successfully treated single-gene defects belong to this category. A prime example is provided by congenital hypothyroidism, of which 10% Figure 14.3 Substrate augmentation. (Upper panel) A treatment for SLC35A2-CDG is high-dose galactose supplementation, where oral supplementation of galactose (yellow circle) increases UDP-galactose supplies and thus transport across the defective UDP-galactose transporter SLC35A2. (Lower panel) Defects in SLC39A8 lead to a deficiency in manganese (Mn 2+). Lack of this cofactor impairs the function of galactosyltransferases (Gal T). Cofactor supplementation leads to an improved Gal T function and thus normalized glycosylation in SLC39A8-CDG (CDG type IIn). (Adapted from Park JH, Marquardt T: Treatment options in congenital disorders of glycosylation, Front Genet 12:735348, 2021. https://doi.org/10.3389/fgene.2021.735348.) ▶

CHAPTER 14 — The Treatment of Genetic Disease 297 the patient as well as for the family. Nutrients such as 20 essential amino acids cannot be withheld entirely, however; their intake must be sufficien...

Ch14 · Pt6 298 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE to 15% of cases are monogenic in origin. Monogenic congenital hypothyroidism can result from pathogenic variants in any one of numerous genes encoding proteins required for the development of the thyroid gland or the biosynthesis or metabolism of thyroxine. Because congenital hypothyroidism from all causes is common (~1 in 4000 neonates), neonatal screening is conducted in many countries so that thyroxine administration may be initiated soon after birth to prevent the severe intellectual deficits that are otherwise inevitable (see Chapter 19). Diversion Diversion therapy is the enhanced use of alternative metabolic pathways to reduce the concentration of a harmful metabolite. A major use of this strategy is in the treatment of the urea cycle disorders (Fig. 14.4). The function of the urea cycle is to convert ammonia, which is neurotoxic, to urea, a benign end product of protein catabolism excreted in urine. If the cycle is disrupted by an enzyme defect such as ornithine transcarbamylase deficiency (Case 36), the consequent hyperammonemia cannot be controlled by dietary protein restriction alone. Blood ammonia levels can be reduced to normal, however, by the diversion of excess nitrogen to metabolic pathways that are normally of minor significance, leading to the synthesis of harmless compounds. Thus the administration to hyperammonemic patients of large quantities of sodium benzoate forces the ligation of ammonia with glycine to form hippurate, which is excreted in urine (see Fig. 14.4). Glycine synthesis is thereby increased, and for each mole of glycine formed, one mole of ammonia is consumed. Additional compounds, sodium phenylbutyrate and glycerol phenylbutyrate, are metabolized to phenylacetate, which then conjugates with glutamine and is excreted as phenylacetylglutamine (PAGN). In this case, each mole of PAGN removes two moles of nitrogen, preventing ammonia accumulation. A comparable approach is used to reduce cholesterol levels in heterozygotes for familial hypercholesterolemia (Case 16) (see Chapter 13). If bile acids are sequestered in the intestine by the oral administration of a compound such as colesevelam and then excreted in feces rather than being reabsorbed, bile acid synthesis from cholesterol increases (Fig. 14.5). The reduction in hepatic cholesterol levels leads to increased production of low-density lipoprotein (LDL) receptors from their single normal LDL receptor gene, increased hepatic uptake of LDL-bound cholesterol, and lower levels of plasma LDL cholesterol. This treatment significantly reduces plasma cholesterol levels because 70% of all LDL receptor uptake of cholesterol occurs in the liver. An important general principle is illustrated by this example: Autosomal dominant diseases may sometimes be treated by increasing the expression of the normal allele. Enzyme Inhibition The pharmacologic inhibition of enzymes is sometimes used to reduce the impact of metabolic abnormalities in treating inborn errors. This principle is also illustrated by the treatment of heterozygotes of familial Figure 14.4 The strategy of metabolite diversion. In this example, ammonia cannot be removed by the urea cycle because of a genetic defect of a urea cycle enzyme. The administration of sodium benzoate diverts ammonia to glycine synthesis, and the nitrogen moiety is subsequently excreted as hippurate. The administration of sodium phenylacetate, or it precursors, sodium phenylbutyrate or glycerol phenylbutyrate, diverts ammonia to glutamate synthesis and two nitrogen moieties are then excreted as phenylacetylglutamine. ▶

298 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE to 15% of cases are monogenic in origin. Monogenic congenital hypothyroidism can result from pathogenic variants in any one of numerous gene...

Ch14 · Pt7 CHAPTER 14 — The Treatment of Genetic Disease 299 hypercholesterolemia. If a statin, a class of drugs that are powerful inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, or HMG Co A reductase (the ratelimiting enzyme of cholesterol synthesis), is used to decrease hepatic de novo cholesterol synthesis in these patients, the liver compensates by increasing the synthesis of LDL receptors from the remaining intact LDL receptor allele. The increase in LDL receptors typically lowers plasma LDL cholesterol levels by 40% to 60% in familial hypercholesterolemia heterozygotes; used together with colesevelam, the effect is synergistic, and even greater decreases can be achieved (see Fig. 14.5). Inhibition of PCSK9 prevents degradation of LDL receptors, increasing their numbers and thereby further reducing plasma cholesterol by 50% to 60%. PCSK9 inhibitors, approved by the US Food and Drug Administration (FDA) since 2015, are effective as adjunctive therapy in individuals who need additional reduction in cholesterol and are particularly important for those who cannot tolerate statin therapy (5–10%) due to muscle pain. Receptor Antagonism In some instances the pathophysiology of an inherited disease results from the increased and inappropriate activation of a biochemical or signaling pathway. In such cases, one therapeutic approach is to antagonize critical steps in the pathway. A powerful example is provided by treatment of an autosomal dominant connective tissue disorder, Marfan syndrome (Case 30). The disease results from pathogenic variants in FBN1, the gene that encodes fibrillin 1, an important structural component of the extracellular matrix. The syndrome is characterized by many connective tissue abnormalities, such as aortic aneurysm, pulmonary emphysema, and eye lens dislocation (Fig. 14.6). Unexpectedly, the pathophysiology of Marfan syndrome is only partially explained by the impact of the reduction in fibrillin-1 microfibrils on the structure of the extracellular matrix. Rather, it has been found that a major function of microfibrils is to regulate signaling by the transforming growth factor β (TGFβ), by binding TGFβ to the large latent protein complex of TGFβ. The decreased abundance of microfibrils in Marfan syndrome leads to an increase in the local abundance of unbound TGFβ and in local activation of TGFβ signaling. This increased TGFβ signaling has been suggested to underlie the pathogenesis of many of the phenotypes of Marfan syndrome, particularly the progressive dilation of the aortic root, and aortic aneurysm and dissection, the major cause of death in this disorder. Moreover, a recently recognized group of other vasculopathies, such as nonsyndromic forms of thoracic aortic aneurysm, has also proved to be driven by altered TGFβ signaling. Angiotensin II signaling is known to increase TGFβ activity, and the angiotensin II type 1 receptor antagonist, losartan, a widely used antihypertensive agent, has been shown to attenuate TGFβ signaling by decreasing the transcription of genes encoding TGFβ ligands, receptor subunits, and activators. Treatment with losartan has been found to decrease substantially the rate of aortic root dilation in clinical trials of Marfan syndrome patients, an effect that appears to be largely due to decreased TGFβ signaling. The novel use of an FDA-approved drug, losartan, to treat a rare inherited disease, Marfan syndrome, represents a paradigm that will be repeated regularly in the future, as small molecule chemical screens to identify compounds with therapeutic potential – including the thousands of FDA-approved drugs – are undertaken to identify safe, effective treatments for other uncommon genetic disorders. No drugs Bile acid depletion Reductase inhibitor + bile acid depletion PCSK9 inhibitor + reductase inhibitor + bile acid depletion LDL LDL LDL LDL LDL LDL LDL LDL LDL LDL LDL LDL PCSK9 PCSK9 PCSK9 PCSK9 LDL LDL LDL LDL Plasma Liver Intestine HMG Co A reductase Cholesterol Bile acids HMG Co A reductase Bile acids HMG Co A reductase Cholesterol Bile acids HMG Co A reductase Cholesterol Bile acids Cholesterol Figure 14.5 Rationale for the combined use of a reagent that sequesters bile acids, such as colesevelam, together with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG Co A reductase) such as a statin, and a PCSK9 inhibitor (green structures) in the treatment of familial hypercholesterolemia heterozygotes. LDL, Low-density lipoprotein. (Adapted from Brown MS, Goldstein JL: A receptor-mediated pathway for cholesterol homeostasis, Science 232:4, 1986. Copyright by the Nobel Foundation.) ▶

CHAPTER 14 — The Treatment of Genetic Disease 299 hypercholesterolemia. If a statin, a class of drugs that are powerful inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, or HMG Co A reduc...

Ch14 · Pt8 300 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Depletion Genetic diseases characterized by the accumulation of a harmful compound are sometimes treated by direct removal of the compound from the body. This principle is illustrated by the treatment of homozygous familial hypercholesterolemia. In this instance, for patients whose LDL levels cannot be lowered by other approaches, a procedure called apheresis is used to remove LDL from the circulation. Whole blood is removed from the patient, LDL is removed from plasma by any one of several methods, and the plasma and blood cells are returned to the patient. The use of phlebotomy to alleviate the iron accumulation of hereditary hemochromatosis (Case 20) provides another example of depletion therapy. Chelation of copper using any of several different agents is effective in treating Wilson disease. TREATMENT TO INCREASE THE FUNCTION OF THE AFFECTED GENE OR PROTEIN The growth in knowledge of the molecular pathophysiology of monogenic diseases has been accompanied by a small but promising increase in therapies that – at the level of DNA, RNA, or protein – increase the function of the gene affected by the variant. Some of the novel treatments have led to striking improvement in the lives of affected individuals, an outcome that, until recently, would have seemed fanciful. An overview of the molecular treatment of single-gene diseases is presented in Fig. 14.7. These molecular therapies represent one facet of the important paradigm embraced by the concept of individualized or precision medicine, which is a general one used to describe the diagnosis, prevention, and treatment of a disease – tailored to individual patients – based on a profound understanding of the mechanisms that underlie its etiology and pathogenesis. Treatment at the Level of the Protein In many situations, if a mutant protein product is made, it may be possible to increase its function. For example, the stability or function of a mutant protein with some residual function may be further increased. With enzymopathies, the improvement in function obtained by this approach is usually very small, on the order of a few percent, but this increment is often all that is required to restore biochemical homeostasis. Enhancement of Mutant Protein Function With Small Molecule Therapy Small molecules are compounds with molecular weights in the few hundreds to thousands. They include vitamins, nonpeptide hormones, and indeed most drugs, whether synthesized by organic chemists or isolated from nature. A strategy for identifying potential drugs is to use highthroughput screening of chemical compound libraries, often containing tens of thousands of known chemicals, against a drug target, such as the protein whose function is disrupted by a variant. As we will discuss, three drugs that are now FDA approved for the treatment of most patients with CF were discovered using such high-throughput screens. Progress in the development of these drugs represents a new frontier with great potential for the treatment of genetic disease. Figure 14.6 Computerized tomography angiograms of the aorta from a control (left) and an individual with Marfan syndrome (right). The aortic root diameter is indicated by the arrow. (Courtesy H. Dietz, Johns Hopkins University.) ▶

300 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Depletion Genetic diseases characterized by the accumulation of a harmful compound are sometimes treated by direct removal of the compound f...

Ch14 · Pt9 CHAPTER 14 — The Treatment of Genetic Disease 301 Small Molecule Therapy to Allow Skipping Over Nonsense Codons. Nonsense variants account for 11% of deleterious alterations in the human genome. Thousands of small molecules are being examined in laboratories around the world to identify novel nontoxic compounds that facilitate the skipping of nonsense codons, not only for the treatment of CF but also for Duchenne muscular dystrophy (DMD) patients carrying nonsense codons, as well as other diseases. Safe, effective drugs of this type will have a major impact on the treatment of inherited disease. Small Molecules to Increase the Function of Correctly Trafficked Mutant Membrane Proteins. Amino acid substitutions in membrane proteins may not disrupt the trafficking of the mutant polypeptide to the plasma membrane, but rather interfere with its function at the cell surface. Small molecule screens for new treatments for CF have led this area of drug discovery. Screens for potentiators – molecules that could enhance the function of mutant CFTR proteins that are correctly positioned at the cell surface – identified ivacaftor, which improves the Cl− transport of some mutant CFTR proteins, such as the p. Gly 551Asp CFTR missense variant (see Fig. 13.14) that inactivates anion transport; this allele is carried by 4% to 5% of all CF patients. In one clinical trial, patients carrying at least one p. Gly 551Asp allele experienced a significant improvement in lung function, weight gain, respiratory symptoms, and a decline in sweat Cl−. Ivacaftor is presently FDA approved for the treatment of eight other CFTR missense variants. Although fewer than 200 CF patients in the United States have one of these eight alleles, the allele-specific indications for ivacaftor treatment highlight both the benefits and dilemmas of personalized medicine for genetic disease: Effective drugs can be discovered, but they may be effective only in a relatively small numbers of individuals. Moreover, at present ivacaftor is extremely expensive, costing ~$300,000 per year. Figure 14.7 The molecular treatment of inherited disease. Each molecular therapy is discussed in the text. ADA, Adenosine deaminase; ASO, antisense oligonucleotide; ERT, enzyme replacement therapy; mRNA, messenger RNA; MSD, membrane-spanning domain; NBD, nucleotide-binding domain; PEG, polyethylene glycol; SCID, severe combined immunodeficiency; siRNA, small interfering RNA: SMA, spinal muscular atrophy. Molecular treatment The Molecular Treatment of Genetic Disease Disease example ASOs to induce skipping of a mutant exon to restore the reading frame of an mRNA Gene therapy RNA interference (RNAi) to decrease the abundance of an mRNA encoding a dominant mutant protein Molecular chaperones to facilitate the folding of mutant proteins Molecular potentiators to enhance the function of mutant proteins Protein replacement with the native protein Replacement using a modified protein Cofactor therapy to increase the function or stability of a mutant protein siRNA CFTR ASO NH3 Promoter COOH 3' 5' mRNA Unfolded protein Folded protein Gene Cofactor Transcribed sequence of gene CH3 Cell membrane NBD 1 NBD 2 MSD 2 MSD 1 N R-domain C ASOs to induce skipping of exon 51 of the dystrophin gene in Duchenne muscular dystrophy Pyridoxine in classic homocystinuria X-linked SCID; ADA deficiency, SMA Transthyretin amyloidosis Ivacaftor, to improve the function of CFTR with the Gly 551Asp missense mutation or in combination with tezacaftor and elexacaftor for any stable CFTR protein Hemophilia, using factors VIII or IX; ERT for Gaucher and Fabry disease PEG-ADA for adenosine-deaminase deficient SCID ▶

CHAPTER 14 — The Treatment of Genetic Disease 301 Small Molecule Therapy to Allow Skipping Over Nonsense Codons. Nonsense variants account for 11% of deleterious alterations in the human genome. Thous...

Ch14 · Pt10 302 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Small Molecules to Correct the Folding of Mutant Membrane Proteins: Pharmacologic Chaperones. Some variants in membrane proteins may disrupt their ability to fold, pass through the endoplasmic reticulum (ER), and be trafficked to the plasma membrane. These mutant proteins are recognized by the cellular protein quality control machinery, trapped in the ER, and prematurely degraded by the proteasome. The p. Phe 508del(F508del) variant of the CFTR protein – which constitutes 68% of all CF causing variants worldwide – is perhaps the best-known example (see Fig. 13.14) of a variant that impairs trafficking of a membrane protein. Over the past decade continuous efforts, including small molecule screens and clinical trials, identified a combination of three modulators, elexacaftor/tezacaftor/ivacaftor, that can correct the folding and trafficking of CFTR carrying the F508del variant or ~200 other CFTR variants. Ivacaftor is a potentiator that improves the function of the CFTR channel once it has made it to the cell membrane. This drug alone was effective in ameliorating lung disease in CF patients with one of nine variants. Elexacaftor and tezacaftor are modulators that improve cellular processing and trafficking of mutant CFTR protein (see Fig. 14.8). Remarkably, this combination is effective in 90% of CF patients (only those who are homozygous or compound heterozygous for nonsense variants where no CFTR protein is made cannot be treated with this regimen). Patients with CF treated with elexacaftor/tezacaftor/ivacaftor experience a 70-point decrease in the sweat chloride concentrations and more remarkably a 10% to 14% increase in predicted pulmonary function testing, accompanied by a 63% decline in pulmonary exacerbations. While this treatment is not a cure, it is life changing for individuals with CF. This example is a milestone in medical genetics because it establishes the principle that molecular chaperones can have dramatic clinical benefits in the treatment of monogenic disease. Nevertheless, the cost of this combination is still ~$300,000 per year. Small Molecules to Enhance the Function of Mutant Enzymes: Vitamin-Responsive Inborn Errors of Metabolism. The biochemical abnormalities of a number of inherited metabolic diseases may respond, sometimes dramatically, to the administration of large amounts of the vitamin cofactor of the enzyme impaired by the pathogenic variant (Table 14.2). In fact, the vitamin-responsive inborn errors are among the most successfully treated of all genetic diseases. The vitamins used are remarkably nontoxic, generally allowing the safe administration of amounts 100 to 500 times greater than those required for normal nutrition. In homocystinuria due to cystathionine synthase deficiency (see Fig. 13.7), for example, ~50% of patients respond to the administration of high doses of pyridoxine (vitamin B6, the precursor of pyridoxal phosphate, the cofactor for the enzyme), an example – as we saw earlier in the case of BH4 administration in PKU – of cofactor responsiveness in a metabolic disease. In most of these responsive patients, free homocysteine completely disappears from the plasma, even though the increase in hepatic cystathionine synthase activity is usually only a few fold, from 1.5% to 4.5% of control activity. The increased pyridoxal phosphate concentrations may stabilize the mutant enzyme or overcome reduced affinity of the mutant enzyme for the cofactor (Fig. 14.9). In any case, vitamin B6 treatment substantially improves the clinical course of the disease in responsive patients. Nonresponsive patients generally carry null alleles and HCO3 _ Cl _ +ivacaftor +elexacaftor F508delCFTR Endoplasmic reticulum Golgi trans Golgi +tezacaftor HCO3 _ Cl _ Wildtype CFTR Figure 14.8 Modulators and potentiators to treat cystic fibrosis. Tezacaftor and elexacaftor allow CFTR carrying the F508del variant to traverse the endoplasmic reticulum and Golgi apparatus to reach the cell surface, where ivacaftor prolongs channel open time to allows more chloride ions to leave the cells. Ivacaftor works alone for nine CFTR variants in which channel gating is the main defect. The threedrug combination (marketed in the United States as Trikafta) improves lung function and lowers sweat chloride dramatically. ▶

302 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Small Molecules to Correct the Folding of Mutant Membrane Proteins: Pharmacologic Chaperones. Some variants in membrane proteins may disrupt...

Ch14 · Pt11 CHAPTER 14 — The Treatment of Genetic Disease 303 TABLE 14.2 Treatment of Genetic Disease at the Level of the Mutant Protein Strategy Example Status Enhancement of Mutant Protein Function Small molecule “correctors” that increase the trafficking of the mutant protein through the ER to the plasma membrane Tezacaftor and elexacaftor to increase the abundance of the F508del-CFTR protein at the apical membrane of epithelial cells in CF patients FDA approved and used in combination with ivacaftor; expensive Small molecule “potentiators” that increase the function at the cell membrane of correctly trafficked membrane proteins Ivacaftor (VX-770) used alone to enhance the function of specific variant CFTR proteins at the epithelial apical membrane FDA approved for the treatment of CF patients carrying specific alleles; most effective when used in combination with tezacaftor and elexacaftor; expensive Vitamin cofactor administration to increase the residual activity of the mutant enzyme Vitamin B6 for pyridoxine-responsive homocystinuria Treatment of choice in the 50% of cystathionine synthase patients who are responsive: inexpensive Protein Augmentation Replacement of an extracellular protein Factor VIII in hemophilia A Well-established, effective, safe Extracellular replacement of an intracellular protein Polyethylene glycol–modified adenosine deaminase (PEG-ADA) in ADA deficiency Well-established, safe, and effective, but costly; now used principally to stabilize patients before gene therapy or HLAmatched bone marrow transplantation Replacement of an intracellular protein – cell targeting β-glucocerebrosidase in nonneuronal Gaucher disease Well-established; biochemically and clinically effective; expensive ADA, Adenosine deaminase; CF, cystic fibrosis; ER, endoplasmic reticulum; FDA, US Food and Drug Administration; HLA, human leukocyte antigen; PEG, polyethylene glycol. Vitamin cofactor Exogenous vitamin cofactor Inactive apoenzyme Mutant apoenzyme with defective cofactor binding site Active holoenzyme Partially active holoenzyme Figure 14.9 The mechanism of response of a mutant apoenzyme to the administration of its cofactor at high doses. Vitamin-responsive enzyme defects are often due to variants that reduce the normal affinity (top) of the enzyme protein (apoenzyme) for the cofactor needed to activate it. In the presence of the high concentrations of the cofactor that result from the administration of up to 500 times the normal daily requirement, the mutant enzyme acquires a small amount of activity sufficient to restore biochemical normalcy. (Redrawn from Valle D: Genetic disease: an overview of current therapy, Hosp Pract 22:167–182, 1987.) therefore have no residual cystathionine synthase activity to augment. Small Molecules to Stabilize Mutant Proteins. This class of therapeutics has increased dramatically in recent years and is exemplified not only by known cofactors but also by pharmacologic chaperones. One example is migalastat, which is approved for the treatment of adults with Fabry disease with specific pathogenic variants in GLA that have been shown to be responsive to migalastat in in vitro assays of enzyme activity. Other examples include miglustat and eliglustat for Gaucher disease (see Fig. 14.2 and Table 14.1). Small Molecules to Stabilize Mutant Proteins and Prevent Aggregation. Many conditions are ▶

CHAPTER 14 — The Treatment of Genetic Disease 303 TABLE 14.2 Treatment of Genetic Disease at the Level of the Mutant Protein Strategy Example Status Enhancement of Mutant Protein Function Small molecu...

