Approach to Single-Gene Disorders

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Single-gene disorders have astraightforward inheritance pattern, and the genetic causes can be traced to changes in specific individual genes. Aparticular disorder could be rare; however, as agroup, single-gene disorders are responsible for asignificant percentage of pediatric diseases. Autosomes refer to the numbered chromosomes (chromosome 1–22), as opposed to the sex chromosomes, Xand Y. Every individual carries two copies of each autosome and, therefore, also has two copies of every gene carried on those chromosomes, one inherited from each parent. Based on the location of the relevant genes, single-gene traits can be divided into autosomal inheritance and sex-linked. Autosomal inheritance, depending on whether one or two mutant alleles are required to cause aphenotype, can be divided into autosomal dominant or autosomal recessive. Based on Mendel’s laws, the two alleles segregate and pass to different offspring, as shown in Fig. 2.1 ; Aand A’ will pass to different individuals, as will aand a’.

Figure 2.1

Mendel’s law of segregation. The father (left) is heterozygous for Aand A’; the mother (right) for aand a’. Offspring can receive either of the father’s alleles and either of the mother’s alleles, giving four possible combinations, shown at the bottom

Terminology

• Allele: Specific version of agene.

• Mutation: Nucleotide change in agene. This may result in arecognizable phenotype, including agenetic disorder.

• Phenotype: Observable physical manifestations of agenotype.

• Genotype: Genetic constitution of an individual.

• Homozygote: Genotype consisting of identical alleles of aparticular gene.

• Heterozygote: Genotype consisting of different alleles for the same gene.

• Compound heterozygote: Genotype consisting of two different mutations at each allele.

• Penetrance: Proportion of individuals carrying amutation who exhibit aspecific phenotype.

• Expressivity: Variations of aphenotype in individuals carrying aparticular genotype.

• Pleiotropy: Asingle-gene mutation that can affect several traits.

• Age-dependent penetrance: Aphenomenon in which aphenotype is increasingly expressed with age. For example, in Huntington disease, amajority of individuals with the mutation will not express aclinical phenotype until late age (Fig. 2.2a ).

• Anticipation: Aphenomenon in which clinical symptoms become apparent at an earlier age and are more severe as agene is passed from generation to generation. For example, in myotonic dystrophy, as the mutated gene is passed on, the severity of muscle weakness can become more severe and the age of onset earlier (Fig. 2.2b ).

• Genetic heterogeneity: The occurrence of similar phenotypes resulting either from distinct mutations in the same gene (allelic heterogeneity) or mutations in different genes (locus heterogeneity).

• Germline mosaicism or gonadal mosaicism: Asituation in which precursor cells of the ova and the spermatozoa are amixture of wild type and mutant cells (Fig. 2.3 ).

• Sex-influenced phenotype: Preferential expression of atrait in aparticular sex. For example, alopecia is more common in males, and in contrast, breast cancer is more common in females. Although the disease genes are found on the autosomes, in these situations, the phenotypes are influenced by the sex.

Figure 2.2

Age-dependent penetrance (a), aphenotype is increasingly expressed with age; and anticipation (b), clinical symptoms become apparent at an earlier age and are more severe as agene is passed from generation to generation

Figure 2.3

Germline mosaicism. The spermatozoa are amixture of wild type and mutant cells

Autosomal Dominant Inheritance

Autosomal dominant inheritance is the most common form of inheritance. Here, mutation on one allele is sufficient to cause an individual to express the phenotype. Areview of the family history will typically demonstrate affected individuals in every generation. The risks for all members who carry agenetic mutation would be 50% for each offspring to be affected (see Fig. 2.4a and b ). In some families, however, adominant trait may appear to skip an individual or ageneration. This could be the result of someone who has not exhibited symptoms yet (incomplete penetrance) or who has not been diagnosed because of being mildly affected (variable expression). Some of the more common dominantly inherited conditions include Marfan syndrome (Fig. 2.5 ), Huntington disease, tuberous sclerosis complex, and neurofibromatosis (Table 2.1 ).

