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InternalMed_Harrison. test is a function of the number of probes or DNA sequences present on the array. Arrays may use probes of different sizes (ranging from 50 to 200,000 base pairs of DNA) and different probe densities depending on the requirements of the application. Low-resolution platforms can have hundreds of probes, targeted to known disease regions, whereas high-resolution platforms can have millions of probes spread across the entire genome. Depending on the size of the probes and the probe placement across the genome, array-based testing may be able to detect single exon deletions or duplications.


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InternalMed_Harrison. Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymorphism (SNP) Analysis CGH and SNP-based genotyping arrays can both be used for the analysis of genomic deletions and duplications. For both techniques, oligonucleotide probes are placed onto a slide or chip in a grid format. Each of these probes is specific for a particular genomic region. In array CGH, the amount of DNA from a patient is compared to that in a clinically normal control, or pool of controls, for each of the probes present on the array. DNA from a patient is fluorescently labeled with a dye of one color, and DNA from a control individual is labeled with another color. These DNA samples are then hybridized at the same time to the array. The resulting fluorescent signal will vary depending on whether both the control and patient DNA are present in equal amounts or if one has a different copy number than the other. SNP platforms use arrays targeting SNPs that are distributed across the genome. SNP arrays


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InternalMed_Harrison. control and patient DNA are present in equal amounts or if one has a different copy number than the other. SNP platforms use arrays targeting SNPs that are distributed across the genome. SNP arrays vary in density of markers and in the technology used for genotyping, depending on the manufacturer of the array. SNP arrays were initially designed to determine genotypes at a biallelic, polymorphic base (e.g., CC, CT, or TT) and have been increasingly used in genome-wide association studies to identify disease susceptibility genes. SNP arrays were subsequently adapted to identify genomic deletions and duplications (Fig. 83e-2). SNP arrays, in addition to identifying copy number changes, can also detect regions of the genome that have an excess of homozygous genotypes and absence of heterozygous genotypes (e.g., CC and TT genotypes only, with no CT genotypes). Absence of heterozygosity is sometimes associated with uniparental disomy (discussed later in this chapter) but is also observed


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InternalMed_Harrison. genotypes (e.g., CC and TT genotypes only, with no CT genotypes). Absence of heterozygosity is sometimes associated with uniparental disomy (discussed later in this chapter) but is also observed when an individual’s parents are related to one another (identity by descent). Regions of homozygosity have been used to help identify genes in which homozygous mutations result in disease phenotypes in families with known consanguinity.


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InternalMed_Harrison. Array-based techniques (which we will now refer to as cytogenomic analysis) have proven superior to chromosome analysis in the identification of clinically significant deletions or duplications. It is estimated that for a deletion or duplication to be visualized by standard cytogenetics it must be minimally between 5 and 10 million base pairs in size. In almost all cases, deletions and duplications of this size contain multiple genes, and these deletions and duplications are disease causing. However, utilization of array-based cytogenomic testing, which can routinely identify deletions and duplications smaller than 50,000 base pairs, reveals that clinically normal individuals all have some deletions and duplications. This presents a dilemma for the analyst to discern which smaller copy number variations (CNVs) are disease causing (pathogenic) and which are likely benign polymorphisms. Although initially burdensome, the cytogenomics community has been curating these CNVs for almost a


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InternalMed_Harrison. variations (CNVs) are disease causing (pathogenic) and which are likely benign polymorphisms. Although initially burdensome, the cytogenomics community has been curating these CNVs for almost a decade, and databases have been created reporting CNVs routinely seen in clinically normal individuals and those routinely seen in individuals with clinical abnormalities. Nevertheless, each copy number variant that is identified in an individual undergoing genomic testing must be evaluated for gene content and overlap with CNVs in other patients and in controls.


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InternalMed_Harrison. Array technologies are DNA based, unlike cytogenetic technologies, which are cell based. Although resolution of gains and losses are greatly increased with array technology, this technique cannot identify structural changes. When DNA is extracted for array studies, chromosomal structure is lost because the DNA is fragmented for better hybridization to the slides. As an example, the array may be able to detect a duplication of a small region of a chromosome, but no information on the location of this extra material can be determined from this test. The location of this extra copy in the genome may be critical, as the chromosomal material may be involved in a translocation, insertion, marker, or other complex rearrangement. Depending on the chromosomal position of this extra material, the patient may have different clinical outcomes, and recurrence risks for the family can be significantly different. Often, combinations of array-based and cytogenetic-based techniques are required to


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InternalMed_Harrison. patient may have different clinical outcomes, and recurrence risks for the family can be significantly different. Often, combinations of array-based and cytogenetic-based techniques are required to fully characterize chromosomal abnormalities (see Table 83e-1 for comparison of these technologies).