Ch14 · Pt12 304 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE characterized by accumulation of misfolded proteins that aggregate and interfere with cell and tissue function. One example is hereditary amyloidosis due to pathogenic variants in TTR, the gene for transthyretin. This condition has highly variable presentation with either polyneuropathy or cardiomyopathy due to amyloid deposition (occasionally both and renal deposition is also possible). Tafamidis is a small molecule that binds to thyroxine binding sites on transthyretin tetramers and inhibits dissociation (>90%) and aggregate formation. This oral agent requires only daily dosing and is most effective when used early in the course of the disease, as it is unable to reverse damage done. Small Molecules to Drive Enzymatic Reactions. Carg lumic acid is a synthetic analogue of N-acetylglutamate, which drives expression of carbamoylphosphate synthetase, the first step in the urea cycle. Already approved to treat N-acetylglutamate synthetase deficiency, a very rare cause of hyperammonemia, carglumic acid was recently shown to be beneficial in treating acute hyperammonemia in patients with propionic acidemia and methylmalonic acidemia (MMA), where it drives the urea cycle despite the lack of known endogenous defect. The etiology of hyperammonemia in these patients is not clear and is quite unpredictable, but its response to carglumic acid, an oral agent, is welcome when compared with either intravenous sodium benzoate/sodium phenylacetate (Ammonul®) or hemodialysis, which have significant risks. Protein Augmentation The principal types of protein augmentation are summarized in Table 14.2. Protein augmentation is a routine therapeutic approach in only a few diseases, all involving proteins whose principal site of action is in the plasma or extracellular fluid. The prime example is the prevention or arrest of bleeding episodes in patients with hemophilia (Case 21) by the infusion of plasma fractions enriched for the appropriate factor or with the use of recombinant factor. The decades of experience with this disease illustrate the problems that can be anticipated as new strategies for replacing other, particularly intracellular, polypeptides are attempted. These problems include the difficulty and cost of procuring sufficient amounts of the protein to treat all patients at the optimal frequency, the need to administer the protein at a frequency consistent with its half-life (only 8–10 hours for factor VIII), and the formation of neutralizing antibodies in some patients (5% of classic hemophiliacs). Enzyme Replacement Therapy: Extracellular Admini stration of an Intracellular Enzyme Adenosine Deaminase Deficiency. Adenosine deaminase (ADA) is a critical enzyme of purine metabolism that catalyzes the deamination of adenosine to inosine and of deoxyadenosine to deoxyinosine (Fig. 14.10). The pathology of ADA deficiency, an autosomal recessive disease, results entirely from the accumulation of toxic purines, particularly deoxyadenosine, in lymphocytes. A profound failure of both cell-mediated (T-cell) and humoral (B-cell) immunity results, making ADA deficiency one cause of severe combined immunodeficiency (SCID). Untreated patients die of infection within the first 2 years of life. The long-term treatment of ADA deficiency is rapidly evolving, with gene therapy (see later section) now a strong alternative to bone marrow transplantation from a fully human leukocyte antigen (HLA) compatible donor. The administration of a modified form of the bovine ADA enzyme, described in the next section, is no longer a first choice for long-term management, but it is an effective stabilizing measure in the short term until these other treatments can be used. Modified Adenosine Deaminase. The infusion of bovine ADA modified by the covalent attachment of an inert polymer, polyethylene glycol (PEG), is superior in several ways to the use of the unmodified ADA enzyme. First, PEG-ADA largely protects the patient from a neutralizing antibody response (which would remove the ADA from plasma). Second, the modified enzyme remains in the extracellular fluid where it can degrade toxic purines. Third, the plasma half-life of PEG-ADA is Adenosine deaminase (ADA) deficiency DNA degradation deoxyadenosine deoxyadenosine T and B cell lymphotoxicity SCID adenosine inosine deoxyinosine Figure 14.10 Adenosine deaminase (ADA) converts adenosine to inosine and deoxyadenosine to deoxyinosine. In ADA deficiency, deoxyadenosine accumulation in lymphocytes is lymphotoxic, killing the cells by impairing DNA replication and cell division to cause severe combined immunodeficiency (SCID). ▶

304 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE characterized by accumulation of misfolded proteins that aggregate and interfere with cell and tissue function. One example is hereditary am...

Ch14 · Pt13 CHAPTER 14 — The Treatment of Genetic Disease 305 3 to 6 days, much longer than the half-life of unmodified ADA. Although the near-normalization of purine metabolism obtained with PEG-ADA does not completely correct immune function (most patients remain T lymphopenic), immunoprotection is restored, with dramatic clinical improvement. The general principles exemplified by the use of PEGADA are that (1) proteins can be chemically modified to improve their effectiveness as pharmacologic reagents, and (2) an enzyme that is normally located inside the cell can be effective extracellularly if its substrate is in equilibrium with the extracellular fluid and if its product can be taken up by the cells that require it. A similar approach is used to treat some adults with classic PKU, unresponsive to BH4 supplementation. Pegvaliase is modified phenylalanine ammonia lyase with PEG added to stabilize. Because it is a bacterial enzyme, it elicits a potent immune response, and the dose must be very slowly titrated up with premedications and an absolute requirement for epinephrine to be available for the daily injection. Pegvaliase has a profound effect on plasma phenylalanine levels lowering them to below treatment range, so careful monitoring is essential. It is contraindicated in pregnancy. Enzyme Replacement Therapy: Targeted Augmentation of an Intracellular Enzyme. Enzyme replacement therapy (ERT) is now established therapy for nine lysosomal storage diseases, with clinical trials being conducted for several others. Nonneuronal (type 1) Gaucher disease was the first lysosomal storage disease for which ERT was shown to be effective. It is the most prevalent lysosomal storage disorder, affecting up to 1 in 450 Ashkenazi Jews and 1 in 40,000 to 100,000 individuals in other populations (Case 18). This autosomal recessive condition results from deficiency of β-glucocerebrosidase. Loss of this enzyme activity leads to the accumulation of its substrate, the complex lipid glucocerebroside, in the lysosome, where it is normally degraded. The lysosomal accumulation of glucocerebroside, particularly in the macrophages and monocytes of the reticuloendothelial system, leads to gross enlargement of the liver and spleen. Bone marrow is slowly replaced by lipid-laden macrophages (Gaucher cells), leading to anemia and thrombocytopenia. The bone lesions cause episodic pain, osteonecrosis, and substantial morbidity. Thousands of patients with nonneuronal Gaucher disease have been treated worldwide with β-glucocerebrosidase ERT, with dramatic clinical benefits. ERT results in rapid resolution of anemia and normalization of platelet counts. There is more gradual normalization of liver and spleen size. In severely affected children with failure to thrive, catchup growth occurs after ERT initiation. ERT improves the characteristic skeletal abnormalities and bone density. Early treatment is most effective in preventing irreversible damage to bones. The success of ERT for nonneuronopathic Gaucher disease provided guidance to the development of enzyme and protein replacement therapy for other lysosomal storage disorders, and other classes of diseases as well, for several reasons. First, this use of ERT highlights the importance of understanding the biology of the relevant cell types. As demonstrated by I-cell disease (see Chapter 13), lysosomal hydrolases such as β-glucocerebrosidase contain posttranslationally added mannose sugars that target the enzyme to the macrophage through a mannose receptor on the plasma membrane. Once bound, the enzyme is internalized and delivered to the lysosome. Thus β-glucocerebrosidase ERT in Gaucher disease targets the protein both to a particular relevant cell and to a specific intracellular address, in this case the macrophage and the lysosome, respectively. Second, the human enzyme can be produced in abundance from cultured cells expressing the glucocerebrosidase gene, a key factor because this treatment, given as biweekly infusions, must be continuous. Only ~1% to 5% of the normal intracellular enzyme activity is required to correct the biochemical abnormalities in this and other lysosomal storage disorders. Third, the administered β-glucocerebrosidase is not recognized as a foreign antigen because patients with nonneuronal Gaucher disease have small amounts of residual enzyme activity. Unfortunately, however, because β-glucocerebrosidase does not cross the blood-brain barrier, ERT cannot treat the neuronopathic forms of Gaucher disease. Although ERT for any lysosomal disease is very expensive, its success has been a tremendous advance in the treatment of monogenic disorders. It has established the feasibility of directing an intracellular enzyme to its physiologically relevant location to produce clinically significant effects. Modulation of Gene Expression Decades ago, the idea that one might treat a genetic disease through the use of drugs that modulate gene expression would have seemed improbable. Increasing knowledge of the normal and pathologic bases of gene expression, however, has made this approach feasible. Indeed, it seems likely that this strategy will become only more widely used as our understanding of gene expression, and how it might be manipulated, increases. Splicing Modification of the Survival Motor Neuron Gene in Spinal Muscular Atrophy Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases with progressive weakness of skeletal and respiratory muscles, leading to significant disability. The disorder is caused by pathogenic variants in the survival motor neuron 1 (SMN1) gene and a consequent decrease in the SMN protein leading to lower motor neuron degeneration (see Chapter 13). Therapeutic approaches can be subdivided in survival motor neuron (SMN)–dependent gene therapies, which act to modify splicing of SMN2 (nusinersen, small ▶

CHAPTER 14 — The Treatment of Genetic Disease 305 3 to 6 days, much longer than the half-life of unmodified ADA. Although the near-normalization of purine metabolism obtained with PEG-ADA does not com...

Ch14 · Pt14 306 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE molecules) or replacing the SMN1 gene (onasemnogene abeparvovec, see later). Antisense Oligonucleotides to Alter Gene Expression: Nusinersen Nusinersen was the first drug approved by the FDA in December 2016 and by the European Medical Agency (EMA) in June 2017 for the treatment of SMA. Nusinersen is an antisense oligonucleotide (ASO) that promotes the inclusion of exon 7 in mRNA transcripts of SMN2 (see Chapter 13). It binds to an intronic splicesilencing site in intron 7 of SMN2 and inhibits the action of other splice factors, promoting exon 7 incorporation into the mRNA (Fig. 14.11). This mechanism allows the translation of a higher level of fully functional SMN protein and was shown to improve survival and pathology in different SMA preclinical studies. Clinical trials for nusinersen demonstrated at times significant efficacy without any major drug-related adverse event. Importantly, ASOs do not cross the blood-brain barrier, so nusinersen must be administered intrathecally. The treatment regimen consists of four injections over 2 months initially, followed by injections every 4 months. Recently, new data from clinical trials for nusinersen demonstrate long-term safety and efficacy in all patient groups and a significant improvement of survival and motor function. Nearly 100% of SMA type 2 patients were able to sit unsupported after 3 years, with some being able to walk with additional support and 76% of SMA type 3 walking independently. mRNA Therapy: Risdiplam Risdiplam (RG7916) is a small molecule splice modulator that was approved by the FDA in 2020 and the EMA in 2021. Risdiplam is an SMN2 splice modulator and binds to SMN2 pre-mRNA at two sites (an exon enhancer sequence and the 5′ splicing site of exon 7). This leads to stabilization of the ribonucleoprotein complex subsequently promoting exon 7 inclusion and full-length SMN protein production. Risdiplam is a SMN-C class of splice modulators and has been shown to increase full-length SMN protein in preclinical studies of both severe and mild SMA, leading to improved survival and motor phenotypes. Importantly, risdiplam can be administered orally. Increasing Gene Expression From the Wild-Type or Mutant Locus Several histone deacetylase inhibitors have been approved by the FDA for treatment of different forms of cancer, including T-cell lymphoma and multiple myeloma. Their use in monogenic disease is still in the early phases of development, but it is an exciting area of research, especially for epigenetic disorders (see Chapter 8). Reducing the Expression of a Dominant Mutant Gene Product: Small Interfering RNAs The pathology of some inherited diseases results from the presence of a mutant protein that is toxic to the cell, as seen with proteins with expanded polyglutamine tracts (see Chapter 13), as in Huntington disease (Case 24), Figure 14.11 The effect of Nusinersen, an antisense oligonucleotide that suppresses exon 7 skipping from the SMN2 gene, allowing higher levels of full length gene expression. (From Chiriboga CA, Swoboda KJ, Darras BT, et al: Results from a phase 1 study of nusinersen (ISIS-SMN[Rx]) in children with spinal muscular atrophy, Neurology 86(10):890–897, 2016. https://doi.org/10.1212/ WNL.0000000000002445.) ▶

306 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE molecules) or replacing the SMN1 gene (onasemnogene abeparvovec, see later). Antisense Oligonucleotides to Alter Gene Expression: Nusinersen...

Ch14 · Pt15 CHAPTER 14 — The Treatment of Genetic Disease 307 or with disorders such as the inherited amyloidoses. The autosomal dominant disorder transthyretin amyloidosis is the result of any of more than 100 missense variants in transthyretin, a protein produced mainly in liver, that transports retinol (one form of vitamin A) and thyroxine in body fluids. The major phenotypes are amyloidotic polyneuropathy, due to deposition of the amyloid in peripheral nerves (causing intractable peripheral sensory neuropathy and autonomic neuropathy), and amyloidotic cardiomyopathy, due to its deposition in the heart. Both disorders greatly shorten the life span, and the only prior treatment was hepatic transplantation. One new approach is provided by a technology called RNA interference (RNAi), which can mediate the degradation of a specific target RNA, such as that encoding transthyretin. Briefly, short RNAs that correspond to specific sequences of the targeted RNA (see Fig. 14.7) – termed small interfering RNAs (siRNAs) – are introduced into cells by, for example, lipid nanoparticles or viral vectors. Strands of the interfering RNA, ~21 nucleotides long, bind to the target RNA and initiate its cleavage. Phase III clinical trials of two compounds, inotersen and patirisen, using an siRNA (encapsulated in injected lipid nanoparticles) directed against transthyretin led to sustained reduction in transthyretin levels and clinical improvements in patients with TTR-polyneuropathy with no significant toxicity. Both agents are now approved for clinical use in many countries and are firstline treatments. The concept of RNAi treatment of an inherited disease is being applied to other diseases where elimination of the mutant gene product is the goal. Induction of Exon Skipping Exon skipping refers to the use of molecular interventions to exclude an exon from a pre-mRNA that encodes a reading frame–disrupting variant, thereby rescuing expression of the mutant gene. If the number of nucleotides in the excluded exon is a multiple of three, no frame shift will occur and, if the resulting polypeptide with the deleted amino acids retains sufficient function, a therapeutic benefit will result. The most widely studied method of inducing exon skipping is through the use of ASOs, which are synthetic 15- to 35-nucleotide single-stranded molecules that can hybridize to specific corresponding sequences in a pre-mRNA (see Fig. 14.7). The clearest example of the potential of this strategy is provided by DMD (see Chapter 13) (Case 14). The goal of exon skipping in DMD is to convert a DMD pathogenic variant into an in-frame counterpart that generates a functional dystrophin, just as the deletions that allow the production of a partially functioning dystrophin are associated with the milder phenotype of Becker muscular dystrophy (see Fig. 13.15). The distribution of DMD variants is nonrandomly distributed in the gene (see Chapter 13), and thus, remarkably, the skipping of just exon 51 alone would restore the dystrophin reading frame of an estimated 13% of all DMD patients (Fig. 14.12). This exon has therefore been the Exon 49 Exon 49 DMD deletion at exon 50 Pre-mRNA Pre-mRNA Splicing Out-of-frame mRNA In-frame mRNA Splicing No dystrophin BMD-like dystrophin PRO051 Exon 51 Exon 49 Exon 51 Exon 52 Intron 49/50 Intron 51 Exon 49 Exon 52 Intron 49/50 Intron 51 Exon 49 Exon 52 A B Exon 51 Figure 14.12 Schematic representation of exon skipping. In a patient with Duchenne muscular dystrophy (DMD) who has a deletion of exon 50, an out-of-frame transcript is generated in which exon 49 is spliced to exon 51 (A). As a result, a stop codon is generated in exon 51, which prematurely aborts dystrophin synthesis. The sequence-specific binding of the exon-internal antisense oligonucleotide PRO051 interferes with the correct inclusion of exon 51 during splicing so that the exon is actually skipped (B). This restores the open reading frame of the transcript and allows the synthesis of a dystrophin similar to that in patients with Becker muscular dystrophy (BMD). mRNA, Messenger RNA. (From van Deutekom JC, Janson AA, Ginjaar IB, et al: Local dystrophin restoration with antisense oligonucleotide PRO051, N Engl J Med 357:2677–2686, 2007.) ▶

CHAPTER 14 — The Treatment of Genetic Disease 307 or with disorders such as the inherited amyloidoses. The autosomal dominant disorder transthyretin amyloidosis is the result of any of more than 100 m...

Ch14 · Pt16 308 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE major focus of exon-skipping drug development. Several clinical trials have established that ASOs that cause skipping of exon 51 can produce significant increases in the number of dystrophin-positive muscle fibers of DMD patients. Moreover, one trial demonstrated stabilization of patient walking ability, but the treatment group was small, so this must be studied in a larger number of subjects. Irrespective of the specific challenges posed by DMD, it will be surprising if exon-skipping strategies do not ultimately play a significant role in the therapy of some inherited disorders. Creation of a bespoke antisense oligonucleotide, called milasen, was used to treat a single patient with Batten disease to skip an extra exon created by activation of a cryptic splice site due to insertion of a retrotranspon (see Chapter 4) in CLN7. This approach is in clinical trials to treat two types of severe early-onset seizure disorders due to variants in sodium channels. Genome Editing Over the last decade molecular biologists have developed methods to introduce site-specific genomic sequence changes into the DNA of intact organisms, including primates. The correction of a mutant gene sequence in its natural DNA context, in a sufficient number of target cells, would be an ideal treatment. This new technology, termed genome editing, uses engineered endonucleases containing a DNA-binding domain that will recognize a specific sequence in the genome, such as the sequence in which a missense variant is embedded (Fig. 14.13). Subsequently, a nuclease domain creates a double-stranded break, and cellular mechanisms for homology-directed repair (HDR) then repair the break (see Chapter 4), introducing the wild-type nucleotide to replace the mutant one. The template for the HDR must be based on a matching homologous wild-type DNA template that is introduced into the target cells before editing. The most widely used editing approach at present is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system, commonly referred to as CRISPR/Cas 9 (see Fig. 14.13). In humans, genome editing offers possibilities for the correction of genetic defects in their natural genomic landscape without the risks associated with the semirandom vector integration of some viral vectors used in gene therapy (see later section). Genome Editing Approaches for β-thalassemia and Sickle Cell Disease. Genome editing in hematopoietic stem cells (HSCs) can be used as a method to delete an erythroid enhancer of the BCL11A gene, thereby blocking its expression in the erythroid cell lineage. As a result, the change from hemoglobin Hb F to Hb A does not occur. Therefore patients retain Hb F instead of the hemoglobin containing a β-thalassemia variant or sickle cell allele (see Chapter 12). The hemoglobinopathies are the most common genetic defects in the world. These diseases are incurable unless an HSC transplantation is performed from a matched donor. Thus development of effective, safe, and affordable gene therapy for these disorders, the most common being sickle cell disease (SCD) and transfusiondependent β-thalassemia (TDT), presents an exciting opportunity. Genome-wide association studies have identified SNVs associated with increased expression of fetal hemoglobin in adults. Some of these SNVs are located in the BCL11A locus on chromosome 2 and have been shown to cause milder phenotypes in both TDT and SCD. BCL11A is a zinc finger–containing transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells; the SNVs that are associated with fetal hemoglobin are in an erythroid-specific enhancer Figure 14.13 The mechanism by which CRISPR/Cas 9 works to target, cut, and repair DNA to allow specific genome editing. In this example, the erythroid enhancer of BCL11 is altered to allow continued expression of γ-globin and sustained fetal hemoglobin, which ameliorates sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT). ▶

308 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE major focus of exon-skipping drug development. Several clinical trials have established that ASOs that cause skipping of exon 51 can produce...