Figure 2.4

(a) Autosomal dominant trait. The risks for all members who carry agenetic mutation would be 50% for each offspring to be affected. (b) Autosomal dominant pedigree. Affected individuals in every generation

Figure 2.5

Boy with Marfan syndrome at age 12 with tall stature and joint hypermobility

Table 2.1 Common conditions of Mendelian Inheritance

The severity and clinical presentation of Marfan syndrome can vary greatly. Another factor that can increase the difficulty in making adiagnosis is that up to 50% of cases are the result of spontaneous mutations in the affected child and, therefore, will not be associated with apositive family history. It is also possible for aparent to be so mildly affected that adiagnosis is not made previous to having amore severely affected child. Advances in molecular diagnosis and the ability to sequence the FBN1 gene, which encodes the protein fibrillin and is the site of mutations in individuals with Marfan syndrome, have allowed more families to obtain an accurate diagnosis. It is also important to confirm the presence of amutation in affected children to allow for identification of additional at-risk individuals in the family. Once amutation has been identified in an affected child, it becomes easy to cost-effectively screen other family members.

The Features of Autosomal Dominant Inheritance

• One mutated allele at alocus is sufficient to cause aphenotype

• Inherited from parent to child

• Fifty percent risk to each offspring of an affected individual

• Those who do not inherit the gene cannot transmit it

• Males and females equally likely to be affected

Case Presentation

Mr. Williams is a 22-year-old man who was previously healthy. He presents to a primary care physician with a 2-day history of chest pain. A physical examination shows that Mr. Williams’ weight is 150 lbs and his height 6′8″. He has long fingers, severe scoliosis, and near-sightedness. The rest of his physical exam is unremarkable. Mr. Williams is referred to a cardiologist to rule out coronary arterial disease. The cardiologist tests him on the treadmill. After the treadmill exercise, Mr. Williams’ echocardiogram shows dilated aorta and aortic aneurysm. He is admitted to hospital for emergency surgery. It is found that he has a nearly dissected aorta, with leaking of the blood into the chest. He recovers well after emergency surgery. He is referred to a genetics clinic to rule out Marfan syndrome. His clinical features meet the diagnostic criteria of Marfan syndrome, and molecular tests confirm the diagnosis. He has three children, two girls and one boy. All look healthy and their physical examinations are unremarkable. Diagnostic molecular test results reveal that the two girls carry the same mutation in the fibrillin gene. Therefore, a diagnosis of familial Marfan syndrome is established.

Autosomal Recessive Inheritance

Autosomal recessive inheritance means that both copies of aparticular gene must carry amutation in order for the phenotype to be expressed. If only one copy is inherited, that person is referred to as acarrier, and, in general, will not express the phenotype (see Fig. 2.6 ). Some commonautosomal recessive conditions are illustrated in Table 2.1 . Most commonly, achild is affected with arecessive condition if both parents are carriers and each transmits amutated gene to the child. The likelihood of this occurring when both parents are carriers would be 25% (see Fig. 2.6 , aa). Some of the more common diseases inherited in this fashion include cystic fibrosis and sickle-cell anemia, which are also routinely included in most state newborn screening programs in the United States.

Figure 2.6

Autosomal recessive inheritance. “Horizontal inheritance” – siblings affected, generally not parents or children; males and females equally likely to be affected

The Features of Autosomal Recessive Inheritance

• Both alleles must be mutated to produce a phenotype (homozygous), one from each parent (who are heterozygous carriers)

• “Horizontal inheritance” – siblings affected, generally not parents or children

• Males and females equally likely to be affected

Case Presentation

A 6-month-old male infant presents in the clinic with achronic cough. Review of the history reveals that he was treated for an upper respiratory infection on two other occasions at 2 and 4 months, respectively. At birth he was 75th percentile in both height and weight but he has trended downward since then and is currently in the 50th percentile of height and only 10th percentile in weight. The remainder of the physical exam is unremarkable and he appears to be meeting all milestones of development. He is treated for another respiratory infection, and it is recommended that he return to clinic in 2 months to assess his growth. He returned to the clinic in 3 weeks, however, with the same chronic cough and inability to clear mucus from the lungs. Areview of the chart indicates that he had anegative newborn screening test for cystic fibrosis. Given the symptoms, afecal elastase and sweat test are ordered. The fecal elastase is normal and the sweat test comes back in the borderline range. ACFTR DNA mutation panel is ordered and also comes back negative. Based on these findings, cystic fibrosis is no longer considered by his physician. The parents seek another opinion since there is still apersistent chronic cough. At this visit adeep throat culture is obtained and the patient is confirmed to be growing Pseudomonas aeruginosa and is treated with tobramycin. In addition, acomprehensive CFTR sequence analysis is ordered which reveals two known cystic-fibrosis-disease-causing mutations. The parents are then tested and each is found to carry one of these mutations, thereby confirming the diagnosis of cystic fibrosis.