Ch14 · Pt17 CHAPTER 14 — The Treatment of Genetic Disease 309 and are known to downregulate BCL11A expression and increase the expression of fetal hemoglobin. Based on these observations, the CRISPR-Cas 9 gene editing system was used in hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A to reduce BCL11A expression in erythroid-lineage cells and thereby restore γ-globin synthesis to reactivate production of fetal hemoglobin. A number of currently ongoing clinical trials use electroporation of CD34+ HSPCs that are obtained from healthy donors and subsequently modified with CRISPR-Cas 9 targeting the BCL11A erythroid-specific enhancer. The initial reports from these clinical trials demonstrated modification of 80% of the alleles at this locus without evidence of off-target gene editing. Results from two patients, one with TDT and the other with SCD, who were injected with autologous CD34+ cells edited with CRISPR-Cas 9 targeting the same BCL11A enhancer have shown high levels of allelic editing in bone marrow and blood of both patients associated with increased pancellular expression of fetal hemoglobin. These patients were subsequently transfusion independent. The patient with SCD did not show any signs of vasoocclusive episodes suggesting that this approach is safe and effective and presents a novel therapeutic avenue for these patients. Modification of the Somatic Genome by Transplantation Transplanted cells retain the genotype of the donor, and consequently transplantation can be regarded as a form of gene transfer therapy because it leads to a modification of the somatic genome. There are two general indications for the use of transplantation in the treatment of genetic disease. First, cells or organs may be transplanted to introduce wild-type copies of a gene into a patient with pathogenic variants in that gene. This is the case, for example, in homozygous familial hypercholesterolemia (see Chapter 13), for which liver transplantation is an effective but high-risk procedure. The second and more common indication is for cell replacement to compensate for an organ damaged by genetic disease (e.g., a liver that has become cirrhotic in a patient with α1-antitrypsin deficiency). Some examples of the uses of transplantation in genetic disease are provided in Table 14.3. Stem Cell Transplantation Stem cells are defined by two properties: (1) their ability to proliferate to form the differentiated cell types of a tissue in vivo, and (2) their ability to self-renew (i.e., to form another stem cell). Embryonic stem cells, which can give rise to the whole organism, are discussed in Chapter 15. Only three types of stem cells are in clinical use at present: HSCs, which can reconstitute the blood system after bone marrow transplantation; corneal stem cells, which are used to regenerate the corneal epithelium, and skin stem cells. These cells are derived from immunologically compatible donors. The possibility that other types of stem cells will be used clinically in the future is enormous because stem cell research is one of the most active and promising areas of biomedical investigation. Although it is easy to overstate the potential of such treatment, optimism about the long-term future of stem cell therapy is justified. Hematopoietic Stem Cell Transplantation in Nonstorage Diseases. In addition to its extensive application in the management of cancer, HSC transplantation using bone marrow stem cells is the treatment of choice for a selected group of monogenic immune deficiency disorders, including SCID of any type. Its role in the management TABLE 14.3 Treatment by Modification of the Genome or Its Expression Type of Modification Example Status RNA interference (RNAi) to reduce the abundance of a toxic or dominant negative protein RNAi for transthyretin amyloidosis Safe, effective, expensive Induction of exon skipping Use of antisense oligonucleotides to induce skipping of exon 7 in spinal muscular atrophy type I Safe, effective, very expensive Gene editing CRISPR/Cas 9 inactivation of the BCL11 gene in hematopoetic stem cells from individuals with Investigational; phase II trial successful Partial modification of the somatic genotype Bone marrow transplantation in β-thalassemia Curative with HLAmatched donor; good results overall By transplantation Bone marrow transplantation in storage diseases (e.g., Hurler syndrome) Excellent results in some diseases, even if the brain is affected, such as Hurler syndrome Cord blood stem cell transplantation for Hurler syndrome Excellent results if transplanted before age 2 (the earlier the better) Liver transplantation in α1-antitrypsin deficiency Up to 80% survival over 5 yr for genetic liver disease By gene transfer into somatic tissues (see Table 14.4) See Table 14.4 See Table 14.4 Cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; Hb F, fetal hemoglobin; HLA, human leukocyte antigen. ▶

CHAPTER 14 — The Treatment of Genetic Disease 309 and are known to downregulate BCL11A expression and increase the expression of fetal hemoglobin. Based on these observations, the CRISPR-Cas 9 gene ed...

Ch14 · Pt18 310 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of genetic disease in general, however, is less certain and under careful evaluation. For example, excellent outcomes have been obtained with allogeneic HSC transplantation in the treatment of children with β-thalassemia and sickle cell disease. Nevertheless, for each disease that bone marrow transplantation might benefit, its outcomes must be evaluated for many years and weighed against the results obtained with other therapies. Hematopoietic Stem Cell Transplantation for Lysosomal Storage Diseases Transplantation of Hematopoietic Stem Cells from Bone Marrow. Bone marrow stem cell transplants are effective in correcting lysosomal storage in many tissues, including (in some diseases) the brain, through the two mechanisms depicted in Fig. 14.14. First, the transplanted cells are a source of lysosomal enzymes that can be transferred to other cells through the extracellular fluid, as discussed in Chapter 13 for I-cell disease. Because bone marrow–derived cells constitute ~10% of the total cell mass of the body, the quantitative impact of enzymes transferred from them may be significant. Second, the mononuclear phagocyte system in tissues is derived from bone marrow stem cells so that, after bone marrow transplantation, this system is of donor origin throughout the body. Of special note are the brain perivascular microglial cells, whose bone marrow origin may partially account for the correction of nervous system abnormalities by bone marrow transplantation in some storage disorders, as we will see next in the case of Hurler syndrome, a lysosomal storage disease due to α-l-iduronidase deficiency. Bone marrow transplantation corrects or reduces the visceral abnormalities of many storage diseases. For example, a normalization or reduction in the size of the enlarged liver, spleen, and heart seen in Hurler syndrome can be achieved; improvements in upper airway obstruction, joint mobility, and corneal clouding are also obtained. Most rewarding, however, has been the impact of transplantation on the neurologic component of this disease. Patients who have good developmental indices before transplantation and who receive transplants before 24 months of age continue to develop cognitively after transplantation, in contrast to the inexorable loss of intellectual function that otherwise occurs. Interestingly, a gene dosage effect is manifested in the donor marrow; children who receive cells from homozygous normal donors appear to be more likely to retain fully normal intelligence than do the recipients of heterozygous donor cells. Allogeneic donor bone marrow stem cells naturally expressing all lysosomal enzyme proteins Patient’s bone marrow stem cells transduced with the normal gene encoding the lysosomal enzyme mutant in the patient Bone marrow transplantation into patient Mechanism 1 Replacement of tissue macrophages by donor cells throughout body Mechanism 2 Enzyme released from donor cells throughout body Lysosomal enzyme in body fluids Recipient cells Recipient cells after lysosomal enzyme uptake Treatment A Treatment B Figure 14.14 The two major mechanisms by which bone marrow transplantation or gene transfer into bone marrow may reduce the substrate accumulation in lysosomal storage diseases. In the case of either treatment, bone marrow transplantation from an allogeneic donor (A) or genetic correction of the patient’s own bone marrow stem cells by gene transfer (B), the bone marrow stem cell progeny, now expressing the relevant lysosomal enzyme, expand to repopulate the monocyte-macrophage system of the patient (mechanism 1). In addition, lysosomal enzymes are released from the bone marrow cells derived from the donor or from the genetically modified marrow cells of the patient and taken up by enzyme-deficient cells from the extracellular fluid (mechanism 2). ▶

310 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of genetic disease in general, however, is less certain and under careful evaluation. For example, excellent outcomes have been obtained wit...

Ch14 · Pt19 CHAPTER 14 — The Treatment of Genetic Disease 311 Transplantation of Hematopoietic Stem Cells from Placental Cord Blood. The discovery that placental cord blood is a rich source of HSCs has made a substantial impact on the treatment of genetic disease. The use of placental cord blood has three great advantages over bone marrow as a source of transplantable HSCs. First, recipients are more tolerant of histoincompatible placental blood than of other allogeneic donor cells. Thus engraftment occurs even if as many as three HLA antigens, cell surface markers encoded by the major histocompatibility complex (see Chapter 9), are mismatched between the donor and the recipient. Second, the wide availability of placental cord blood, together with the increased tolerance of histoincompatible donor cells, greatly expands the number of potential donors for any recipient. This feature is of particular significance to patients from minority populations for whom the pool of potential donors is relatively small. Third, the risk for graft-versus-host disease is substantially reduced with use of placental cord blood cells. Cord blood transplantation from unrelated donors appears to be as effective as bone marrow transplantation from a matched donor for the treatment of Hurler syndrome (Fig. 14.15). Liver Transplantation. For some metabolic liver diseases, liver transplantation is the only treatment of known benefit. For example, the chronic liver disease associated with CF or α1AT deficiency can be treated only by liver transplantation, and together these two disorders account for a large fraction of all the liver transplants performed in the pediatric population. Liver transplantation has now been undertaken for more than two dozen genetic diseases. At present, the 5-year survival rate of all children who receive liver transplants is in the range of 75% to 85%. For almost all of these patients, the quality of life is generally much improved, the specific metabolic abnormality necessitating the transplant is corrected, and in those conditions in which hepatic damage has occurred (such as α1AT deficiency), the provision of healthy hepatic tissue restores growth and normal pubertal development. Liver transplantation is also undertaken as a form of gene replacement in patients with certain inborn errors of metabolism who are at high risk for metabolic decompensation and sudden brain damage or death, but who have no active liver disease. Examples include individuals with urea cycle defects, maple syrup urine disease, propionic acidemia, and MMA. Reports to date indicate a greater than 90% survival and no subsequent metabolic decompensations, although brain and other organ damage sustained prior to transplant does not improve. Cognitive Adaptive Gross motor Fine motor Receptive language 20 18 16 8 10 12 14 6 4 2 0 0 2 4 6 8 10 12 14 16 Expressive language Typical development Calendar age (years) Developmental age (years) Figure 14.15 Preservation of neurocognitive development in children with Hurler syndrome treated by cord blood transplantation. Neurodevelopmental function of children with Hurler syndrome after umbilical cord blood transplantation compared to that of unaffected children. Age-equivalent scores were used to compare and monitor developmental progress. The colored lines depict the mean developmental curves (i.e., cognitive, adaptive, gross motor, fine motor, receptive language, and expressive language) of the surviving patients. These lines were plotted against the mean typical cognitive growth curve (gray continuous line) and approximate variability (95%; gray area) observed in typically developing children. Untreated children suffer relentless loss of neurologic and cognitive function with death late in the first or early in the second decade. (From Coletti HY, Aldenhoven M, Yelin K, et al: Long-term functional outcomes of children with Hurler syndrome treated with unrelated umbilical cord blood transplantation, JIMD Rep 20:77–86, 2015. https://doi. org/10.1007/8904_2014_395.) ▶

CHAPTER 14 — The Treatment of Genetic Disease 311 Transplantation of Hematopoietic Stem Cells from Placental Cord Blood. The discovery that placental cord blood is a rich source of HSCs has made a sub...

Ch14 · Pt20 312 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The Problems and the Future of Transplantation. Two major problems limit the wider use of transplantation for the treatment of genetic disease. First, the mortality after transplantation is still significant, and the morbidity from superimposed infection due to the requirement for immunosuppression and graft-versushost disease is substantial. Nevertheless, the ultimate goal of transplantation research – transplantation without immunosuppression – comes incrementally closer. The increased tolerance of the recipient to cord blood transplants, compared with bone marrow–derived donor cells, exemplifies the advances in this area. The second problem with transplantation is the finite supply of organs, cord blood being a singular exception. For example, for all indications, including genetic disease, more than 8000 liver transplants are performed annually in the United States alone, but more than double that number are added to the waiting list each year. In addition, it remains to be demonstrated that transplanted organs are generally capable of functioning normally for a lifetime. One solution to these difficulties involves the combination of stem cell and either genome editing or gene therapy. Here, a patient’s own stem cells would be cultured in vitro and either transfected by gene therapy with the gene of interest or corrected by CRISPR/Cas 9 editing and returned to the patient to repopulate the affected tissue with genetically restored cells. The identification of stem cells in a variety of adult human tissues and recent advances in gene transfer therapy offer great hope for this strategy. Induced Pluripotent Stem Cells. The ability to induce the formation of pluripotent stem cells (i PSCs) from somatic cells has the potential to provide the optimal solution to both challenges of transplantation posed earlier. In this approach somatic cells, such as skin fibroblasts, would be taken from a patient in need of a transplant and induced to form differentiated cells of the organ of interest. For example, the loss-of-function variant in the α1-antitrypsin gene in the fibroblasts cultured from a patient with α1AT deficiency (see Chapter 13) could be corrected either by gene editing (see earlier section) or gene therapy (see later section); the corrected cells could then be induced to form liverspecific i PSCs, which could then be transplanted into the liver of the patient to differentiate into hepatocytes. Alternatively, mature hepatocytes derived in vitro from the genetically corrected i PSCs could be transplanted. The great merit of this approach is that the genetically corrected liver cells are derived from the patient’s own genome, thus evading immunologic rejection of the transplanted cells as well as graft-versus-host disease. Experimental work in animal models has established that this strategy is capable of correcting inherited disorders. Substantial hurdles with i PSCs must first be overcome, however, including establishing the safety of transplanting cells derived by i PSC methodology and preventing epigenetic modifications in the derived cell type that are not characteristic of wild-type cells of the tissue of interest. GENE THERAPY Gene therapy is the introduction of a biologically active gene into a cell to achieve a therapeutic benefit. In 2012, the first gene therapy product was licensed in the United States and Europe for the treatment of lipoprotein lipase deficiency, and gene therapy has now been approved for the treatment of several more disorders; the number in late-stage clinical trials exceed a dozen, some of which are outlined in Table 14.4. These recent successes firmly establish that the treatment of genetic disease at its most fundamental level – the gene – will be increasingly feasible. The goal of gene therapy is to transfer the therapeutic gene early enough in the life of the patient to prevent the pathogenetic events that damage cells. Moreover, correction of the reversible features of genetic diseases should also be possible for many conditions. In this section, we outline the potential, methods, and probable limitations of gene transfer for the treatment of human genetic disease. The minimal requirements that must be met before the use of gene transfer can be considered for the treatment of a genetic disorder are presented (see Box 14.1). General Considerations for Gene Therapy In the treatment of inherited disease, the most common use of gene therapy will be the introduction of functional copies of the relevant gene into the appropriate target cells of a patient with a loss-of-function variant (because most genetic diseases result from such variants). In these instances, precisely where the transferred gene inserts into the genome of a cell would, in principle, generally not be important (see later discussion). If gene editing (see earlier discussion and Table 14.3) to treat inherited disease becomes routinely possible, then correction of the defect in the mutant gene in its normal genomic context would be ideal and would alleviate concerns such as the activation of a nearby oncogene by the regulatory activity of a viral vector or the inactivation of a tumor suppressor due to insertional mutagenesis by the vector. In some long-lived types of cells, stable, long-term expression may not require integration of the introduced gene into the host genome. For example, if the transferred gene is stabilized in the form of an episome (a stable nuclear but nonchromosomal DNA molecule, such as that formed by an adenoassociated viral vector, discussed later), and if the target cell is long-lived (e.g., T cells, neurons, myocytes, hepatocytes), then long-term expression can occur without integration. ▶

312 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The Problems and the Future of Transplantation. Two major problems limit the wider use of transplantation for the treatment of genetic disea...

Ch14 · Pt21 CHAPTER 14 — The Treatment of Genetic Disease 313 TABLE 14.4 Examples of Inherited Diseases Treated by Gene Therapy of Somatic Tissues Disease Affected Protein (Gene) Vector, Cell Transduced Outcome X-linked SCID γc-cytokine receptor subunit of several interleukin receptors (IL2RG) Retroviral vector Allogenic hematopoietic stem cells; new self-inactivating (SIN) vectors that do not promote oncogene expression Significant clinical improvement in 27 of 32 patients, 5 of whom developed a leukemia-like disorder that was treatable in 4; subsequent efficacy of SIN vectors in short-term follow-up of clinical trials SCID due to ADA deficiency Adenosine deaminase (ADA) Retroviral vector Allogenic hematopoietic stem cells 29 of 40 treated patients are off PEG-ADA enzyme replacement therapy X-linked adrenoleukodystrophy A peroxisomal adenosine triphosphate–binding cassette transporter (ABCD1) Lentiviral vector Autologous hematopoietic stem cells Apparent arrest of cerebral demyelination in the 17 of 19 boys studied Spinal muscular atrophy Survival motor neuron (SMN1) Adeno-associated virus vector injected IV Marked improvement in respiratory and skeletal muscle strength in >1800 patients; FDA approved; extremely expensive Hemophilia B Factor IX (F9) Adeno-associated virus vector Patients received a single IV injection Stable expression of factor IX at 1–7% of normal levels up to 3 yr posttreatment; >20 patients able to stop prophylactic factor IX treatment Leber congenital amaurosis or earlyonset severe retinal dystrophy (one form) RPE65, a protein required for the cycling of retinoids (vitamin A metabolites) to photoreceptors (RPE65) Adeno-associated virus vector Retinal pigment epithelial cells FDA approved for treatment of individuals age 12 mo–65 yr with either congenital or early-onset retinal dystrophy due to pathogenic variants in RPE65 ADA, Adenosine deaminase; Hb, hemoglobin; IV, intravenous; PEG, polyethylene glycol; SCID, severe combined immunodeficiency. BOX 14.1 ESSENTIAL REQUIREMENTS OF GENE THERAPY FOR AN INHERITED DISORDER Identity of the molecular defect The identity of the affected gene must be known. A functional copy of the gene A complementary DNA (cDNA) clone of the gene or the gene itself must be available. If the gene or cDNA is too large for the current generation of vectors, a functional version of the gene from which nonessential components have been removed to reduce its size may suffice. An appropriate vector The most commonly used vectors at present are derived from the adeno-associated viruses (AAVs) or retroviruses, including lentivirus. Knowledge of the pathophysiologic mechanism Knowledge of the pathophysiologic mechanism of the disease must be sufficient to suggest that the gene transfer will ameliorate or correct the pathologic process and prevent, slow, or reverse critical phenotypic abnormalities. Loss-of-function variants require replacement with a functional gene; for diseases due to dominant negative alleles, inactivation of the mutant gene or its products will be necessary. Favorable risk-to-benefit ratio A substantial disease burden and a favorable risk-tobenefit ratio, in comparison with alternative therapies, must be present. Appropriate regulatory components for the transferred gene Tight regulation of the level of gene expression is relatively unimportant in some diseases and critical in others. In thalassemia, for example, overexpression of the transferred gene would cause a new imbalance of globin chains in red blood cells, whereas low levels of expression would be ineffective. In some enzymopathies, a few percent of normal expression may be therapeutic, and abnormally high levels of expression may have no adverse effect. An appropriate target cell Ideally, the target cell must have a long half-life or good replicative potential in vivo. It must also be accessible for direct introduction of the gene or, alternatively, it must be possible to deliver sufficient copies of the gene to it (e.g., through the bloodstream) to attain a therapeutic benefit. The feasibility of gene therapy is often enhanced if the target cell can be cultured in vitro to facilitate gene transfer into it; in this case, it must be possible to introduce a sufficient number of the recipient cells into the patient and have them functionally integrate into the relevant organ. Strong evidence of efficacy and safety Cultured cell and animal studies must indicate that the vector and gene construct are both effective and safe. The ideal precedent is to show that the gene therapy is effective, benign, and enduring in a large animal genetic model of the disease in question. At present, however, large animal models exist for only a few monogenic diseases. Genetically engineered or spontaneous mutant mouse models are much more widely available. Regulatory approval Protocol review and approval by an institutional review board are essential. In most countries, human gene therapy trials are also subject to oversight by a governmental agency. ▶

CHAPTER 14 — The Treatment of Genetic Disease 313 TABLE 14.4 Examples of Inherited Diseases Treated by Gene Therapy of Somatic Tissues Disease Affected Protein (Gene) Vector, Cell Transduced Outcome X...

Ch14 · Pt22 314 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Gene therapy may also be undertaken to inactivate the product of a dominant mutant allele whose abnormal product causes the disease. For example, vectors carrying siRNAs (see earlier section) could, in principle, be used to mediate the selective degradation of a mutant mRNA encoding a dominant negative proα1(I) collagen that causes osteogenesis imperfecta (see Chapter 13). Gene Transfer Strategies An appropriately engineered gene may be transferred into target cells by one of two general strategies (Fig. 14.16). The first involves introduction of the gene into cells that have been cultured from the patient ex vivo (i.e., outside the body) and then reintroduction of the cells to the patient after the gene transfer. In the second approach, the gene is injected directly in vivo into the tissue or extracellular fluid of interest (from which it is taken up by the target cells). In some cases, it may be desirable to target the vector to a specific cell type; this is usually achieved by modifying the coat of a viral vector so that only the designated cells bind the viral particles. The Target Cell The ideal target cells are stem cells (which are selfreplicating) or progenitor cells taken from the patient (thereby eliminating the risk for graft-versus-host disease); both cell types have substantial replication potential. Introduction of the gene into stem cells can result in the expression of the transferred gene in a large population of daughter cells. At present, bone marrow is the only tissue whose stem cells have been successfully targeted as recipients of transferred genes. Genetically modified bone marrow stem cells have been used to cure two forms of SCID, as discussed later. Gene transfer therapy into blood stem cells is also likely to be effective for the treatment of hemoglobinopathies and storage diseases for which bone marrow transplantation has been effective, as discussed earlier. An important logistical consideration is the number of cells into which the gene must be introduced to have a significant therapeutic effect. To treat PKU, for example, the approximate number of liver cells into which the phenylalanine hydroxylase gene would have to be transferred is ~5% of the hepatocyte mass, or ~1010 cells, although this number could be much less if the level of expression of the transferred gene is higher than wild-type. A much greater challenge is gene therapy for muscular dystrophies, for which the gene must be inserted into a significant fraction of the huge number of myocytes in the body to have therapeutic efficacy. Protein-coding sequences (cDNA) Promoter/ enhancer Functional human "gene" Functional human "gene" Functional gene in a packaged viral vector Plasmid Regulatory elements determining: - amount of expression - tissue specificity - timing of expression Protein coding sequence Viral DNA Viral DNA Plasmid Patient's cells in culture Patient Regulatory elements Most commonly, a retroviral vector or an adeno-associated viral vector or Figure 14.16 The two major strategies used to transfer a gene to a patient. For patients with a genetic disease, the most common approach is to construct a viral vector containing the human complementary DNA (cDNA) of interest and to introduce it directly into the patient or into cells cultured from the patient that are then returned to the patient. The viral components at the ends of the molecule are required for the integration of the vector into the host genome. In some instances, the gene of interest is placed in a plasmid, which is then used for the gene transfer. ▶

314 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Gene therapy may also be undertaken to inactivate the product of a dominant mutant allele whose abnormal product causes the disease. For exa...

Ch14 · Pt23 CHAPTER 14 — The Treatment of Genetic Disease 315 DNA Transfer Into Cells: Viral Vectors The ideal vector for gene therapy would be safe, readily made, and easily introduced into the appropriate target tissue, and it would express the gene of interest for life. Indeed, no single vector is satisfactory in all respects for all types of gene therapy, and a repertoire of vectors is required. Here, we briefly review three of the most widely used classes of viral vectors, those derived from retroviruses, adeno-associated viruses (AAVs), and adenoviruses. One of the most widely used classes of vectors is derived from retroviruses, simple RNA viruses that can integrate into the host genome. They contain only three structural genes, which can be removed and replaced with the gene to be transferred (see Fig. 14.16). The current generation of retroviral vectors has been engineered to render them incapable of replication. In addition, they are nontoxic to the cell, and only a low number of copies of the viral DNA (with the transferred gene) integrate into the host genome. Moreover, the integrated DNA is stable and can accommodate up to 8 kb of added DNA, commodious enough for many genes that might be transferred. A major limitation of many retroviral vectors, however, is that the target cell must undergo division for integration of the virus into the host DNA, limiting the use of such vectors in nondividing cells such as neurons. In contrast, lentiviruses, the class of retroviruses that includes HIV, are capable of DNA integration in nondividing cells, including neurons. Lentiviruses have the additional advantage of not showing preferential integration into any specific gene locus, thus reducing the chances of activating an oncogene in a large number of cells. AAVs do not elicit strong immunologic responses, a great advantage that enhances the longevity of their expression. Moreover, they infect dividing or nondividing cells to remain in a predominantly episomal form that is stable and confers long-term expression of the transduced gene. A disadvantage is that the current AAV vectors can accommodate inserts of up to only 5 kb, which is smaller than many genes in their natural context. The third group of viral vectors, adenovirus-derived vectors, can be obtained at high titer, will infect a wide variety of dividing or nondividing cell types, and can accommodate inserts of 30 to 35 kb. However, in addition to other limitations, they have been associated with at least one death in a gene therapy trial through the elicitation of a strong immune response. At present their use is restricted to gene therapy for cancer. Risks of Gene Therapy Gene therapy for the treatment of human disease has risks of three general types: Adverse response to the vector or vector-disease combination. Principal among the concerns is that the patient will have an adverse reaction to the vector or the transferred gene. Such problems should be largely anticipated with appropriate animal and preliminary human studies. Insertional mutagenesis causing malignancy. The second concern is insertional mutagenesis – that is, the transferred gene will integrate into the patient’s DNA and activate a protooncogene or disrupt a tumor suppressor gene, leading possibly to cancer (see Chapter 16). The illicit expression of an oncogene is less likely to occur with the current generation of viral vectors, which have been altered to minimize the ability of their promoters to activate the expression of adjacent host genes. Insertional inactivation of a tumor suppressor gene is likely to be infrequent and, as such, is an acceptable risk in diseases for which there is no therapeutic alternative. Insertional inactivation of an essential gene. A third risk – that insertional inactivation could disrupt a gene essential for viability – will, in general, be without significant effect because such lethal mutations are expected to be rare and will kill only single cells. Although vectors appear to somewhat favor insertion into transcribed genes, the chance that the same gene will be disrupted in more than a few cells is extremely low. The one exception to this statement applies to the germline; an insertion into a gene in the germline could create a dominant disease-causing mutation that might manifest in the treated patient’s offspring. Such events, however, are likely to be rare and the risk acceptable because it would be difficult to justify withholding, on this basis, carefully planned and reviewed trials of gene therapy from patients who have no other recourse. Moreover, the problem of germline modification by disease treatment is not confined to gene therapy. For example, most chemotherapy used in the treatment of malignant disease is mutagenic, but this risk is accepted because of the therapeutic benefits. Diseases That Have Been Amenable to Gene Therapy More than two dozen single-gene diseases have been shown to improve with gene therapy, and a large number of other monogenic disorders are potential candidates for this strategy, including retinal degenerations, hematopoietic conditions such as sickle cell anemia and thalassemia, and disorders affecting liver proteins such as PKU, urea cycle disorders, familial hypercholesterolemia, and α1AT deficiency. Here we discuss several disorders in which gene therapy has been clearly effective, and we highlight some of the challenges associated with this therapeutic approach. X-linked Severe Combined Immunodeficiency The SCIDs are due to pathogenic variants in genes required for lymphocyte maturation. Affected individuals fail to thrive and die early in life of infection because ▶

CHAPTER 14 — The Treatment of Genetic Disease 315 DNA Transfer Into Cells: Viral Vectors The ideal vector for gene therapy would be safe, readily made, and easily introduced into the appropriate targe...

Ch14 · Pt24 316 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE they lack functional B and T lymphocytes. The most common form of the disease, X-linked SCID, results from variations in the X-linked gene IL2RG, encoding the γc-cytokine receptor subunit of several interleukin receptors. The receptor deficiency causes an early block in T- and natural killer–lymphocyte growth, survival, and differentiation and is associated with severe infections, failure to thrive, and death in infancy or early childhood if left untreated. This condition was chosen for a gene therapy trial for two principal reasons. First, bone marrow transplantation cures the disease, indicating that the restoration of lymphocyte expression of IL2RG can reverse the pathophysiologic changes. Second, it was believed that transduced cells carrying the transferred gene would have a selective survival advantage over untransduced cells. The outcome of trials of X-linked SCID has been dramatic and resulted, in 2000, in the first gene therapy cure of a patient with a genetic disease. Subsequent confirmation has been obtained in most patients in subsequent clinical trials (see Table 14.4). Bone marrow stem cells from the patients were infected in culture (ex vivo) with a retroviral vector that expressed the γc cytokine subunit cDNA. A selective advantage was conferred on the transduced cells by the gene transfer. Transduced T cells and natural killer cells populated the blood of treated patients, and the T cells appeared to behave normally. Although the frequency of transduced B cells was low, adequate levels of serum immunoglobulin and antibody levels were obtained. Dramatic clinical improvement occurred, with resolution of protracted diarrhea and skin lesions and restoration of normal growth and development. These initial trials demonstrated the great potential of gene therapy for the correction of inherited disease. This highly promising outcome, however, came at the cost of induction of a leukemia-like disorder in 5 of the 20 treated patients, who developed an extreme lymphocytosis resembling T-cell acute lymphocytic leukemia; 4 of them are now well after treatment of the leukemia. The malignancy was due to insertional mutagenesis: The retroviral vector inserted into the LMO2 locus, causing aberrant expression of the LMO2 mRNA, which encodes a component of a transcription factor complex that mediates hematopoietic development. Consequently, trials using integrating vectors in hematopoietic cells must now monitor insertion sites and survey for clonal proliferation. Current-generation vectors are designed to avoid this mutagenic effect by using strategies such as including a self-inactivating or “suicide” gene cassette in the vector to eliminate clones of malignant cells. At this point, bone marrow stem cell transplantation remains the treatment of choice for those children with SCID fortunate enough to have a donor with an HLA-identical match. For patients without such a match, autologous transplantation of HSPCs, in which the genetic defect has been corrected by gene therapy, offers a lifesaving alternative, but one that may not be without risk. Spinal Muscular Atrophy SMA is a monogenic defect associated with the loss of SMN protein and became an excellent candidate for gene replacement therapies when it was discovered that systemic delivery of AAV9-based gene transfer via intravenous injection can cross the blood-brain barrier and efficiently transform target cells in the central nervous system, including motor neurons in the spinal cord in mice and nonhuman primates. This changed the assumption that neurologic disorders may not be candidates for gene therapies. Development of the self-complementary AAV9 (sc AAV9) vector further improved the efficiency and speed of gene transcription in a number of preclinical experiments. The first gene therapy trial for SMA included 15 infants, 3 with low dose and 12 with high dose. All 15 patients survived to 20 months and did not require respiratory support. Eleven patients reached the motor milestone of sitting unassisted, and two patients were able to walk independently. These data plus additional data released from the successive phase II/III trial led to FDA approval in 2019. The main benefits of this method are that a single, one-time injection is required, and the SMN protein becomes systemically expressed. Safety and tolerability, however, have to be strictly monitored as acute hepatotoxicity and sensory neuron toxicity were reported in primates and piglets following high-dose intravenous administration of AAV vectors expressing human SMN. As a result, most individuals require prednisolone therapy to minimize the hepatotoxicity. Another consideration is the reported presence of preexisting anti-AAV9 antibodies in SMA patients, which might influence tolerability and efficacy. RPE65-associated Retinal Dystrophy Leber congenital amaurosis (LCA), an autosomal recessive condition, comprises a heterogeneous group of eye diseases that cause nystagmus and significant visual impairment in early infancy and total blindness by the third to fourth decades of life. LCA2 (also known as RPE65-LCA) is associated with pathogenic variants of the RPE65 gene encoding the retinoid isomerohydrolase in the retinal pigment epithelium (RPE), which result in rod-cone–type retinal dystrophy. The gene therapy can be injected directly into the eye’s subretinal space, which is an immunoprivileged organ. The majority of studies are conducted on one eye, while the other serves as a control. For RPE65-associated retinal dystrophy voretigene neparvovec is a gene therapy that packages the RPE65 cDNA in an AAV2 vector. This therapy was approved by the FDA in 2017. Follow-up of the original group through 4 years has shown sustained benefit, and additional gene therapy trials are currently underway to treat several other types of LCA. ▶

316 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE they lack functional B and T lymphocytes. The most common form of the disease, X-linked SCID, results from variations in the X-linked gene I...

Ch14 · Pt25 CHAPTER 14 — The Treatment of Genetic Disease 317 Hemophilia B Hemophilia B is an X-linked disorder of coagulation caused by pathogenic variants in the F9 gene, leading to a deficiency or dysfunction of clotting factor IX (Case 21). The disease is characterized by bleeding into soft tissues, muscles, and weight-bearing joints and occurs within hours to days after trauma. Severely affected subjects, with less than 1% of normal levels of factor IX, have frequent bleeding that causes crippling joint disease and early death. Prophylactic – but not curative – treatment with intravenous factor IX concentrate several times a week is expensive and leads to the generation of inhibitory antibodies. In 2011, the first successful gene therapy treatment of hemophilia B was reported in six patients using an AAV8 vector that is tropic for hepatocytes, where factor IX is normally produced. After a single infusion of the AAV8-F9 vector, four patients were able to discontinue prophylactic factor IX infusions, whereas the other two tolerated longer intervals between infusions. The two patients who received the highest dose of the vector had transient asymptomatic increases in liver enzyme levels – which resolved with steroid treatment – indicating that immune-related side effects must remain a concern in future studies. Unfortunately, the AAV vectors cannot accommodate the gene for factor VIII, so other vectors will have to be developed for hemophilia A patients. Apart from this limitation of cargo size, however, AAV-mediated gene therapy targeted to hepatocytes may be applicable to any genetic disease in which production of the protein in the liver is the desired goal. The Prospects for Gene Therapy To date, more than 5000 clinical gene therapy trials (the majority are for cancer) have been undertaken worldwide to evaluate both the safety and efficacy of this long-promised and conceptually promising technology. Approximately 775 of these trials were for the treatment of monogenic diseases. The exciting results obtained with gene therapy to date, albeit with small numbers of patients and only a few diseases, validate the optimism behind this immense effort. Although the breadth of applications remains uncertain, it is to be hoped that over the next few decades, gene therapy for both monogenic and genetically complex diseases will contribute to the management of many disorders, both common and rare. PRECISION MEDICINE: THE PRESENT AND FUTURE OF THE TREATMENT OF MENDELIAN DISEASE The treatment of single-gene diseases embodies the concept of precision medicine tailored to the individual patient as deeply as any other area of medical treatment. Knowledge of the specific sequence in an individual is central to many of the targeted therapies described in this chapter. The promise of gene therapy for an individual with a mendelian disorder must be based on the identification of the responsible gene in each affected individual and on the design of a vector that will deliver the therapeutic gene to the targeted tissue. Similarly, approaches based on gene editing require knowledge of the specific variant to be corrected. Beyond this, however, precision medicine will frequently require knowledge of the precise allele and of its specific effect on the mRNA and protein. In many cases, the exact nature of the variant will define the drug that will bind to a specific regulatory sequence to enhance or reduce the expression of a gene. In other cases, the variant will dictate the sequence of an allele-specific oligonucleotide to mediate the skipping of an exon with a premature termination codon, or of an siRNA to suppress a dominant negative allele. A compendium of small molecules will gradually become available to act as chaperones that will rescue mutant proteins from misfolding and proteasomal degradation, or to modulate the activity of mutant proteins. Genetic treatment is not only becoming more creative but also more precise. The future promises a longer and vastly improved quality life for many patients. GENERAL REFERENCES Valle D, Beaudet AL, Vogelstein B, et al, editors: The online metabolic and molecular bases of inherited disease, 2019. Vernon HJ, Manoli I: Milestones in treatments for inborn errors of metabolism: reflections on where chemistry and medicine meet, Am J Med Genet Part A 185A:3350–3358, 2021. REFERENCES FOR SPECIFIC TOPICS Arora N, Daley GQ: Pluripotent stem cells in research and treatment of hemoglobinopathies, Cold Spring Harb Perspect Med 2:a 011841, 2012. Bélanger-Quintana A, Burlina A, Harding CO, et al: Up to date knowledge on different treatment strategies for phenylketonuria, Mol Genet Metabolism 104:S19–S25, 2011. Biffi A, Montini E, Lorioli L, et al: Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy, Science 341:1233158, 2013. https://doi.org/10.1126/science.1233158 Birnkrant DJ, Bushby K, Bann CM, et al: Diagnosis and management of Duchenne muscular dystrophy, part 1: Diagnosis, and neuromuscular, rehabilitation, endocrine, and gastrointestinal and nutritional management, Lancet Neurol 17:251–267, 2018. Birnkrant DJ, Bushby K, Bann CM, et al: Diagnosis and management of Duchenne muscular dystrophy, part 2: Respiratory, cardiac, bone health, and orthopaedic management, Lancet Neurol 17:347–361, 2018. Birnkrant DJ, Bushby K, Bann CM, et al: Diagnosis and management of Duchenne muscular dystrophy, part 3: Primary care, emergency management, psychosocial care, and transitions of care across the lifespan, Lancet Neurol 17:445–455, 2018. Cathomen T, Ehl S: Translating the genomic revolution – targeted genome editing in primates, N Engl J Med 370:2342–2345, 2014. Coelho T, Adams D, Silva A, et al: Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med 369(9):818–829, 2013. Daley GQ: The promise and perils of stem cell therapeutics, Cell Stem Cell 10:740–749, 2012. Desnick RJ, Schuchman EH: Enzyme replacement therapy for lysosomal diseases: Lessons from 20 years of experience and remaining challenges, Ann Rev Genomics Hum Genet 13:307–335, 2012. de Souza N: Primer: Genome editing with engineered nucleases, Nat Methods 9:27, 2012. ▶

CHAPTER 14 — The Treatment of Genetic Disease 317 Hemophilia B Hemophilia B is an X-linked disorder of coagulation caused by pathogenic variants in the F9 gene, leading to a deficiency or dysfunction...

Ch14 · Pt26 318 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Dong A, Rivella S, Breda L: Gene therapy for hemoglobinopathies: Progress and challenges, Trans Res 161:293–306, 2013. Duan D, Goemans N, Takeda S, et al: Duchenne muscular dystrophy, Nat Rev Dis Primers 7:13, 2021. Gaspar HB, Qasim W, Davies EG, et al: How I treat severe combined immunodeficiency, Blood 122:3749–3758, 2013. Gaziev J, Lucarelli G: Hematopoietic stem cell transplantation for thalassemia, Curr Stem Cell Res Ther 6:162–169, 2011. Goemans NM, Tulinius M, van den Akker JT: Systemic administration of PRO051 in Duchenne’s muscular dystrophy, N Engl J Med 364: 1513–1522, 2011. Groenink M, den Hartog AW, Franken R, et al: Losartan reduces aortic dilatation rate in adults with Marfan syndrome: A randomized controlled trial, Eur Heart J 34:3491–3500, 2013. Hanna JH, Saha K, Jaenisch R: Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues, Cell 143:508–525, 2010. Hanrahan JW, Sampson HM, Thomas DY: Novel pharmacological strategies to treat cystic fibrosis, Trends Pharmacol Sci 34:119–125, 2013. High KA: Gene therapy in clinical medicine. In Longo D, Fauci A, Kasper D, editors: Harrison's principles of internal medicine, ed 19, New York, 2015, Mc Graw-Hill. Huang R, Southall N, Wang Y, et al: The NCGC Pharmaceutical Collection: A comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics, Sci Transl Med 3:80ps 16, 2011. Iftikhar M, Frey J, Shohan MJ, et al: Current and emerging therapies for Duchenne muscular dystrophy and spinal muscular atrophy, Pharmacol Ther 220:107719, 2021. Jarmin S, Kymalainen H, Popplewell L, et al: New developments in the use of gene therapy to treat Duchenne muscular dystrophy, Expert Opin Biol Ther 14:209–230, 2014. Johnson SM, Connelly S, Fearns C, et al: The transthyretin amyloidoses: from delineating the molecular mechanism of aggregation linked to pathology to a regulatory agency approved drug, J Mol Biol 421: 185–203, 2012. Kim J, Hu C, El Achkar MC, et al: Patient-customized oligonucleotide therapy for a rare genetic disease, N Engl J Med 381(17): 1644–1652, 2019. https://doi.org/10.1056/NEJMoa 1813279 Li M, Suzuki K, Kim NY, et al: A cut above the rest: Targeted genome editing technologies in human pluripotent stem cells, J Biol Chem 289:4594–4599, 2014. Mallack EJ, Turk B, Yan H, et al: The landscape of hematopoietic stem cell transplant and gene therapy for X-linked adrenoleukodystrophy, Curr Treat Options Neurol 21(12):61, 2019. https://doi. org/10.1007/s 11940-019-0605-y Mukherjee S, Thrasher AJ: Gene therapy for primary immunodeficiency disorders: Progress, pitfalls and prospects, Gene 525:174– 181, 2013. Nathwani AC, Tuddenham EGD, Rangarajan S: Adenovirus-associated virus vector–mediated gene transfer in hemophilia B, N Engl J Med 365:2357–2365, 2011. Nicolau S, Waldrop MA, Connolly AM, et al: Spinal muscular atrophy, Semin Pediatr Neurol 37:100878, 2021. Okam MM, Ebert BL: Novel approaches to the treatment of sickle cell disease: the potential of histone deacetylase inhibitors, Expert Rev Hematol 5:303–311, 2012. Otsuru S, Gordon PL, Shimono K, et al: Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms, Blood 120: 1933–1941, 2012. Peltz SW, Morsy M, Welch EW, et al: Ataluren as an agent for therapeutic nonsense suppression, Ann Rev Med 64:407–425, 2013. Perrine SP, Pace BS, Faller DV: Targeted fetal hemoglobin induction for treatment of beta hemoglobinopathies, Hematol Oncol Clin North Am 28:233–248, 2014. Pillai NR, Stroup BM, Poliner A, et al: Liver transplantation in propionic and methylmalonic acidemia: A single center study with literature review, Mol Genet Metab 128(4):431–443, 2019. https://doi. org/10.1016/j.ymgme.2019.11.001 Prasad VK, Kurtzberg J: Cord blood and bone marrow transplantation in inherited metabolic diseases: Scientific basis, current status and future directions, Br J Haematol 148:356–372, 2009. Pritchard AB, Izumi K, Payan-Walters I, et al: Inborn error of metabolism patients after liver transplantation: Outcomes of 35 patients over 27 years in one pediatric quaternary hospital, Am J Med Genet A 188(5):1443–1447, 2022. https://doi.org/10.1002/ajmg.a.62659 Ramdas S, Servais L: New treatments in spinal muscular atrophy: An overview of currently available data, Exp Opin Pharmacother 21:307–315, 2020. Ramsey BW, Davies J, Mc Elvaney NG, et al: A CFTR potentiator in patients with cystic fibrosis and the G551D mutation, N Engl J Med 365:1663–1672, 2011. Robinton DA, Daley GQ: The promise of induced pluripotent stem cells in research and therapy, Nature 481:295–305, 2012. Salmaninejad A, Abarghan JY, Qomi BS, et al: Common therapeutic advances for Duchenne muscular dystrophy (DMD), Int J Neurosci 131:370–389, 2021. Sander JD, Joung JK: CRISPR-Cas systems for editing, regulating and targeting genomes, Nat Biotechnol 32:347–355, 2014. Schorling DC, Pechmann A, Kirschner J: Advances in treatment of spinal muscular atrophy – new phenotypes, new challenges, new implications for care, J Neuromuscul Dis 7:1–13, 2020. Sheikh O, Yokota T: Developing DMD therapeutics: A review of the effectiveness of small molecules, stop-codon readthrough, dystrophin gene replacement, and exon-skipping therapies, Expert Opin Investig Drugs 30:167–176, 2021. Sosicka P, Ng BG, Freeze HH: Chemical therapies for congenital disorders of glycosylation, ACS Chem Biol, 2021. https://doi.org/10.1021/ acschembio.1c00601 Southwell AL, Skotte NH, Bennett CF, et al: Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases, Trends Mol Med 18:634–643, 2012. Tebas P, Stein D, Tang WW, et al: Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV, N Engl J Med 370:901–910, 2014. van Ommen G-JB, Aartsma-Rus A: Advances in therapeutic RNAtargeting, Trends Mol Med 18:634–643, 2012. Verhaart IEC, Aarsma-Rus A: Therapeutic developments for Duchenne muscular dystrophy, Nat Rev Neurol 15:373–386, 2019. Verma IM: Gene therapy that works, Science 341:853–855, 2013. Xu J, Peng C, Sankaran VG, et al: Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing, Science 334:993–996, 2011. USEFUL WEBSITES Registry and results database of publicly and privately supported clinical studies of human participants conducted around the world: https://clinicaltrials.gov/ Gene Therapy Clinical Trials Worldwide: http://www.wiley.com/ legacy/wileychi/genmed/clinical/ PROBLEMS 1. X-linked chronic granulomatous disease (CGD) is characterized by a defect in host defense that leads to severe, recurrent, and often fatal pyogenic infections beginning in early childhood. The X-linked CGD locus encodes the heavy chain of cytochrome b, a component of the oxidase that generates superoxide in phagocytes. Because interferon-γ (IFN-γ) enhances the oxidase activity of normal phagocytes, IFN-γ was administered to boys with X-linked CGD to see whether their oxidase activity increased. Before treatment, unlike those of severely affected patients, the phagocytes of some less severely affected patients had small but detectable bursts of oxidase activity, suggesting that their increased activity resulted from greater production of cytochrome b from the affected locus. In these cases, IFN-γ increased the cytochrome b content, superoxide production, and killing continued ▶

318 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Dong A, Rivella S, Breda L: Gene therapy for hemoglobinopathies: Progress and challenges, Trans Res 161:293–306, 2013. Duan D, Goemans N, Ta...