Sex-Linked Traits

Sex-linked traits are associated with mutations in genes thatare located on the sex chromosomes. Males have a Y-chromosome and only one X-chromosome, whereas females have two X-chromosomes without aY; therefore, the inheritance pattern for this group of traits is quite different from those that are due to mutation in genes on theautosomes. If the mutant gene is located on the X-chromosome and the father is affected, he will transmit this mutant allele to all of his daughters but none of his sons. In contrast, if the mutated gene is located on the Y-chromosome it will be passed to all of his sons. In females, the mutated gene will be transmitted to both sons and daughters 50% of the time (Fig. 2.7 ). Some common X-linked conditions are illustrated in Table 2.1 .

Figure 2.7

(a) X-linked recessive traits. The clinical phenotypes are always expressed in males, but are usually not expressed or only mildly expressed in females unless homozygous and compound heterozygous mutations occur. (b) Pedigree demonstrating X-linked recessive traits skipping generations through unaffected, carrier women

X-Inactivation

Since males have only one X-chromosome and the Y-chromosome is much smaller and has far fewer genes than the X-chromosome, it seems that males and females are genetically imbalanced. This dosage difference is compensated for by X-inactivation. X-inactivation, also known as Lyonization, is aprocess in which one of the two copies ofthe X-chromosome in females is inactivated early in development. As shown in Fig. 2.8 , for an individual cell, which of the two copies of the X-chromosome isinactivated is random. Once an X-chromosome is inactivated, however, it will remain inactive. If an individual cell has more than two X-chromosomes, still only one will be active. X-inactivation is controlled by the X-inactivation center (XIC), which is necessary and sufficient to cause X-chromosome inactivation. The inactivated X-chromosome is packed into ahigh density of heterochromatin and can be seen in the nucleus as a“Barr body.” Asmall number of genes on the inactivated X-chromosome are expressed. The ends of the Xand Ycontain homologous genes that escape inactivation on the X. These regions are referred to as pseudo-autosomal. Both sexes will have two copies of every gene from the pseudo-autosomal region.

Figure 2.8

X-chromosome inactivation. In females, one of the two copies of the X-chromosome is inactivated randomly. OnceanX-chromosome is inactivated, however, it will remain inactive. X-inactivation is controlled by the X-inactivation center(XIC), which is necessary and sufficient to cause X-chromosome inactivation

X-linked conditions can be classified into X-linked dominant or X-linked recessive. Aclassic example of anX-linked recessive disorder is Duchenne muscular dystrophy (DMD) (Table 2.1 ). DMD is characterized by progressive muscle degeneration and weakness. Symptoms typically manifest in amale at an early age. Muscle weakness progresses from proximal to distal. Psuedo-hypertrophy of the calves is often seen early in the course. As the disease progresses, muscle loss occurs and myocytesare eventually replaced by fat and fibrotic tissue. Before reaching the teenage years, the majority of patients have difficulty walking. Most become wheelchair bound by early adolescence. Skeletal deformities can occursecondary to muscle weakness. Some patients with DMD also develop cardiomyopathy; some will have intellectual impairment. DMD is caused by amutation of the dystrophin gene on the X-chromosome. Dystrophin is acomponent of the protein-complex in skeletal muscle (Fig. 2.9 ). Loss of dystrophin results in an increased calcium release in the sarcolemma, and then cell death.