Ch14 · Pt27 CHAPTER 14 — The Treatment of Genetic Disease 319 of Staphylococcus aureus in the granulocytes. The IFN-γ effect was associated with a definite increase in the abundance of the cytochrome b heavy chain. Presumably, the cytochrome b polypeptide of these patients is partially functional, and its increased expression improved the physiological defect. Describe the genetic differences that might account for the differential response of phagocytes of patients with X-linked CGD to IFN-γ in vitro. 2. Identify some of the limitations on the types of proteins that can be considered for extracellular enzyme replacement therapy, as exemplified by polyethylene glycol– adenosine deaminase (PEG-ADA) for ADA deficiency. What makes this approach inappropriate for phenylalanine hydroxylase deficiency? If Tay-Sachs disease caused only liver disease, would this strategy succeed? If not, why? Might Lesch-Nyhan disease be a candidate for this approach? 3. A 3-year-old girl, Rhonda, has familial hypercholesterolemia due to a deletion of the 5′ end of each of her lowdensity lipoprotein (LDL) receptor genes that removed the promoter and the first two exons. (Rhonda’s parents are second cousins.) You explain to the parents that she will require plasmapheresis every 1 to 2 weeks for years. At the clinic, however, they meet another family with a 5-year-old boy with the same disease. The boy has been treated with drugs with some success. Rhonda’s parents want to know why she has not been offered similar pharmacologic therapy. Explain. 4. What classes of variants are likely to be found in individuals with homocystinuria who are not responsive to the administration of large doses of pyridoxine (vitamin B6)? How might you explain the fact that Tom is completely responsive, whereas his first cousin Allan has only a partial reduction in plasma homocysteine levels when he is given the same amount of vitamin B6? 5. You have isolated the gene for phenylalanine hydroxylase (PAH) and wish ultimately to introduce it into patients with PKU. Your approach will be to culture cells from the patient, introduce a functional version of the gene into the cells, and reintroduce the cells into the patient. a. What DNA components do you need to make a functional PAH protein in a gene transfer experiment? b. Which tissues would you choose in which to express the enzyme, and why? How does this choice affect your gene construct in (a)? c. You introduce your version of the gene into fibroblasts cultured from a skin biopsy specimen from the patient. Northern (RNA) blot analysis shows that the messenger RNA (mRNA) is present in normal amounts and is the correct size. However, no PAH protein can be detected in the cells. What kinds of abnormalities in the transferred gene would explain this finding? d. You have corrected all the problems identified in (c). On introducing the new version of the gene into the cultured cells, you now find that the PAH protein is present in great abundance, and when you harvest the cells and assay the enzyme (in the presence of all the required components), normal activity is obtained. However, when you add 3H-labeled phenylalanine to the cells in culture, no 3H-labeled tyrosine is formed (in contrast, some cultured liver cells produce a large quantity of 3H-labeled tyrosine in this situation). What are the most likely explanations for the failure to form 3H-tyrosine? How does this result affect your gene therapy approach to patients? e. You have developed a method to introduce your functional version of the gene directly into a large proportion of the hepatocytes of patients with PAH deficiency. Unexpectedly, you find that much lower levels of PAH enzymatic activity are obtained in patients in whom significant amounts of the inactive PAH homodimer were detectable in hepatocytes before treatment than in patients who had no detectable PAH polypeptide before treatment. How can you explain this result? How might you overcome the problem? 6. Both variant alleles of an autosomal gene in your patient produce a protein that is decreased in abundance but has residual function. What therapeutic strategies might you consider in such a situation? 7. A Phase III clinical trial is undertaken to evaluate the effectiveness of a small molecule drug that facilitates skipping over nonsense codons. The drug had been shown in earlier trials to have a modest but significant clinical effect in individuals with cystic fibrosis (CF) with at least one CFTR nonsense variant. Two individuals with CF each have a nonsense variant in one CFTR allele, but at different locations in the reading frame. One patient responds to the drug, whereas the other does not. Discuss how the location of the nonsense variant could account for this differential response. ▶

PROBLEMS—CONT’D .

Chapter 15: Developmental Genetics and Birth Defects

Ch15 · Pt1 chapter 15 Developmental Genetics and Birth Defects Anthony Wynshaw-Boris ▶

Ophir Klein Knowledge of the principles and concepts of developmental genetics, including the mechanisms and pathways responsible for normal human development in utero, is essential for the practition...

Ch15 · Pt2 322 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE rehabilitation medicine, and the allied health professions to provide ongoing care for children with serious birth defects. Malformations, Deformations, and Disruptions Medical geneticists divide birth defects into three major categories: malformations, deformations, and disruptions. We will illustrate the difference between these three categories with examples of three distinct birth defects, all involving the limbs. Malformations result from intrinsic abnormalities in one or more genetic programs operating in development. An example of a malformation is the extra fingers in the disorder known as Greig cephalopolysyndactyly (Fig. 15.1). This syndrome, discussed later in the chapter, results from loss-of-function variants in a gene for a transcription factor, GLI3, which is one component of a complex network of transcription factors and signaling molecules that interact to cause the distal end of the human upper limb bud to develop into a hand with five digits. Because malformations arise from intrinsic defects in genes that specify a series of developmental steps or programs, and because such programs are often used more than once in different parts of the embryo or fetus at different stages of development, a malformation in one part of the body is often but not always associated with malformations elsewhere as well. In contrast to malformations, deformations are caused by extrinsic factors impinging physically on the fetus during development. They are especially common during the second trimester of development when the fetus is constrained within the amniotic sac and uterus. For example, contractions of the joints of the extremities, known as arthrogryposes, in combination with deformation of the developing skull, occasionally accompany constraint of the fetus due to twin or triplet gestations or prolonged leakage of amniotic fluid (Fig. 15.2). Arthrogryposes have many causes, and some of them are intrinsic malformations due to pathogenic variants. Most deformations apparent at birth either resolve spontaneously or can be treated by external fixation devices to reverse the effects of the instigating cause. Disruptions, the third category of birth defect, result from destruction of irreplaceable normal fetal tissue. Disruptions are more difficult to treat than deformations because they involve actual loss of normal tissue. Disruptions may be the result of vascular insufficiency, trauma, or teratogens. One example is amnion disruption, the partial amputation of a fetal limb associated with strands of amniotic tissue. Amnion disruption is A B Figure 15.1 Polydactyly and syndactyly malformations. (A) Insertional polydactyly. This patient has heptadactyly with insertion of a digit in the central ray of the hand and a supernumerary postaxial digit. This malformation is typically associated with metacarpal fusion of the third and fourth digits. Insertional polydactyly is common in patients with Pallister-Hall syndrome. (B) Postaxial polydactyly with severe cutaneous syndactyly of digits 2 through 5. This type of malformation is seen in patients with Greig cephalopolysyndactyly syndrome. (Images courtesy Dr. Leslie Biesecker, Bethesda, Maryland.) Figure 15.2 Deformation known as congenital arthrogryposis seen with a condition referred to as amyoplasia. There are multiple, symmetric joint contractures due to abnormal muscle development caused by severe fetal constraint in a pregnancy complicated by oligohydramnios. Intelligence is generally normal, and orthopedic rehabilitation is often successful. (Image courtesy Dr. Judith Hall, University of British Columbia, Vancouver, Canada.) ▶

322 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE rehabilitation medicine, and the allied health professions to provide ongoing care for children with serious birth defects. Malformations, D...

Ch15 · Pt3 CHAPTER 15 — Developmental Genetics and Birth Defects 323 often recognized clinically by the presence of partial and irregular digit amputations in conjunction with constriction rings (Fig. 15.3). The pathophysiologic concepts of malformations, deformations, and disruptions are useful clinical guides to the recognition, diagnosis, and treatment of birth defects, but they sometimes overlap. For example, vascular malformations may lead to disruption of distal structures, and urogenital malformations that cause oligohydramnios can cause fetal deformations. Thus a given constellation of birth defects in an individual may represent combinations of malformations, deformations, and disruptions. Genetic, Genomic, and Environmental Causes of Malformations Malformations have many causes (Fig. 15.4). Chromo some imbalance accounts for ~25%, of which autosomal trisomies for chromosomes 21, 18, and 13 (see Chapter 6) are some of the most common. The clinical application of genome-wide arrays in comparative genomic hybridization (CGH or array-CGH; see Chapter 5) has highlighted small, de novo submicroscopic deletions and/or duplications, also known as copy number variants (CNVs), in as many as 10% of individuals with birth defects. An additional 20% are caused by variants in single genes, which are being discovered at an increasing pace with the advent of whole exome and genome sequencing. Some malformations, such as achondroplasia or Waardenburg syndrome, are inherited as autosomal dominant traits. Many heterozygotes with birth defects, however, represent new mutations that are so severe that they are genetic lethals and are therefore often found to be isolated cases within families (see Chapter 7). Other malformation syndromes are inherited in an autosomal or X-linked recessive pattern, such as the Smith-Lemli-Opitz syndrome or the Lowe syndrome, respectively. Figure 15.3 Disruption of limb development associated with amniotic bands. This 26-week fetus shows nearly complete disruption of the thumb with only a nubbin remaining. The third and fifth fingers have constriction rings of the middle and distal phalanges, respectively. The fourth digit is amputated distally with a small fragment of amnion attached to the tip. (Image courtesy Dr. Mason Barr, Jr., University of Michigan, Ann Arbor, Michigan.) Teratogen 5% Multifactorial 40% Chromosome imbalance 25% Single-gene defects 20% Copy number variants 10% Figure 15.4 The relative contribution of single-gene defects, chromosome abnormalities, copy number variants, multifactorial traits, and teratogens to birth defects. Another ~40% of major birth defects have no identifiable cause but recur in families of affected children with a greater frequency than would be expected on the basis of the population frequency and are thus considered to be multifactorial diseases (see Chapter 9). This category includes well-recognized birth defects ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 323 often recognized clinically by the presence of partial and irregular digit amputations in conjunction with constriction rings (Fig. 15.3). The...

Ch15 · Pt4 324 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE such as cleft lip with or without cleft palate, and congenital heart defects. The remaining 5% of birth defects are thought to result from exposure to certain environmental agents— drugs, infections, alcohol, chemicals, or radiation—or from maternal metabolic disorders such as poorly controlled maternal diabetes mellitus or maternal phenylketonuria (see Chapter 13). Such agents are called teratogens (derived, inelegantly, from the Greek word for monster plus -gen, meaning cause) because of their ability to cause malformations (discussed later in this chapter). Pleiotropy: Syndromes and Sequences A birth defect resulting from a single underlying causative agent may result in abnormalities of more than one organ system in different parts of the embryo or in multiple structures that arise at different times during intrauterine life, a phenomenon referred to as pleiotropy. The agent responsible for the malformation could be either a mutant gene or a teratogen. Pleiotropic birth defects come about in two different ways, depending on the mechanism by which the causative agent produces its effect. When the causative agent causes multiple abnormalities in parallel, the collection of abnormalities is referred to as a syndrome. If, however, a mutant gene or teratogen affects only a single organ system at one point in time, and it is the perturbation of that organ system that causes the rest of the constellation of pleiotropic defects to occur as secondary effects, the malformation is referred to as a sequence. Pleiotropic Syndromes. The autosomal dominant branchio-oto-renal dysplasia syndrome exemplifies a pleiotropic syndrome. It has long been recognized that patients with branchial arch anomalies affecting development of the ear and neck structures are at high risk for having renal anomalies. The branchio-oto-renal dysplasia syndrome, for example, consists of abnormal cochlear and external ear development, cysts and fistulas in the neck, renal dysplasia, and renal collecting duct malformations. The mechanism of this association is that a conserved set of genes and proteins are used by mammals to form both the ear and the kidney. The syndrome is caused by pathogenic variant in one of three genes—EYA1, SIX1, or SIX5—which encode transcriptional regulators that function in both ear and kidney development. Similarly, the Rubinstein-Taybi syndrome, caused by loss of function in a transcriptional coactivator, results in abnormalities in the transcription of many genes that depend on this coactivator being present in a transcription complex for normal expression (Fig. 15.5). Sequences. In contrast, an example of a sequence is the U-shaped cleft palate and small mandible referred to as the Robin sequence (Fig. 15.6). This sequence comes about because a restriction of mandibular growth before the ninth week of gestation causes the tongue to lie more posteriorly than is normal, interfering with normal closure of the palatal shelves, thereby causing a cleft palate. The Robin sequence can be an isolated birth defect of unknown cause or can be due to extrinsic impingement on the developing mandible by a twin in utero. This A B Figure 15.5 Physical characteristics of patients with Rubinstein-Taybi syndrome, a highly variable and pleiotropic syndrome of developmental delay, distinctive facial appearance, broad thumbs and large toes, and congenital heart defects. The syndrome is caused by loss-of-function variants in one of two different but closely related transcriptional coactivators, CBP or EP300. (A) Distinctive facial features. (B) Appearance of hands and feet. (Reprinted with permission from Jones KL, Jones MC, del Campo M: Smith’s recognizable patterns of human malformation, ed 7, Philadelphia, 2013, WB Saunders.) ▶

324 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE such as cleft lip with or without cleft palate, and congenital heart defects. The remaining 5% of birth defects are thought to result from e...

Ch15 · Pt5 CHAPTER 15 — Developmental Genetics and Birth Defects 325 phenotype can also be one of several features of a condition known as Stickler syndrome, in which pathogenic variants in one of six genes encoding subunits of collagen result in an abnormally small mandible as well as other defects in stature, joints, and eyes. The Robin sequence in Stickler syndrome is thought to be a sequence because the mutant collagen gene itself is likely not responsible for the failure of palatal closure; rather, the cleft palate appears to be secondary to the primary defect in jaw growth. Whatever the cause, it is useful to distinguish a cleft palate due to the Robin sequence from other types of cleft palate, which can have differing prognoses and implications for the child and family. Knowledge of dysmorphology and developmental genetic principles is thus necessary to properly diagnose each condition and to recognize that different prognoses are associated with the different primary causes. INTRODUCTION TO DEVELOPMENTAL BIOLOGY The examples introduced briefly in the previous section serve to illustrate the principle that the clinical practice of medical genetics rests on a foundation of the basic science of developmental biology. For this reason, it behooves practitioners to have a working knowledge of Normal Robin sequence Robin sequence (“U-shaped”) Primary cleft palate (“V-shaped”) Palate Tongue A D C B Figure 15.6 (A) Hypoplasia of the mandible and resulting posterior displacement of the tongue lead to the Robin sequence, in which the tongue obstructs palatal closure. (B) Posterior placement of the tongue in the Robin sequence causes a deformation of the palate during development, leading to the constellation of a small chin and a U-shaped cleft palate involving the soft palate and extending into the hard palate. (C) In contrast, primary cleft palate resulting from failure of closure of maxillary ridges is a malformation that begins in the anterior region of the maxilla and extends posteriorly to involve first the hard palate and then the soft palate, and it is often V shaped. (D) The delay in jaw development can be observed by serial three-dimensional fetal scans, from as early as 17 weeks (left) to 20 weeks (middle) and 29 weeks (right). (A–C, Adapted from Wolpert L: Principles of development, New York, 2002, Oxford University Press; D, from Pooh RK, Kurjak A: Recent advances in 3D assessment of various fetal anomalies, J Ultrasound Obstet Gynecol 3:1–23, 2009.) ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 325 phenotype can also be one of several features of a condition known as Stickler syndrome, in which pathogenic variants in one of six genes enco...

Ch15 · Pt6 326 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE some of the basic principles of developmental biology and to be familiar with the ways that abnormal function of genes and pathways affect development and, ultimately, their patients. Developmental biology is concerned with a single, unifying question: How can a single cell transform itself into a mature organism? In humans, this transformation occurs each time a single fertilized egg develops into a human being with more than 1013 to 1014 cells, several hundred recognizably distinct cell types, and dozens of tissues. This process must occur in a reliable and predictable pattern and time frame. Developmental biology has its roots in embryology, which was based on observing and surgically manipulating developing organisms. Early animal embryologic studies, carried out in the 19th and early 20th centuries with readily accessible amphibian and avian embryos, determined that embryos developed from single cells and defined many of the fundamental processes of development. Much more recently, the application of molecular biology, genetics, and genomics to embryology has transformed the field by allowing scientists to study and manipulate development by a broad range of powerful biochemical and molecular techniques. Of note, the rapid advancement of next generation sequencing has allowed the development of single-cell RNA sequencing technologies (sc RNAseq), which has underscored the transcriptional diversity of cells throughout development and in the adult. Development and Evolution A critically important theme in developmental biology is its relationship to the study of evolution. Early in development, the embryos of many species have important similarities. As development progresses, the features shared between species are successively transformed into more specialized features that are, in turn, shared by successively fewer but more closely related species. A comparison of embryologic characteristics among and within evolutionarily related organisms shows that developmental attributes (e.g., fingers) specific to certain groups of animals (e.g., primates) are built on a foundation of less specific attributes common to a larger group of animals (e.g., mammals), which are in turn related to structures seen in an even larger group of animals (e.g., vertebrates). Structures in different organisms are termed homologous if they evolved from a structure present in a common ancestor (Fig. 15.7). In the case of the forelimb, the various ancestral lineages of the three species shown in Fig. 15.7, tracing back to their common predecessor, share a common attribute: a functional forelimb. The molecular developmental mechanism that created those limb structures is shared across all three of the contemporary species. Not all similarity is due to homology, however. Evolutionary studies also recognize the existence of analogous structures, those that appear similar but arose independently of one another, through different lineages that cannot be traced back to a common ancestor with that structure. The molecular pathways that generate analogous structures are often not evolutionarily conserved. In the example shown in Fig. 15.7, the wing structures of the bat and the birds arose independently in evolution to facilitate the task of aerial movement. The evolutionary lineages of these two animals do not share a common ancestor with a primitive winglike structure from which both bats and birds inherited wings. On the contrary, one can readily see that the birds developed posterior extensions from the limb to form a wing, whereas bats evolved wings by spreading the digits of their forelimbs and connecting them with syndactylous tissue. This situation is termed convergent evolution. The evolutionary conservation of developmental processes is critically important to studies of human development because the vast majority of such research cannot (for important ethical reasons) be performed in humans (see Chapter 20). Thus, to understand a developmental observation, scientists use animal models to investigate normal and abnormal developmental processes. The ability to extend the results to humans is completely dependent on the evolutionary conservation of mechanisms of development and homologous structures. GENES AND ENVIRONMENT IN DEVELOPMENT Developmental Genetics Development results from the action of genes interacting with environmental cues. The gene products involved include transcriptional regulators, growth factors (diffusible signals that interact with cells and direct them toward specific developmental pathways), the receptors for such factors, structural proteins, intracellular signaling molecules, and many others. It is therefore not surprising that most of the numerous developmental disorders that occur in humans are caused by chromosomal, subchromosomal, or gene variants. Even though the genome is clearly the primary source of information that controls and specifies human development, the role of genes in development is often mistakenly described as a “master blueprint.” In reality, however, the genome does not resemble an architect’s blueprint that specifies precisely how the materials are to be used, how they are to be assembled, and their final dimensions; it is not a literal description of the final form that all embryologic and fetal structures will take. Rather, the genome specifies a set of interacting proteins and noncoding RNAs (see Chapter 3) that set in motion the processes of growth, migration, differentiation, and apoptosis that ultimately result, with a high degree of ▶

326 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE some of the basic principles of developmental biology and to be familiar with the ways that abnormal function of genes and pathways affect d...

Ch15 · Pt7 CHAPTER 15 — Developmental Genetics and Birth Defects 327 Human arm Bird wing Bat wing Upper arm Lower arm Carpals Metacarpal Phalanges Figure 15.7 Diagram of the upper limb of three species: human, bird, and bat. Despite the superficially dissimilar appearance of the human arm and hand, the avian wing, and the bat wing, the similarity in their underlying bone structure and functionality reveals the homology of the forelimbs of all three species. In contrast, the two superficially similar wings in the bird and bat are analogous, not homologous structures. Although both the bird and bat wings are used for flying, they are constructed quite differently and did not evolve from a winglike structure in a common ancestor. (Redrawn from Hauk R: Frequently asked questions about bats, 2011, Western National Parks Association.) probability, in the correct mature structures. Thus, for example, there are no genetic instructions directing that the phalanx of a digit adopt an hourglass shape or that the eye be spherical. These shapes arise as an implicit consequence of developmental processes, thereby generating correctly structured cells, tissues, and organs. Probability Although genes are the primary regulators of development, other processes must also play a role. That development is regulated but not determined by the genome is underscored by the important role that probability plays in normal development. For example, in the mouse, a pathogenic variant in the Dishevelled-2 gene produces congenital heart defects in only ~50% of mice who carry the variant, even when such carriers are from inbred strains of mice that are genetically identical. Thus the 50% penetrance of the Dishevelled-2 variant cannot be explained by different modifying gene variants in the mice affected with heart defects versus the mice who are unaffected. Instead, it appears likely that the Dishevelled-2 variant shifts the balance of some developmental process by increasing the probability that a threshold for causing congenital heart defects is exceeded, much as we explored in Chapter 9 when discussing complex patterns of inheritance in humans. Thus carrying a Dishevelled-2 pathogenic variant will not always lead to heart defects, but it sometimes will, and neither the rest of the genome nor nongenetic factors are responsible for development of the defect in only a minority of animals. Probabilistic processes provide a rich source of interindividual variation that can lead to a range of developmental outcomes, some normal and some not. ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 327 Human arm Bird wing Bat wing Upper arm Lower arm Carpals Metacarpal Phalanges Figure 15.7 Diagram of the upper limb of three species: human, b...

Ch15 · Pt8 328 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Environmental Factors As indicated earlier, the local environment in which a cell or tissue finds itself plays a central role in providing a normal developmental context. It is therefore not unexpected that drugs or other agents introduced from the environment can be teratogens, often because they interfere with intrinsic molecules that mediate the actions of genes. Identification of the mechanism of teratogenesis has obvious implications not only for clinical medicine and public health but also for basic science; understanding how teratogens cause birth defects can provide insight into the underlying developmental pathways that have been disturbed and result in a defect. Because the molecular and cellular pathways used during development are often not employed in similar developmental processes after adulthood, teratogens that cause serious birth defects may have few or no side effects in adult patients. One important example of this concept is fetal retinoid syndrome, seen in fetuses of pregnant women who took the drug isotretinoin during pregnancy. Isotretinoin is an oral retinoid that is used systemically for the treatment of severe acne. It causes major birth defects when taken by a pregnant woman because it mimics the action of endogenous retinoic acid, a substance that in the developing embryo and fetus diffuses through tissues and interacts with cells, causing them to follow particular developmental pathways. Different teratogens often cause very specific patterns of birth defects, the risk for which depends critically on the gestational age at the time of exposure, the vulnerability of different tissues to the teratogen, and the level of exposure during pregnancy. One of the best examples is thalidomide syndrome. Thalidomide, a sedative widely used in the 1950s, was later found to cause a high incidence of malformed limbs in fetuses exposed between 4 and 8 weeks of gestation because of its effect on the vasculature of the developing limb. Another example is the fetal alcohol syndrome. Alcohol causes a particular pattern of birth defects involving primarily the central nervous system because it is relatively more toxic to the developing brain and related craniofacial structures than to other tissues. Some teratogens, such as x-rays, are also mutagens. A fundamental distinction between teratogens and mutagens is that mutagens cause damage by creating heritable alterations in genetic material, whereas teratogens act directly and transiently on developing embryonic tissue. Thus fetal exposure to a mutagen can cause an increased risk for birth defects or other diseases (e.g., cancer) throughout the life of the exposed individual and even in his or her offspring, whereas exposure to a teratogen increases the risk for birth defects for current but not for subsequent pregnancies. BASIC CONCEPTS OF DEVELOPMENTAL BIOLOGY Overview of Embryologic Development Developmental biology has its own set of core concepts and terminology that may be confusing or foreign to the student of genetics. We therefore provide a brief summary of a number of key concepts and terms used in this chapter (see Box 15.1). BOX 15.1 CORE CONCEPTS AND TERMINOLOGY IN HUMAN DEVELOPMENTAL BIOLOGY Blastocyst: a stage in embryogenesis after the morula, in which cells on the outer surface of the morula secrete fluid and form a fluid-filled internal cavity within which is a separate group of cells, the inner cell mass, which will become the fetus itself (see Fig. 15.8 and 15.9). Chimera: an embryo made up of two or more cell lines that differ in their genotype. Contrast with mosaic. Chorion: membrane that develops from the outer cells of the blastocyst and goes on to form the placenta and the outer layer of the sac in which the fetus develops. Determination: the stage in development in which cells are irreversibly committed to forming a particular tissue. Dichorionic twins: monozygotic twins arising from splitting of the embryo into two parts, before formation of the blastocyst, so that two independent blastocysts develop. Differentiation: the acquisition by a cell of novel characteristics specific for a particular cell type or tissue. Ectoderm: the primary embryonic germ layer that gives rise to the nervous system and skin. Embryo: the stage of a developing human organism between fertilization and 9 weeks of gestation, when separation into placental and embryonic tissues occurs. Embryogenesis: the development of the embryo. Embryonic stem cells: cells derived from the inner cell mass that under appropriate conditions can differentiate into all of the cell types and tissues of an embryo and form a complete, normal fetus. Endoderm: the primary embryonic germ layer that gives rise to many of the visceral organs and lining of the gut. Epiblast: a differentiated portion of the inner cell mass that gives rise to the embryo proper. Human embryonic stem cells are considered to be epiblast stem cells. Fate: the ultimate destination for a cell that has traveled down a developmental pathway. Fetus: the stage of the developing human between 9 weeks of gestation and birth. Gastrulation: the stage of development just after implantation in which the cells of the inner cell mass rearrange themselves into the three germ layers. Germ cell: the cells that are the progenitors of the gametes. These cells are allocated early in development and undergo sex-specific differentiation. Germ layers: three distinct layers of cells that arise in the inner cell mass, the ectoderm, mesoderm, and endoderm, which develop into distinctly different tissues in the embryo. Hypoblast: the differentiated portion of the inner cell mass that contributes to fetal membranes (amnion). Inner cell mass: a group of cells inside the blastocyst destined to become the fetus. continued ▶

328 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Environmental Factors As indicated earlier, the local environment in which a cell or tissue finds itself plays a central role in providing a...

Ch15 · Pt9 CHAPTER 15 — Developmental Genetics and Birth Defects 329 Mesoderm: the primary embryonic germ layer that gives rise to connective tissue, muscles, bones, vasculature, and the lymphatic and hematopoietic systems. Monoamniotic twins: monozygotic twins resulting from cleavage of part of the inner cell mass (epiblast) but without cleavage of the part of the inner cell mass that forms the amniotic membrane (hypoblast). Monochorionic twins: monozygotic twins resulting from cleavage of the inner cell mass without cleavage of the cells on the outside of the blastocyst. Monozygotic twins: twins arising from a single fertilized egg, resulting from cleavage during embryogenesis in the interval between the first cell division of the zygote and gastrulation. Morphogen: a substance produced by cells in a particular region of an embryo that diffuses from its point of origin through the tissues of the embryo to form a concentration gradient. Cells undergo specification and then determination to different fates, depending on the concentration of morphogen they experience. Morphogenesis: the creation of various structures during embryogenesis. Morula: a compact ball of 16 cells produced after four cell divisions of the zygote. Mosaic: an individual who develops from a single fertilized egg but in whom mutation after conception results in cells with two or more genotypes. Contrast with chimera. Mosaic development: a stage in development in which cells have already become committed to the point that removal of a portion of an embryo will not allow normal embryonic development. Multipotent stem cell: a stem cell capable of self-renewal as well as of developing into many different types of cells in a tissue, but not an entire organism. These are often called adult stem cells or tissue progenitor cells. Organogenesis: the creation of individual organs during embryogenesis. Pluripotent cell: an early stem cell capable of self-renewal as well as of becoming any cell in any embryonic tissue, including the germ cells. Embryonic stem cells are pluripotent. Progenitor cell: a cell that is traversing a developmental pathway on its way to becoming a fully differentiated cell. Regulative development: a stage in development in which cells have not yet become determined so that the cells that remain after removal of a portion of an embryo can still form a complete organism. Specification: a step along the path of differentiation in which cells acquire certain specialized attributes characteristic of a particular tissue but can still be influenced by external cues to develop into a different type of cell or tissue. Stem cell: a cell that is capable both of generating another stem cell (self-renewal) and of differentiating into specialized cells within a tissue or an entire organism. Totipotent cell: a very early stem cell that can form all cell types in a body, plus the extraembryonic, or placental, cells. Embryonic cells within the first couple of divisions after fertilization are the only cells that are totipotent. Zygote: the fertilized egg, the first step in embryogenesis. BOX 15.1 CORE CONCEPTS AND TERMINOLOGY IN HUMAN DEVELOPMENTAL BIOLOGY—CONT’D Cellular Processes During Development During development, cells divide (proliferate), acquire novel functions or structures (differentiate), move within the embryo (migrate), and undergo programmed cell death (often through apoptosis). These four basic cellular processes act in various combinations and in different ways to allow growth and morphogenesis (literally, the “creation of form”), thereby creating an embryo of normal size and shape, containing organs of the appropriate size, shape, and location, and consisting of tissues and cells with the correct architecture, structure, and function. Although growth may seem too obvious to discuss, growth itself is carefully regulated in mammalian development, and unregulated growth is disastrous. The mere doubling (one extra round of cell division) of cell number (hyperplasia) or an increase of cell size (hypertrophy) in an organism can be fatal. Dysregulation of growth of segments of the body can cause severe deformity and dysfunction, such as in hemihyperplasia and other segmental overgrowth disorders. Furthermore, the exquisite differential regulation of growth can change the shape of a tissue or an organ. Morphogenesis is accomplished in the developing organism by the coordinated interplay of the mechanisms introduced in this section. In some contexts, morphogenesis is used as a general term to describe all of development, but this is formally incorrect because morphogenesis has to be coupled to the process of growth discussed here to generate a normally shaped and functioning tissue or organ. Human Embryogenesis This description of human development begins where Chapter 2 ends, with fertilization. After fertilization, the embryo undergoes a series of cell divisions without overall growth, termed cleavage. The single fertilized egg undergoes four divisions to yield the 16-cell morula by day 4 (Fig. 15.8). At day 5, the embryo transitions to become a blastocyst, in which cells that give rise to the placenta form a wall, inside of which the cells that will make the embryo itself aggregate to one side into what is referred to as the inner cell mass. This is the point at which the embryo acquires its first obvious manifestation of polarity, an axis of asymmetry that divides the inner cell mass (most of which goes on to form the mature organism) from the embryonic tissues that will go on to form the chorion, an extraembryonic tissue (e.g., placenta) (Fig. 15.9). The inner cell mass then separates again into the epiblast, which will make the embryo proper, and the hypoblast, which will form the amniotic membrane. The embryo implants in the endometrial wall of the uterus in the interval between days 7 and 12 after fertilization. After implantation, gastrulation occurs, in which ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 329 Mesoderm: the primary embryonic germ layer that gives rise to connective tissue, muscles, bones, vasculature, and the lymphatic and hematopoie...

Ch15 · Pt10 330 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE B C D F G H A E Figure 15.8 Human development begins with cleavage of the fertilized egg. (A) The fertilized egg at day 0 with two pronuclei and the polar bodies. (B) A two-cell embryo at day 1 after fertilization. (C) A four-cell embryo at day 2. (D) The eight-cell embryo at day 3. (E) The 16-cell stage later in day 3, followed by the phenomenon of compaction, whereby the embryo is now termed a morula (F, day 4). (G) The formation of the blastocyst at day 5, with the inner cell mass indicated by the arrow. Finally, the embryo (arrow) hatches from the zona pellucida (H). (Reprinted with permission from Jones KL: Smith’s recognizable patterns of human malformation, ed 6, Philadelphia, 2005, WB Saunders.) Endometrial gland Endometrial capillary Endometrial epithelium Inner cell mass Trophoblast Endometrial connective tissue Glandular secretion Syncytiotrophoblast Inner cell mass Cytotrophoblast Hypoblast (primary endoderm) Uterine cavity Blastocyst cavity Blastocyst cavity Uterine gland Endometrial capillary Syncytiotrophoblast Amnion Endometrial epithelium Epiblast Cytotrophoblast Hypoblast Exocoelomic membrane Exocoelomic cavity Amniotic cavity Embryonic pole A B C Figure 15.9 Cell lineage and fate during preimplantation development. Embryonic age is given in time after fertilization in humans: (A) 6 days, (B) 7 days, (C) 8 days postfertilization. (Reprinted with permission from Moore KL, Persaud TVN: The developing human: clinically oriented embryology, ed 6, Philadelphia, 1998, WB Saunders.) ▶

330 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE B C D F G H A E Figure 15.8 Human development begins with cleavage of the fertilized egg. (A) The fertilized egg at day 0 with two pronuclei...

Ch15 · Pt11 CHAPTER 15 — Developmental Genetics and Birth Defects 331 cells rearrange themselves into a structure consisting of three cellular compartments, termed the germ layers, comprising the ectoderm, mesoderm, and endoderm. The three germ layers give rise to different structures. The endodermal lineage forms the central visceral core of the organism. This includes the cells lining the main gut cavity, the airways of the respiratory system, and other similar structures. The mesodermal lineage gives rise to kidneys, heart, vasculature, and structural or supportive functions in the organism. Bone and muscle are nearly exclusively mesodermal and have the two main functions of structure (physical support) and providing physical and nutritive support of the hematopoietic system. The ectoderm gives rise to the central and peripheral nervous systems and the skin. During the complicated movements that occur in gastrulation, the embryo also establishes the major axes of the final body plan: anterior-posterior (cranial-caudal), dorsal-ventral (back-front), and leftright axes, which are discussed later. The next major stages of development involve the initiation of the nervous system, establishment of the basic body plan, and then organogenesis, which occupies weeks 4 to 8. The position and basic structures of all the organs are now established, and the cellular components necessary for their full development are now in place. It is during this phase of embryonic development that neural tube defects occur, as we explore next. Neural Tube Defects Neural tube defects (NTDs) are among the most common and devastating birth defects. Anencephaly and spina bifida are NTDs that frequently occur together in families and are considered to have a common pathogenesis. In anencephaly, the forebrain, overlying meninges, vault of the skull, and skin are all absent. Many infants with anencephaly are stillborn, and those born alive survive a few hours at most. Approximately two-thirds of affected infants are female. In spina bifida, there is failure of fusion of the arches of the vertebrae, typically in the lumbar region. There are varying degrees of severity, ranging from spina bifida occulta, in which the defect is in the bony arch only, to spina bifida aperta, in which a bone defect is also associated with meningocele (protrusion of meninges) or meningomyelocele (protrusion of neural elements as well as meninges through the defect; see Fig. 18.5). As a group, NTDs are a leading cause of stillbirth, death in early infancy, and handicap in surviving children. Their incidence at birth is variable, ranging from almost 1% in Ireland to 0.2% or less in the United States. The frequency also appears to vary with social factors and season of birth and oscillates widely over time (with a marked decrease in recent years; see later discussion). A small proportion of NTDs have known specific causes, for example, amniotic bands (see Fig. 15.3), some single-gene defects with pleiotropic expression, some chromosomal disorders, and some teratogens. Most NTDs, however, are isolated defects of unknown cause. Maternal Folic Acid Deficiency and Neural Tube Defects. NTDs were long believed to follow a multifactorial inheritance pattern determined by multiple genetic and environmental factors, as introduced generally in Chapter 9. It was therefore a stunning discovery to find that the single greatest factor in causing NTDs is a vitamin deficiency. The risk for NTDs was found to be inversely correlated with maternal serum folic acid levels during pregnancy, with a threshold of 200 µg/L, below which the risk for NTD becomes significant. Along with reduced blood folate levels, elevated homocysteine levels were also seen in the mothers of children with NTDs, suggesting that a biochemical abnormality was present at the step of recycling of tetrahydrofolate to methylate homocysteine to methionine (see Fig. 13.7). Folic acid levels are strongly influenced by dietary intake and can become depressed during pregnancy even with a typical intake of ~230 µg/day. The impact of folic acid deficiency is exacerbated by a genetic variant of the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), caused by a common missense variant that makes the enzyme less stable than normal. Instability of this enzyme hinders the recycling of tetrahydrofolate and interferes with the methylation of homocysteine to methionine. The variant allele is so common in many populations that between 5% and 15% of the population is homozygous for the variant. In studies of infants with NTDs and their mothers, it was found that mothers of infants with NTDs were twice as likely as controls to be homozygous for the allele encoding the unstable enzyme. How this enzyme defect contributes to NTDs and whether the abnormality is a direct result of elevated homocysteine levels, depressed methionine levels, or some other metabolic derangement remains undefined. Prevention of Neural Tube Defects. There are two approaches to preventing NTDs. The first is to educate women to supplement their diets with folic acid 1 month before conception and continuing for 2 months after conception during the period when the neural tube forms. Dietary supplementation with 400 to 800 µg of folic acid per day for women who plan their pregnancies has been shown to reduce the incidence of NTDs by more than 75%. Since 1998, the United States has required cereal products labeled as enriched to be supplemented with 140 μg folic acid per 100 g flour as a public health measure to avoid the problem of women failing to supplement their diets individually during pregnancy. The Centers for Disease Control and Prevention estimates that this has reduced the number of infants born with NTDs by 1300 per year. The second approach is to apply prenatal screening for all pregnancies and offer prenatal diagnosis to highrisk pregnancies. Prenatal diagnosis of anencephaly and most cases of open spina bifida relies on detecting excessive levels of α-fetoprotein (AFP) and other fetal substances in the amniotic fluid and by ultrasonographic ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 331 cells rearrange themselves into a structure consisting of three cellular compartments, termed the germ layers, comprising the ectoderm, mesode...

Ch15 · Pt12 332 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE scanning, as we shall discuss further in Chapter 18. However, less than 5% of all patients with NTDs are born to women with previous affected children. For this reason, screening of all pregnant women for NTDs by measurements of AFP and other fetal substances in maternal serum is now widespread. Thus we anticipated that a combination of preventive folic acid therapy and maternal AFP screening would provide major public health benefits by drastically reducing the incidence of NTDs, which it has by 35% compared with presupplementation levels. Human Fetal Development The embryonic phase of development occupies the first 2 months of pregnancy and is followed by the fetal phase of development, which is concerned primarily with the maturation and further differentiation of the components of the organs. For some organ systems, development does not cease at birth. For example, the brain undergoes substantial postnatal development, and limbs undergo epiphyseal growth and ultimately closure after puberty. The Germ Cell: Transmitting Genetic Information In addition to growth and differentiation of somatic tissues, the organism must also specify which cells will go on to become the gametes of the mature adult. The germ cell compartment serves this purpose. As described in Chapter 2, cells in the germ cell compartment become committed to undergoing gametogenesis and meiosis in order that the species can pass on its genetic complement and facilitate the recombination and random assortment of chromosomes. In addition, the sexspecific epigenetic imprint that certain genes require must be reset within the germ cell compartment (see Chapters 3, 6, 7, and 8). The Stem Cell: Maintaining Regenerative Capacity in Tissues In addition to specifying the program of differentiation that is necessary for development, the organism must also set aside tissue-specific stem cells that can regenerate differentiated cells during adult life. The best-characterized example of these cells is in the hematopoietic system. Among the 1011 to 1012 nucleated hematopoietic cells in the adult organism are ~104 to 105 cells that have the potential to generate any of the more specialized blood cells on a continuous basis during a lifetime. Hematopoietic stem cells can be transplanted to other humans and completely reconstitute the hematopoietic system (see Chapter 14). A system of interacting gene products maintains a properly sized pool of hematopoietic stem cells. These regulators permit a balance between the maintenance of stem cells through self-replication and the generation of committed precursor cells that can go on to develop into the various mature cells of the hematopoietic system (Fig. 15.10) (see Box 15.2). Multipotent Myeloid Progenitor Totipotent Stem Cell Multipotent Lymphoid Progenitor T-cell progenitor B-cell progenitor Pre-Pre-B Pre-B B cell Plasma cell T cell RBC Platelets Monocyte Neutrophil Eosinophil Basophil/ mast cell Figure 15.10 The development of blood cells is a continuous process that generates a full complement of cells from a single, totipotent hematopoietic stem cell. This hematopoietic stem cell is a committed stem cell that differentiated from a more primitive mesodermal stem cell. RBC, Red blood cell. (Reprinted with permission from Stamatoyannopoulos G, Nienhuis AW, Majerus PW, et al: The molecular basis of blood diseases, ed 2, Philadelphia, 1994, WB Saunders.) ▶

332 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE scanning, as we shall discuss further in Chapter 18. However, less than 5% of all patients with NTDs are born to women with previous affecte...