Figure 2.9

Dystrophin staining of skeletal muscle from aDMD patient. (Courtesy of Hart G.W. Lidov M.D., Ph.D. Department of Pathology, Children’s Hospital Boston, Boston, MA) (a) H&E stained frozen section of skeletal muscle from a6-year-old boywith Duchenne dystrophy. Fibers are slightly more rounded than usual for age, the diameter of fibers is more variable than normal, there is increased connective tissue between fibers (endomysial fibrosis) and scattered basophilic fibers (regenerating fibers) are present. Degenerating or necrotic fibers and inflammatory cells are sometimes seen in this condition, but are not prominent in this image. (b) Amuscle biopsy from achild, without neuromuscular disease, immunostained for dystrophin. The brown reaction product, indicating the location of dystrophin, is found as alayer alongthe plasma membrane of all muscle fibers, asubsarcolemmal location. (c) Immunoperoxidase staining for dystrophin, in the same patient as above. Dystrophin is completely absent in most fibers, although asmall cluster of dystrophin positivefibers are seen in the lower right, so-called revertant fibers

DMD and Becker muscular dystrophy, or BMD, are allelic disorders. DMD is mainly caused by adeletion or frameshift or stop mutation in the dystrophin gene. In contrast, the milder phenotype of BMD is due to mutations that maintain the dystrophin reading frame. Females who are heterozygous carriers of amutation in the dystrophin gene are usually asymptomatic, though some may experience mild weakness.

X-Linked Recessive Traits

• The clinical phenotypes are always expressed in males, but are usually not expressed or only mildly expressed in females. Therefore, mutated genes may be transmitted through aseries of unaffected females (Fig. 2.8 ).

• All daughters of affected males are unaffected. Female carriers will transmit the mutant gene to 50% of her sons who will be affected, and to 50% of her daughters, who will be carriers.

• New mutations could occur, especially if the mutations are genetically lethal (i.e., not compatible with reproduction). Those mutations will be replaced by novel mutations.

X-Linked Dominant Traits

For X-linked dominant conditions, the phenotypes in females are generally milder than in males. Therefore, daughters and sons of affected females are at 50% risk of being affected. All daughters but no sons of affected males will be affected. An example of an X-linked dominant condition is Rett syndrome (Fig. 2.10 , Table 2.1 ). In afemale, classical Rett syndrome is aprogressive neurodevelopmental disorder. Clinical features include normal psychomotor development during the first 6–18 months of life followed by aperiod of developmental stagnation, then rapid regression in language and motor skills. During the progressive period, the typical clinical signs are repetitive stereotypic hand movements. Other clinical features include screaming, inconsolable crying, and autistic tendencies. Head growth may begin decelerating as early as 3 months of age. Brain size may be smaller than normal, but microcephaly is not an invariant feature of Rett syndrome. Seizures occur in 90% of affected females. Generalized tonic-clonic seizures and partial complex seizures are common. Failure to thrive is also often seen in affected females. It is possible that this may be associated with oropharyngeal and gastroesophageal incoordination, which cause poor feeding. In classical Rett syndrome, the female can survive into adulthood. Most males with Rett syndrome do not survive pregnancy, but some affected males present with severe neonatal encephalopathy, which usually results in death before age two.

Figure 2.10

Five-year-old girl with Rett syndrome. She had normal developmental milestones until 18 months of age and then lost most speech capability at 2½ years

Rett syndrome is caused by mutations of methyl CpG binding protein 2 (MECP2). This protein plays an important role in the function of neuronal cells. The protein binds to methylated DNA, interacting with other proteins to form acomplex that leads to inhibition of gene expression. Methylation occurs in CpG islands, which are frequently found near the promoter region of agene. Once MECP2 binds to DNA, the DNA will condense and become inactive. Recent studies show that MECP2 forms acomplex with histone deactylase (hDac), which catalyzes the removal of an acetyl group on the histone, therefore blocking the transcription of the gene. Rett syndrome is often misdiagnosed as autism, cerebral palsy, or nonspecific developmental delay, and can be afrequent cause of delayed development in girls. Diagnosis is mainly clinical, after excluding neurodegenerative disorders and other causes of delayed development. Confirmatory DNA testing is available in several specialty labs. The treatment is mainly speech therapy and counseling.

Case Presentation

A 3-year-old girl born of non-consanguineous parents presented to the clinic with aggressive behavior since 1 year of age. She had normal developmental milestones until 18 months of age. However, she seemed to plateau in development and then lost most speech capability at 2½ years. There was no history of seizures or hyperventilation. On examination she had hypotonia, unusual hand movements, and autistic behavior. There was no ataxia. Her systemic examination was normal. She was diagnosed as being autistic. At age 5 years, she was reevaluated at a genetics clinic in an effort to determine an underlying cause for her delay. A review of her history confirmed that there had been no developmental progression beyond the 2-year level despite several years of intensive intervention. The history and exam revealed moderate dementia, partial apraxia, and microcephaly. She also had constant wringing movements of hands, patting, clapping, was easily annoyed, and could utter only two to three meaningless words. Based on the clinical findings and history, a diagnosis of Rett syndrome was made and a DNA blood test was sent for confirmation. Results indicated a known mutation in the MECP2 gene, thereby confirming the diagnosis of Rett syndrome.