Ch15 · Pt13 CHAPTER 15 — Developmental Genetics and Birth Defects 333 BOX 15.2 EMBRYONIC STEM CELL TECHNOLOGY Inner cell mass cells are thought to be capable of forming any tissue in the body. This has been proven in mice but is only suspected of being true in humans, in whom it cannot be tested for obvious ethical reasons. The full developmental potential of inner cell mass cells is the basis of the experimental field of embryonic stem cell technology in mice, a technology that is crucial for generating animal models of human genetic disease (Fig. 15.11). In this technique, mouse inner cell mass cells are grown in culture as embryonic stem cells and undergo genetic manipulation to introduce a given mutation into a specific gene. These cells are then injected into the inner cell mass of another early mouse embryo. The mutated cells are incorporated into the inner cell mass of the recipient embryo and contribute to many tissues of that embryo, forming a chimera (a single embryo made up of cells from two different sources). If the mutated cells contribute to the germline in a chimeric animal, the offspring of that animal can inherit the engineered mutations. The ability of the recipient embryo to tolerate the incorporation of these pluripotent cells, which then undergo specification and can contribute to any tissue in a living mouse, is the converse of regulative development, the ability of an embryo to tolerate removal of some cells. Human embryonic stem cells (h ESCs), which probably come from the epiblast, have been made from unused fertilized embryos. h ESCs are the subject of intensive research as well as ethical controversy. Although the use of h ESCs for cloning an entire human being is considered highly unethical and universally banned, current research is directed toward generating particular cell types from h ESCs to provide cellular models of human genetic diseases or to repair damaged tissues and organs, a goal of regenerative medicine. Induced pluripotent stem cells (i PSCs) are another source of early stem cells that can be cultured and differentiated in vitro into particular cell types. Human i PSCs are derived through reprogramming of readily available and ethically uncontroversial somatic cells, such as fibroblasts, to very early stem cells through the introduction of certain transcription factors into the cells (e.g., the transcription factors Oct 4 [Pou 5f1], Sox 2, c Myc, and Klf 4). This technology makes what were previously inaccessible tissues from patients with genetic disorders, such as cardiac myocytes from patients with cardiomyopathies, or central nervous system neurons from patients with neurodegenerative diseases available for research and, ultimately, perhaps tissue-based therapy using their own gene-corrected i PSCs. ▶

Early blastocyst (3.5 d) cultured on fibroblast “feeder” layer Colonies from single cells Chimeric mouse will produce gametes derived from host () or ES cells () Hematopoietic cells Hepatic cells Myog...

Ch15 · Pt14 334 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Chapters 3 and 8). The epigenetic control of gene expression is responsible for the loss of developmental plasticity, as we discuss next. Regulative and Mosaic Development Early in development, cells in many organisms are functionally equivalent and subject to dynamic processes of specification, a phenomenon known as regulative development. In regulative development, removal or ablation of part of an embryo can be compensated for by the remaining similar cells. In contrast, later in development, each of the cells in some parts of the embryo has a distinct fate, and in each of those parts, the embryo only appears to be homogeneous. In this situation, known as mosaic development, loss of a portion of an embryo would lead to the failure of development of the final structures that those cells were fated to become. Thus the developmental plasticity of the embryo generally declines with time. Regulative Development and Twinning That early mammalian development is primarily regulative has been demonstrated by basic embryologic experiments and confirmed by observations in clinical medicine. Identical (monozygotic) twins are the natural experimental evidence that early development is regulative. The most common form of identical twinning occurs in the second half of the first week of development, effectively splitting the inner cell mass into two halves, each of which develops into a normal fetus (Fig. 15.12). Were the embryo even partly regulated by mosaic development at this stage, the twins would develop only partially and consist of complementary parts. This is clearly not the case because twins are generally completely normally developed and eventually attain normal size through prenatal and postnatal growth. The various forms of monozygotic twinning demonstrate regulative development at several different stages. Dichorionic twins result from cleavage at the four-cell stage. Monochorionic twins result from a cleaved inner cell mass. Monoamniotic twins result from an even later cleavage, in this case within the bilayered embryo, which then forms two separate embryos but only one extraembryonic compartment that goes on to make the single amnion. All of these twinning events demonstrate that these cell populations can reprogram their development to form complete embryos from cells that, if cleavage had not occurred, would have contributed to only part of an embryo. The successful application of the technique of preimplantation diagnosis (see Chapter 18) also illustrates that early human development is regulative. In this procedure, male and female gametes are harvested from the presumptive parents and fertilized in vitro (Fig. 15.13). When these fertilized embryos have reached the eightcell stage (at day 3), a biopsy microneedle is used to remove one of these cells. The isolated cell with its clearly visible nucleus can then be examined using a variety of appropriate cytogenetic or genomic tests to ascertain if the embryo is suitable for implantation. Embryos composed of the remaining seven cells that are not affected by the disease can then be selected and implanted in the mother. The capacity of the embryo to recover from the biopsy of one of its eight cells is attributable to regulative development. Were those cells removed by biopsy fated to form a particular part or Dichorionic Monochorionic; diamniotic (common) Monoamniotic Conceptus Inner cell mass Uterine wall Epiblast Timing (days 0–3 4–7 >7 days post conception) Frequency 35% 65% Rare Figure 15.12 The arrangement of placental membranes in monozygotic twins depends on the timing of the twinning event. Dichorionic twins result from a complete splitting of the entire embryo, leading to duplication of all extraembryonic tissues. Monochorionic diamniotic twins are caused by division of the inner cell mass at the blastocyst stage. Monoamniotic twins are caused by division of the epiblast but not the hypoblast. (Reprinted with permission from Ogilvie CM, Braude PR, Scriven PN: Preimplantation diagnosis – An overview, J Histochem Cytochem 53:255–260, 2005.) ▶

334 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Chapters 3 and 8). The epigenetic control of gene expression is responsible for the loss of developmental plasticity, as we discuss next. Re...

Ch15 · Pt15 CHAPTER 15 — Developmental Genetics and Birth Defects 335 segment of the body (i.e., governed by mosaic development), one would predict that these parts of the body would be absent or defective in the mature individual. Instead, the embryo has compensatory mechanisms to replace those cells, which then undergo normal development as specified by their neighboring cells. Mosaic Development Embryonic development generally proceeds from more regulative to more mosaic development. Typical identical twinning early in development, as mentioned earlier, is an illustration of regulative development. However, later embryo cleavage events result in the formation of conjoined twins, which are two fetuses that share body structures and organs because the cleavage occurred after the transition from regulative to mosaic development, too late to allow complete embryos. Interestingly, in some adult nonhuman species, ablation of a specific tissue may not limit development. For example, the mature salamander can regenerate an entire tail when it is cut off, apparently retaining a population of cells that can reestablish the developmental program for the tail after trauma. One of the goals of research in developmental biology is to understand this process in other species and potentially harness it in practice for human regenerative medicine. Axis Specification and Pattern Formation A critical function of the developing organism is to specify the spatial relationships of structures within the embryo. In early development, the organism must determine the relative orientation of a number of body segments and organs, involving the establishment of three axes: The head-to-tail axis, termed the cranial-caudal or anterior-posterior axis, is established very early in embryogenesis and is probably determined in certain species by the entry position of the sperm that fertilizes the egg. (It is referred to as the rostral-caudal axis later in development.) The dorsal-ventral axis is the second dimension, and here, too, a series of interacting proteins and signaling pathways are responsible for determining dorsal and ventral structures. The morphogen sonic hedgehog (discussed later) participates in setting up the axis of dorsal-ventral polarity along the spinal cord. Finally, a left-right axis must be established. The left-right axis is essential for proper heart development and positioning of viscera. It is established by the leftward flow of fluid from motile cilia present in the node where cell migration occurs during gastrulation. Interruption of placement or rotation of these cilia results in randomization of left-right axis A B C D Figure 15.13 Blastomere biopsy of a human cleavage stage embryo. (A) Eight-cell embryo, day 3 after fertilization. (B) Embryo on holding pipette (left) with biopsy pipette (right) breaching the zona pellucida. (C) Blastomere removal by suction. (D) Blastomere removed by biopsy with a clearly visible single nucleus (indicated by arrow). ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 335 segment of the body (i.e., governed by mosaic development), one would predict that these parts of the body would be absent or defective in the...

Ch15 · Pt16 336 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE determination, termed situs inversus, in which some thoracic and abdominal viscera are on the wrong side of the chest and abdomen, as well as infertility in males. Such ciliary defects can also cause cardiac anomalies. The three axes that must be specified in the whole embryo must also be specified early in the developing limb. Within the limb, the organism must specify the proximal-distal axis (shoulder to fingertip), the anterior-posterior axis (thumb to fifth finger), and the dorsal-ventral axis (dorsum to palm). On a cellular scale, individual cells also develop an axis of polarity (e.g., the basal-apical axis of the proximal renal tubular cells or the axons and dendrites of a neuron). Thus specifying axes in the whole embryo, in limbs, and in cells is a fundamental process in development. Once an organismal axis is determined, the embryo then overlays a patterning program onto that axis. Conceptually, if axis formation can be considered as the drawing of a line through an undeveloped mass of cells and specifying which end is to be the head and which end the tail, then patterning is the division of the embryo into segments and the assignment to these segments of an identity, such as head, thorax, or abdomen. The HOX genes (discussed in the next section) have major roles in determining the different structures that develop along the anterior-posterior axis. The end result of these pattern specification programs is that cells or groups of cells are assigned an identity related primarily to their position within the organism. This identity is subsequently used by the cells as an instruction to specify how development should proceed. Pattern Formation and the HOX Gene System The homeobox (HOX) gene system, first described in the fruit fly Drosophila melanogaster, constitutes a paradigm in developmental biology. HOX genes are so named because the proteins they encode are transcription factors that contain a conserved DNA-binding motif called the homeodomain. The segment of the gene encoding the homeodomain is called a homeobox, thus giving the gene family its name, HOX. Many species of animals have HOX genes, and the homeodomains encoded by these genes are similar; however, different species contain different numbers of HOX genes (e.g., fruit flies contain 8 and humans nearly 40). The 40 human HOX genes are organized into four clusters on four different chromosomes. Strikingly, the order of the individual genes within the clusters is conserved across species. The human HOX gene clusters (Fig. 15.14) were generated by a series of gene duplication events, conceptually similar to those described in lab pb (Zen) Dfd Ser Antp abd-A abd-B Ubx a 1 a 2 a 3 a 4 a 5 a 6 a 7 a 9 a 10 a 11 a 13 b 1 b 2 b 3 b 4 b 5 b 6 b 7 b 8 b 9 c 4 c 5 c 6 c 8 c 9 c 10 c 11 c 12 c 13 d 1 d 3 d 4 d 8 d 9 d 10 d 11 d 13 d 12 Drosophila Human or mouse Anterior Posterior Distal Proximal HOXA9 HOXA9-13 HOXA9-11 Anterior Posterior Distal Proximal HOXD9 HOXD9-13 HOXD9-11 A B C 3' 5' 3' 5' Figure 15.14 Action and arrangement of HOX genes. (A) An ancestral HOX gene cluster in a common ancestor of vertebrates and invertebrates has been quadruplicated in mammals, and individual members of the ancestral cluster have been lost. (B) The combination of HOX genes expressed in adjacent regions along the anteroposterior axis of developing embryos selects a unique developmental fate (as color-coded in the segments of the fly and human embryo). (C) In the developing limbs, different combinations of HOXA and HOXD genes are expressed in adjacent zones that help specify developmental fate along the proximal-distal and anterior-posterior axes. (From Wolpert L, Beddington R, Brockes J, et al: Principles of development, New York, 1998, Oxford University Press. Copyright 1998, Oxford University Press.) ▶

336 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE determination, termed situs inversus, in which some thoracic and abdominal viscera are on the wrong side of the chest and abdomen, as well a...

Ch15 · Pt17 CHAPTER 15 — Developmental Genetics and Birth Defects 337 Chapter 12 for the evolution of the globin gene family. Initially, ancient events duplicated the original ancestral HOX gene in tandem along a single chromosome. Subsequent duplications of this single set of HOX genes and relocation of the new gene set to other locations in the genome resulted in four unlinked HOX gene clusters in humans (and other mammals) named HOXA, HOXB, HOXC, and HOXD. Unique combinations of HOX gene expression in small groups of cells, located in particular regions of the embryo, help determine the developmental fate of those regions. Just as specific combinations of HOX genes from the single HOX gene cluster in the fly are expressed along the anterior-posterior axis of the body and regulate different patterns of gene expression and therefore different body structures (see Fig. 15.14), mammals use a number of HOX genes from different clusters to accomplish similar tasks. Early, in the whole embryo, HOX transcription factors specify the anterior-posterior axis: the HOXA and HOXB clusters, for example, act along the rostral-caudal axis to determine the identity of individual vertebrae and somites. Later in development, the HOXA and HOXD clusters determine regional identity along the axes of the developing limb. One interesting aspect of HOX gene expression is that the order of the genes in a cluster parallels the position in the embryo in which that gene is expressed and the time in development when it is expressed (see Fig. 15.14). In other words, the position of a HOX gene in a cluster is collinear with both the timing of expression and the location of expression along the anteriorposterior axis in the embryo. For example, in the HOXB cluster, the genes expressed first and in the anterior portion of the embryo are at one end of the cluster; the order of the rest of the genes in the cluster parallels the order in which they are expressed, both by location along the anterior-posterior axis of the embryo and by timing of expression. Although this gene organization is distinctly unusual and is not a general feature of gene organization in the genome (see Chapter 3), a similar phenomenon is seen within another developmentally regulated human gene family, the globin gene cluster (see Chapter 12). In both cases, the association of spatial organization in the genome with temporal expression in development presumably reflects long-range regulatory elements in the genome that govern the epigenetic packaging and accessibility of different genes at different times in the embryo. The HOX gene family thus illustrates several important principles of developmental biology and evolution: First, a group of genes functions together to accomplish similar general tasks at different times and places in the embryo. Second, homologous structures are generated by sets of homologous transcription factors derived from common evolutionary predecessors. For example, flies and mammals have a similar basic body plan (head anterior to the trunk, with limbs emanating from the trunk, cardiorespiratory organs anterior to digestive), and that body plan is specified by a set of genes that were passed down through common evolutionary predecessors. Third, patterns of expression of these homologues are distinct but overlapping. The intersection of these distinct patterns provides unique combinations of transcription factors that specify cellular diversity. For example, HOXD9-13 genes are expressed in the most distal part of the developing limb bud (see Fig. 15.14) while HOXA9-13 are expressed only in the posterior region of the developing limb bud. Cells that express both HOXD9-13 and HOXA9-13 are specified into posterior distal limb bud and limb, while cells that express HOXD9-13 and HOXA9-11 are specified into more anterior limb bud and limb. And fourth, although it is not usually the case with genes involved in development, the HOX genes show a remarkable genomic organization within a cluster that correlates with their function during development. CELLULAR AND MOLECULAR MECHANISMS IN DEVELOPMENT In this section we review the basic cellular and molecular mechanisms that regulate development (see Box 15.3). We illustrate each mechanism with a human birth defect or disease that results from the failure of each of these normal mechanisms. BOX 15.3 FUNDAMENTAL MECHANISMS OPERATING IN DEVELOPMENT Gene regulation by transcription factors Cell-cell signaling by direct contact and by morphogens Induction of cell shape and polarity Cell movement Programmed cell death ▶

Gene Regulation by Transcription Factors Transcription factors control development by controlling the expression of other genes, some of which are also transcription factors. Groups of transcription f...

Ch15 · Pt18 338 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE as acetylation, and the regulation of histone modifications is accomplished by histone acetyltransferases and deacetylases (see Chapter 3). These epigenetic changes to histones are marks that indicate whether a particular gene is likely to be active or inactive. Regulatory modules control development by causing different combinations of transcription factors to be expressed at different places and at different times to direct the spatiotemporal regulation of development. By directing differential gene expression across space and time, the binding of various transcriptional regulatory modules to transcriptional complexes is controlled by histone modifications and is a central element of the development of the embryo. Transcriptional regulatory complexes are localized into clusters of topologically associating domains (TADs) that allow for loop extrusion of DNA between CTCT sites, bringing otherwise distal gene-regulatory elements (i.e., enhancers and silencers) into 3D proximity of target genes to regulate their expression (see Chapter 3). Such loops regulate the communication between gene-regulatory elements and genes by bringing general transcription factors together with the specific transcription factors that are responsible for creating the selectivity of a transcriptional complex (Fig. 15.15). Most general transcription factors are found in thousands of these transcriptional complexes throughout the genome, and although each is essential, their roles in development are nonspecific. Specific transcription factors bind to enhancers and participate in forming active transcription factor complexes, mostly under the control of epigenetic marks of histone modifications, but only in specific cells or at specific times in development, thereby providing the regulation of gene expression that allows developmental processes to be exquisitely controlled. The importance of transcription factors in normal development is illustrated by an unusual mutation of HOXD13 that causes synpolydactyly, an incompletely dominant condition in which heterozygotes have interphalangeal webbing and extra digits in their hands and feet. Rare homozygotes have similar but more severe abnormalities and have bone malformations of the hands, wrists, feet, and ankles (Fig. 15.16). The HOXD13 variant responsible for synpolydactyly is caused by expansion of a polyalanine tract in the amino-terminal domain of the protein; the normal protein contains 15 alanines, whereas the mutant protein contains 22 to 24 alanines. The polyalanine expansion that causes synpolydactyly is likely to act by a gain-offunction mechanism (see Chapter 12), as heterozygosity for a HOXD13 loss-of-function variant has only a mild effect on limb development, characterized by a rudimentary extra digit between the first and second metatarsals and between the fourth and fifth metatarsals of the feet. Regardless of the exact mechanism, this condition demonstrates that a general function for HOX genes is to determine regional identity along specific body axes during development. Morphogens and Cell-to-Cell Signaling One of the hallmarks of developmental processes is that cells must communicate with each other to develop proper spatial arrangements of tissues and cellular subtypes. This communication occurs through cell signaling mechanisms. These cell-cell communication systems are commonly composed of a cell surface receptor and the molecule, called a ligand, that binds to it. On ligand binding, receptors transmit their signals through intracellular signaling pathways. One of the common ligandreceptor pairs is the fibroblast growth factors and their receptors. There are 23 recognized members of the fibroblast growth factor gene family in the human, and many of them are important in development. The fibroblast growth factors serve as ligands for tyrosine kinase receptors. Abnormalities in fibroblast growth factor receptors cause diseases such as achondroplasia (Case 2) (see Chapter 7) and certain syndromes that involve abnormalities of craniofacial development, referred to as craniosynostoses because they demonstrate premature fusion of cranial sutures in the skull. Enhancer Activators Coactivators General transcription factors Promoter RNA transcript TATA box DNA CTCF CTCF Coding region Repressor RNA polymerase Figure 15.15 Activation of transcription occurs in transcriptional complexes that form loops from topologically associated domains (TADs, see Chapter 3). General transcription factors (blue), and RNA polymerase bind to cis-acting sequences closely adjacent to the messenger RNA (mRNA) transcriptional start site; these cis-acting sequences are collectively referred to as the promoter. More distal enhancer or silencer elements bind specialized and tissue-specific transcription factors. Coactivator proteins facilitate a biochemical interaction between specialized and general transcription factors. (Redrawn from Tjian R: Molecular machines that control genes, Sci Am 272:54–61, 1995.) ▶

338 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE as acetylation, and the regulation of histone modifications is accomplished by histone acetyltransferases and deacetylases (see Chapter 3)....

Ch15 · Pt19 CHAPTER 15 — Developmental Genetics and Birth Defects 339 One of the best examples of a developmental morphogen is hedgehog, originally discovered in Drosophila and named for its ability to alter the orientation of epidermal bristles. Diffusion of the hedgehog protein creates a gradient in which different concentrations of the protein cause surrounding cells to assume different fates. In humans, three genes closely related to Drosophila hedgehog also encode developmental morphogens; one example is the gene sonic hedgehog (SHH). Although the specific programs controlled by hedgehog in Drosophila are very different from those controlled by its mammalian counterparts, the underlying themes and molecular mechanisms are similar. For example, secretion of the SHH protein by the notochord and the floor plate of the developing neural tube generates a gradient that induces and organizes the different types of cells and tissues in the developing brain and spinal cord (Fig. 15.17A). SHH is also produced by a small group of cells in the limb bud known as the zone of polarizing activity, which is responsible for establishing the posterior side of the developing limb bud and the asymmetric pattern of digits within individual limbs (see Fig. 15.17B). Variants that inactivate the SHH gene in humans cause birth defects that may be inherited as autosomal A B C D F E A B C D F E Figure 15.16 An unusual gain-of-function variant in HOXD13 creates an abnormal protein with a dominant negative effect. Photographs and radiographs show the synpolydactyly phenotype. (A, B) Hand and radiograph of an individual heterozygous for a HOXD13 variant. Note the branching metacarpal III and the resulting extra digit IIIa. The syndactyly between digits has been partially corrected by surgical separation of III and IIIa-IV. (C, D) Hand and radiograph of an individual homozygous for a HOXD13 variant. Note syndactyly of digits III, IV, and V and their single knuckle; the transformation of metacarpals I, II, III, and V to short carpal-like bones (stars); two additional carpal bones (asterisks); and short second phalanges. The radius, ulna, and proximal carpal bones appear normal. (E, F) Foot and radiograph of the same homozygous individual. Note the relatively normal size of metatarsal I, the small size of metatarsal II, and the replacement of metatarsals III, IV, and V with a single tarsal-like bone (stars). (Reprinted with permission from Muragaki Y, Mundlos S, Upton J, et al: Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13, Science 272:548–551, 1996.) Posterior Anterior Posterior Anterior Polarizing region Apical ectodermal ridge Digits Concentration of morphogen Limb buds Posterior Anterior Distal Proximal 4 3 2 4 3 2 2 3 4 Notochord Neural crest Motor neurons 4 3 2 4 4 3 3 2 A B Figure 15.17 (A) Transverse section of the developing neural tube. Sonic hedgehog protein released from the notochord diffuses upward to the ventral portion of the developing neural tube (brown); high concentrations immediately above the notochord induce the floor plate, whereas lower concentrations more laterally induce motor neurons. Ectoderm above (dorsal to) the neural tube releases bone morphogenetic proteins that help induce neural crest development at the dorsal edge of the closing neural tube (dark purple). (B) Morphogenetic action of the sonic hedgehog (SHH) protein during limb bud formation. SHH is released from the zone of polarizing activity (labeled polarizing region in B) in the posterior limb bud to produce a gradient (shown with its highest levels as 4, declining to 2). Mutations or transplantation experiments that create an ectopic polarizing region in the anterior limb bud cause a duplication of posterior limb elements. (A, From Lumsden A, Graham A: Neural patterning: A forward role for hedgehog, Curr Biol 5:1347–1350, 1995. Copyright 1995, Elsevier Science; B, from Wolpert L, Beddington R, Brockes J, et al: Principles of development, New York, 1998, Oxford University Press.) ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 339 One of the best examples of a developmental morphogen is hedgehog, originally discovered in Drosophila and named for its ability to alter the...

Ch15 · Pt20 340 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE dominant traits, which demonstrates that a 50% reduction in gene expression is sufficient to produce an abnormal phenotype, presumably by altering the magnitude of the hedgehog protein gradient. Affected individuals usually exhibit holoprosencephaly (failure of the midface and forebrain to develop), leading to absence of forebrain structures and hypotelorism (closely spaced eyes), and they have cleft lip and palate. On occasion, however, the clinical findings are mild or subtle such as, for example, a single central incisor or partial absence of the corpus callosum (Fig. 15.18). Because variable expressivity has been observed in members of the same family, it cannot be due to different variants and instead must reflect the action of modifier genes at other loci, chance, environment, or some combination of all three. Cell Shape and Organization Cells must organize themselves with respect to their position and polarity in their microenvironment. For example, kidney epithelial cells must undergo differential development of the apical and basal aspects of their organelles to effect reabsorption of solutes. The acquisition of polarity by a cell can be viewed as the cellular version of axis determination (as discussed in a previous section) with respect to the development of the overall embryo. Under normal circumstances, each renal tubular cell elaborates on its cell surface a filamentous structure, known as a primary cilium. One hypothesis is that the primary cilium is designed to sense fluid flow in the developing kidney tubule and signal the cell to stop proliferating and to polarize. Another hypothesis is that the primary cilium is a sort of cellular antenna that concentrates signal transduction components to facilitate activation or repression of developmental pathways. There is substantial evidence that the sonic hedgehog signal transduction pathway acts in this fashion. Adult polycystic kidney disease (Case 37) is caused by loss of function of one of two protein components of primary cilia, polycystin 1 or polycystin 2, so that the cells fail to sense fluid flow or to activate or repress signal transduction pathways properly. As a result, they continue to proliferate and do not undergo the appropriate developmental program of polarization, in which they stop dividing and display polarized expression of certain proteins on either the apical or basal aspect of the tubular epithelial cells (Fig. 15.19). The continued cell division leads to the formation of cysts, fluid-filled spaces lined by renal tubular cells. Figure 15.18 Variable expressivity of an SHH variant. The mother and her daughter carry the same missense variant in SHH, but the daughter is severely affected with microcephaly, abnormal brain development, hypotelorism, and a cleft palate, whereas the only manifestation in the mother is a single central upper incisor. (From Roessler E, Belloni E, Gaudenz K, et al: Mutations in the human Sonic Hedgehog gene cause holoprosencephaly, Nat Genet 14:357–360, 1996.) EGFR EGFR EGFR EGFR EGFR EGFR EGFR erb-b 2 EGFR erb-b 2 Normal adult Normal fetus Polycystic kidney disease erb-b 2 Lumen Lumen Lumen erb-b 2 Figure 15.19 Polarization of epidermal growth factor receptor (EGFR) in epithelium from a normal fetus, a normal adult, and a patient with polycystic kidney disease. Fetal cells and epithelial cells from patients with polycystic kidney disease express a heterodimer of EGFR and erb-b 2 at apical cell membranes. In normal adults, tubular epithelia express homodimeric complexes of EGFR at the basolateral membrane. (Modified from Wilson PD: Polycystic kidney disease, N Engl J Med 350:151–164, 2004. Copyright 2004, Massachusetts Medical Society.) ▶

340 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE dominant traits, which demonstrates that a 50% reduction in gene expression is sufficient to produce an abnormal phenotype, presumably by al...

Ch15 · Pt21 CHAPTER 15 — Developmental Genetics and Birth Defects 341 Cell Migration Programmed cell movement is essential in development, and one region where it is critical is the central nervous system. The human central nervous system develops from the neural tube, a cylinder of cells created during weeks 4 to 5 of embryogenesis. Much of our knowledge of early development of the central nervous system derives from the mouse and mouse models of neurodevelopmental disorders. Initially, the neural tube is only a single cell layer thick, a pseudostratified columnar epithelium. Once sufficient neuroepithelial cells are produced by vertical and symmetrical division, these cells divide asymmetrically as neural stem cells. These neural stem cells stretch from the apical surface adjacent to the ventricle to the basal surface. The nucleus of these neural stem cells is adjacent to the apical surface in the ventricular cell layer situated adjacent to the ventricle, and the fiber of these cells stretches to the basal or pial surface as the so-called radial glial cells. When these radial glia (one type of neural stem cells) divide vertically and asymmetrically, they generate new neural stem cells as well as committed neuronal precursors and secondary neural stem cells. These secondary, more basally located neural stem cells can then amplify the number of cells produced from a given radial glial progenitor. Postmitotic neuronal precursors then migrate outward toward the pial surface along the radial glia. The central nervous system is built by waves of migration of these neuronal precursors. The neurons that populate the inner layers of the cortex migrate earlier in development, and each successive wave of neurons passes through the previously deposited, inner layers to form the next outer layer (Fig. 15.20A). The complex interplay of the production of neurons from these neuronal precursors (neurogenesis) and their movement to precise locations (neuronal migration) results in the remarkable and specific wiring of the central nervous system. Ventral Ventricle Dorsal Developing neural tube PS MZ IZ VZ CP SP PS I IV V VI II III Radial glia Pial surface Pial surface Ventricle Normal PS MZ IZ VZ CP SP PS Lissencephaly Heterozygous LIS1 mutation A Figure 15.20 (A) The role of neuronal migration in normal cortical development and the defective migration in individuals heterozygous for a LIS1 mutation causing lissencephaly. (Top) A radial slice is taken from a normal developing neural tube of the mouse, showing the progenitor cells at the ventricular zone (VZ). These cells divide, differentiate into postmitotic cells, and migrate radially along a scaffold made up of glia. The different shapes and colors represent the cells that migrate and form the various cortical layers: IZ, intermediate zone; SP, subplate; CP, cortical plate; MZ, marginal zone; PS, pial surface. The six distinguishable layers of the normal cortex (molecular, external granular, external pyramidal, internal granular, internal pyramidal, multiform) that occupy the region of the cortical plate are labeled I through VI. (Bottom) Aberrant migration and failure of normal cortical development seen in lissencephaly. ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 341 Cell Migration Programmed cell movement is essential in development, and one region where it is critical is the central nervous system. The hu...

Ch15 · Pt22 342 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE B C Figure 15.20, cont’d (B, C) The human brain contains expansion of a novel class of early neural precursors termed basal (or outer subventricular zone) radial glia that are attached to the basal (pial) surface but not the apical surface. These cells are found in the mouse, but in very small numbers, while their numbers are greatly expanded in primates, including humans. (A, Diagram modified from Gupta A, Tsai L-H, Wynshaw-Boris A: Life is a journey: A genetic look at neocortical development, Nat Rev Genet 3:342–355, 2002. B and C, from Lui JH, Hansen DV and Kriegstein AR. Development and evolution of the human neocortex, Cell 146:18–36, 2011.) More recently, it has been possible to observe the early development of the human brain from fetal tissue obtained from elective pregnancy termination. These studies revealed that the human brain contains expansion of a novel class of early neural precursors termed basal (or outer subventricular zone) radial glia that are attached to the basal (pial) surface, but not the apical surface (see Fig. 15.20B and C). These cells are found in the mouse (Fig. 15.21B), but in very small numbers, while their numbers are greatly expanded in primates including humans (see Fig. 15.21C), leading to the idea that the basal radial glial expansion during primate evolution is a major cause of the increased size of the primate and human brain. The basal radial glia divide horizontally, unlike the ventricular zone radial glia. Lissencephaly (literally, “smooth brain”) is a severe abnormality of brain development causing profound intellectual disability. This developmental defect is one component of the Miller-Dieker syndrome (Case 32), which is caused by a contiguous gene deletion syndrome that involves one copy of the LIS1 gene on chromosome 17. When there is heterozygous loss of LIS1 function, there is a disruption of both neurogenesis of ventricular and basal radial glia, as well as defective neuronal migration (see Fig. 15.20A). The result is a thickened, hypercellular cerebral cortex with undefined cellular layers and poorly developed gyri, thereby making the surface of the brain appear smooth. In addition to the neuronal migrations described, another remarkable example of cell migration involves the neural crest, a population of cells that arises from the dorsolateral aspect of the developing neural tube (see Fig. 15.17A). Neural crest cells must migrate from their original location at the dorsal and lateral surface of the neural tube to remarkably distant sites, such as the ventral aspect of the face, the ear, the heart, the gut, and many other tissues, including the skin, where they differentiate into pigmented melanocytes. Population of the gut by neural crest progenitors gives rise to the autonomic innervation of the gut; failure of that migration leads to the aganglionic colon seen in Hirschsprung disease. The genetics of Hirschsprung disease are complex (see Chapter 9), but a number of key signaling molecules have been implicated. One of the best characterized is the RET protooncogene. As discussed in Chapter 9, pathogenic variants in RET have been identified in ~50% of patients with Hirschsprung disease. Another example of defects in neural crest development is the group of birth defects known as the Waardenburg syndrome, which includes defects in skin and hair pigmentation, coloration of the iris, and colon innervation (see Fig. 15.21). This syndrome can be caused by pathogenic variants in at least four different transcription factors, each resulting in abnormalities in neural crest development. ▶

342 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE B C Figure 15.20, cont’d (B, C) The human brain contains expansion of a novel class of early neural precursors termed basal (or outer subven...

Ch15 · Pt23 CHAPTER 15 — Developmental Genetics and Birth Defects 343 Programmed Cell Death Programmed cell death is a critical function in development and is necessary for the morphologic development of many structures. It occurs wherever tissues need to be remodeled during morphogenesis, as during the separation of the individual digits, in perforation of the anal and choanal membranes, or in the establishment of communication between the uterus and vagina. One major form of programmed cell death is apoptosis. Studies of mice with loss-of-function variants in the Foxp 1 gene indicate that apoptosis is required for the remodeling of the tissues that form portions of the ventricular septum and cardiac outflow tract (endocardial cushions), to ensure the normal positioning of the origins of the aortic and pulmonary vessels. By eliminating certain cells, the relative position of the cushions is shifted into their correct location. It is also suspected that defects of apoptosis underlie some other forms of human congenital heart disease (see Chapter 9), such as the conotruncal heart defects of Di George syndrome caused by deletion of the TBX1 gene located in chromosome 22q11 (see Chapter 6). Apoptosis also occurs during development of the immune system to eliminate lymphocyte lineages that react to self, thereby preventing autoimmune disease. A B C Figure 15.21 Patients with type I Waardenburg syndrome. (A) Mother and daughter with white forelocks. (B) A 10-year-old with congenital deafness and white forelock. (C) Brothers, one of whom is deaf. There is no white forelock, but the boy on the right has heterochromatic irides. Pathogenic variants of PAX3, which encodes a transcription factor involved in neural crest development, cause type I Waardenburg syndrome. (A, From Partington MW: An English family with Waardenburg’s syndrome, Arch Dis Child 34:154–157, 1959; B, from Di George AM, Olmsted RW, Harley RD: Waardenburg’s syndrome. A syndrome of heterochromia of the irides, lateral displacement of the medial canthi and lacrimal puncta, congenital deafness, and other characteristic associated defects, J Pediatr 57:649– 669, 1960; C, from Jones KL: Smith’s recognizable patterns of human malformation, ed 6, Philadelphia, 2005, WB Saunders.) ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 343 Programmed Cell Death Programmed cell death is a critical function in development and is necessary for the morphologic development of many str...

Ch15 · Pt24 344 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE INTERACTION OF DEVELOPMENTAL MECHANISMS IN EMBRYOGENESIS Embryogenesis requires the coordination of multiple developmental processes in which proliferation, differentiation, migration, and apoptosis all play a part. For example, many processes must occur to convert a mass of mesoderm into a heart or a layer of neuroectoderm into a spinal cord. To understand how these processes interact and work together, developmental biologists typically study embryogenesis in a model organism, such as fish, frogs, worms, flies, chicks, mice, or other animal species. The general principles elucidated by these more easily manipulated systems can then be applied to understanding developmental processes in humans. The Limb as a Model of Organogenesis The vertebrate limb is a relatively well-studied product of developmental processes. There is no genomic specification for a human arm to be ~1 m long, with one proximal bone, two bones in the forelimb, and 27 bones in the hand. Instead, the limb results from a series of regulated processes that specify development along three axes, the proximal-distal axis, the dorsal-ventral axis, and the anterior-posterior axis (Fig. 15.22). Limbs begin as protrusions of proliferating cells, the limb buds, along the lateral edge of the mesoderm of the human embryo in the fourth week of development. The location of each limb bud along the anterior-posterior axis of the embryo (head-to-tail axis) is associated with the expression of a specific transcription factor at each location, TBX4 for the hindlimbs and TBX5 for the forelimbs, whose expression is induced by various combinations of fibroblast growth factor ligands. Thus the primarily proliferative process of limb bud outgrowth is activated by growth factors and transcription factors. The limb bud grows primarily in an outward, lateral expansion of the proximal-distal axis of the limb (see Fig. 15.17B). Whereas proximal-distal expansion of the limb is the most obvious process, the two other axes are established soon after the onset of limb bud outgrowth. The anterior-posterior axis is set up soon after limb bud outgrowth, with the thumb considered to be an anterior structure because it is on the edge of the limb facing the upper body. The fifth finger is a posterior structure because it is on the side of the limb bud oriented toward the lower part of the body. During limb formation, the morphogen SHH is expressed in the posterior aspect of the developing limb bud, and its expression level forms a gradient A Di V Po Px Do A Di V Po Px Do Figure 15.22 This scanning electron micrograph of a 4-week human embryo illustrates the early budding of the forelimb. Overlaid onto the bud are the three axes of limb specification: DoV, dorsal-ventral (dorsal comes out of the plane of the photo, ventral goes into the plane of the photo); Px-Di, proximal-distal; and A-Po, anterior-posterior. (From Carlson BM: Human embryology and developmental biology, ed 3, Philadelphia, 2004, Mosby.) Anterior Distal Posterior Proximal GLI3R GLI3 GLI3R SHH Zone of polarizing activity Figure 15.23 Schematic diagram of the anterior-posterior and proximal-distal axes of the limb bud and its molecular components. In this diagram, the anterior aspect is up and the distal aspect is to the right. SHH expression occurs in the zone of polarizing activity of the posterior limb bud. SHH inhibits conversion of the GLI3 transcription factor to GLI3R in the posterior regions of the limb bud. However, SHH activity does not extend to anterior regions of the bud. The absence of SHH allows GLI3 to be converted to GLI3R (a transcriptional repressor) in the anterior limb bud. By this mechanism, the anterior-posterior axis of the limb bud is established with a gradient of GLI3 versus GLI3R. (Modified from Gilbert SF: Developmental biology, ed 7, Sunderland, Massachusetts, 2003, Sinauer Associates.) ▶

344 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE INTERACTION OF DEVELOPMENTAL MECHANISMS IN EMBRYOGENESIS Embryogenesis requires the coordination of multiple developmental processes in whic...

Ch15 · Pt25 CHAPTER 15 — Developmental Genetics and Birth Defects 345 that is primarily responsible for setting up the anteriorposterior axis in the developing limb (see Fig. 15.17B). Defects in anterior-posterior patterning in the limb cause excessive digit patterning, manifested as polydactyly, or failure of complete separation of developing digits, manifested as syndactyly. The dorsal-ventral axis is also established, resulting in a palm or sole on the ventral side of the hand and foot, respectively. One can now begin to understand the mechanisms underlying birth defect syndromes by applying knowledge from molecular developmental biology to human disorders. For example, variants in the GLI3 transcription factor gene cause two pleiotropic developmental anomaly syndromes, the Greig cephalopolysyndactyly syndrome (GCPS) and the Pallister-Hall syndrome (see Fig. 15.1). These two syndromes comprise distinct combinations of limb, central nervous system, craniofacial, airway, and genitourinary anomalies that are caused by perturbed balance in the production of two variant forms of GLI3, referred to as GLI3 and GLI3R, as shown in Fig. 15.23. GLI3 is a component of the SHH signaling pathway. SHH signals, in part, through a cell surface receptor encoded by a gene called PTCH1, which is concentrated in the cilium of cells during development. Pathogenic variants in PTCH1 cause the nevoid basal cell carcinoma syndrome. Also known as Gorlin syndrome, this syndrome comprises craniofacial anomalies and occasional polydactyly that are similar to those seen in GCPS, but in addition, Gorlin syndrome manifests dental cysts and susceptibility to basal cell carcinoma. By considering Gorlin syndrome and GCPS, one can appreciate that the two disorders share phenotypic manifestations precisely because the genes that are mutated in the two disorders have overlapping effects in the same developmental genetic pathway. A third protein in the SHH signaling pathway, the CREB-binding protein, or CBP, is a transcriptional coactivator of the GLI3 transcription factor. Pathogenic variants in CBP cause the Rubinstein-Taybi syndrome (see Fig. 15.5), which also shares phenotypic manifestations with GCPS and Gorlin syndrome. CONCLUDING COMMENTS Many other examples of this phenomenon could be cited, but the key points to emphasize are that genes are the primary regulators of developmental processes, their protein products function in developmental genetic pathways, and these pathways are employed in related developmental processes in a number of organ systems. Understanding the molecular basis of gene function, how those functions are organized into modules, and how abnormalities in those modules cause and correlate with malformations and pleiotropic syndromes forms the basis of the modern clinical approach to human birth defects. The understanding of these developmental pathways in great detail may also provide an avenue in the future to devise therapies that target appropriate parts of these pathways. GENERAL REFERENCES Barresi MJF, Gilbert SF: Developmental biology, ed 12, Sunderland, 2020, Oxford University Press. Carlson BM: Human embryology and developmental biology, ed 6, Philadelphia, 2018, WB Saunders. Dye FJ: Dictionary of developmental biology and embryology, ed 2, New York, 2012, Wiley-Blackwell. Erickson RP, Wynshaw-Boris AJ, editors: Epstein’s inborn errors of development: the molecular basis of clinical disorders of morphogenesis ed 3, New York, 2016, Oxford University Press. Wolpert L, Tickle C: Principles of development, ed 4, New York, 2011, Oxford University Press. REFERENCES SPECIFIC TO PARTICULAR TOPICS Acimovic I, Vilotic A, Pesl M, et al: Human pluripotent stem cellderived cardiomyocytes as research and therapeutic tools, Biomed Res Int 2014:512831, 2014. Ross CA, Akimov S: Human induced pluripotent stem cells: Potential for neurodegenerative diseases, Hum Mol Genet 23(R1):R17–R26, 2014. PROBLEMS 1. What is the difference between regulative and mosaic development? What is the significance of these two stages of development for reproductive genetics and prenatal diagnosis? 2. Match the terms in the left-hand column with the terms that best fit in the right-hand column. a. Erasure of imprinting during germ cell development b. Position-dependent development c. Regulative development d. Embryonic stem cells 1. Totipotency 2. Morphogen 3. Epigenetic regulation of gene expression 4. Monozygotic twinning 3. Match the terms in the left-hand column with the terms that best fit in the right-hand column. a. Amniotic band b. Polydactyly c. Inadequate amniotic fluid d. Limb reduction e. Robin sequence 1. U-shaped cleft palate 2. Thalidomide 3. GLI3 mutation 4. Disruption 5. Deformation 4. What type of diploid cells would not be appropriate nucleus donors in an animal cloning experiment and why? 5. For discussion: Why do some pathogenic variants in transcription factors result in developmental defects even when they are present in the heterozygous state? ▶

CHAPTER 15 — Developmental Genetics and Birth Defects 345 that is primarily responsible for setting up the anteriorposterior axis in the developing limb (see Fig. 15.17B). Defects in anterior-posterio...

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Chapter 14: The Treatment of Genetic Disease

Chapter 15: Developmental Genetics and Birth Defects