Y-Linked Disorders

Disorders associated with Y-linked inheritance are relatively few. In this case, the disease-causing gene is on the Y-chromosome. Only males are affected and every son of an affected male is affected. Most Y-linked disorders involved male-sex determination, particularly due to mutation in genes controlling sperm quality and quantity. They are most commonly diagnosed in men who seek evaluation for infertility.

Maternal Inheritance

The mitochondrion is the site in the cell where the majority of the ATP is produced. Each mitochondrion contains multiple copies of acircular double-stranded DNA molecule that encodes 13 protein subunits of the respiratory chain. The vast majority of mitochondria are maternally inherited. Mitochondrial genome mutations can lead to failure of energy metabolism and follow apattern of maternal inheritance. It should be noted, however, that most mitochondrial proteins are encoded by genes in the nucleus, and mutations display typical Mendelian inheritance (usually autosomal recessive). As shown in Fig. 2.11 , amutation at the A3243G position of the mitochondrial genome causes maternal inheritance of diabetes. All affected individuals inherit the mutant mitochondrial genome from their mothers. No affected males pass the mutated genome to their offspring. Since each cell contains hundreds to thousands of mitochondria, each mitochondrion also contains multiple copies of the mitochondrial genomes. Therefore, the mutant and wild type mitochondrial genome can coexist, which is known as heteroplasmy. The degree of heteroplasmy can affect the clinical expression of the mutation.

Figure 2.11

Maternal inheritance in mitochondrial diseases. The mutation in the mitochondrial genome is passed to offspring exclusively through women

Genomic Imprinting

For amajority of autosomal genes, the paternal and maternal alleles are both expressed. However, asmall portion of genes are expressed in aparent-of-origin-specific manner. For example, gene H19 on Chromosome 11 is only expressed from the maternal allele. In contrast, IGF2 on the same chromosome is only expressed from the paternal allele. This phenomenon is referred to as genomic imprinting.

Prader–Willi syndrome is atypical example of a disorder of genomic imprinting. Prader–Willi syndrome is characterized by hypotonia, short stature, polyphagia, obesity, and small hands and feet. Hypogonadism and mental retardation also occur. The disorder results from lack of expression of agene on chromosome 15 that is normally expressed from the paternal copy. Avariety of types of mutation can lead to Prader–Willi syndrome. The most common is deletion of the region on 15q11.2. An alternative mechanism is uniparental disomy, in which both copies of 15 are derived from the mother. This can occur as shown in Fig. 2.12 . Deletion of the maternal copy, or paternal uniparental disomy results in adifferent disorder – Angelman syndrome, characterized by seizures, developmental delay, and poorly coordinated body movements.

Figure 2.12

Genomic imprinting of Prader–Willi syndrome. Anormal sperm is fertilized with an egg with two copies of chromosome 15, and this will result in trisomy 15. Since trisomy 15 is lethal, the cell will often find away to remove one copy of chromosome15. If the paternal chromosome 15 is eliminated, the remaining two copies of chromosome 15 will be both from the mother and result in maternal uniparental disomy. Since some genes are only expressed in paternal alleles and when two copies of gene are the maternal alleles, some of the genes normally expressed from the paternal allele are missing. In this case, this would result in Prader–Willi syndrome

In summary, Mendelian disorders comprise an important component of pediatric genetic disorders. The most critical diagnostic tool is the family history. Being able toaccurately analyze the pattern of inheritance can help making adiagnosis and identifying any at-risk family members. To analyze apedigree, it is veryimportant to know if transmission is vertical and to examine formale–male transmission (X-linked disorders will not have male-to-male transmission). Inaddition, it is also very helpful to examine the pedigreeto determine if all the daughters of an affected father are affected. This could bean evidence of sex-linked-dominant mode of transmission. Does the trait skip ageneration, indicative of non-penetrance? If thepedigree is large enough, determination of the segregation ratio is important (expect half of the offspring of affected individuals to be affected for a dominant trait).