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InternalMed_Harrison. Recent advances in genomic sequencing, known as next-generation sequencing (NGS), have vastly increased the speed and throughput of DNA sequence analysis. NGS is rapidly finding its way into the diagnostic lab for detection of clinically relevant intragenic mutations, and new bioinformatic tools for analysis of genomic deletions and duplications are being developed. It is anticipated that NGS will soon allow the complete analysis of a patient’s genome, with identification of intragenic mutations as well as chromosome abnormalities resulting in gain or loss of genetic material. Identification of completely balanced translocations is the most challenging for NGS, but recent reports of successes in this area suggest that in a matter of time, sequencing will be used for all types of genomic analysis.


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InternalMed_Harrison. Cytogenetic analysis is most commonly used for (1) examination of the fetal chromosomes or genome during pregnancy (prenatal diagnosis) or in the event of a spontaneous miscarriage; (2) examination of chromosomes in the neonatal or pediatric population to look for an underlying diagnosis in the case of congenital or developmental anomalies, including short stature and abnormalities of sexual differentiation or progression; (3) chromosome analysis in adults who are facing fertility problems; or (4) examination of cancer cells to look for alterations that aid in establishing a diagnosis or contributing to the prognosis of a tumor (Table 83e-2).


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InternalMed_Harrison. Prenatal diagnosis is carried out by analysis of samples obtained by four techniques: amniocentesis, chorionic villous sampling, fetal blood sampling, and analysis of cell free DNA from maternal serum. Amniocentesis, which has been the most commonly used test to date, is usually performed between 15 and 17 weeks of gestational age and carries a small but significant risk for miscarriage. Amniocentesis can be performed as early as 12 weeks, but because there is a lower volume of fluid, the risks for fetal injury or miscarriage are greater. Chorionic villous sampling (CVS) or placental biopsy is routinely carried out earlier than amniocentesis, between 10 and 12 weeks, but a reported increase in limb defects when the procedure is carried out earlier than


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InternalMed_Harrison. Timing of Testing Indications for Testing 10 weeks has resulted in reduced use of this test in some centers. Fetal blood sampling (percutaneous umbilical blood sampling [PUBS]) is a riskier procedure that is carried out in the second or third trimester of pregnancy, usually to follow up on an unclear finding from an amniocentesis (such as mosaicism) or an ultrasound abnormality that was detected later in pregnancy. One of the far-reaching recent advances in prenatal diagnosis of chromosome and other genetic disorders is the utilization of cell free fetal DNA that can be identified in maternal serum. The obvious advantages of using fetal DNA obtained from maternal serum is that the DNA can be obtained at minimal risk to the pregnancy, because it requires a maternal blood sample, rather than amniotic fluid which is obtained by puncturing the uterine membranes and carries a risk of miscarriage or infection. Although cell free fetal DNA screening, also called noninvasive prenatal


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InternalMed_Harrison. than amniotic fluid which is obtained by puncturing the uterine membranes and carries a risk of miscarriage or infection. Although cell free fetal DNA screening, also called noninvasive prenatal screening, has started to be offered clinically, it requires further confirmation of fetal tissues when an abnormal result is identified. Furthermore, ethical concerns have been raised, because it is feared that the ease of doing this test may encourage testing for individuals who are not truly prepared to deal with the choices that accompany diagnosis of a genetic disease and this testing may change the ethical implications of prenatal testing. Nevertheless, this is an active of area of research, both in terms of the technology and the utilization and implications.


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InternalMed_Harrison. Common Indications Common indications for prenatal diagnosis by cytogenetic or cytogenomic analysis are (1) advanced maternal age, (2) presence of an abnormality of the fetus on ultrasound examination, and (3) abnormalities in maternal serum screening that reveal an increased risk for chromosome abnormality.


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InternalMed_Harrison. Maternal age is well known to be an important risk factor for having a fetus with trisomy. At a maternal age less than 25 years, 2% of all clinically recognized pregnancies are trisomic, but by a maternal age of 36 years, this figure increases to 10%, and by the maternal age of 42 years, the figure increases to >33%. Based on the risk of having a chromosomally abnormal fetus in comparison to the risk for an adverse event from amniocentesis or CVS, the recommendation is that women over the age of 35 consider prenatal testing if they want to know the chromosomal status of their fetus. The precise mechanism for the maternal age effect is not known, but it is believed that it involves a breakdown in the process of chromosome segregation. A similar effect is not seen for trisomy and paternal age. This difference may reflect the fact that oocytes are generated early in ovary development in the female, whereas spermatogonia are generated continuously after puberty in the male.


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InternalMed_Harrison. Abnormalities on prenatal ultrasound are the second most frequent indication for prenatal genetic screening. Ultrasound screening can reveal structural or functional anomalies in the fetus, which might be associated with chromosome or genomic disorders. Follow-up chromosome studies may therefore be recommended. Maternal serum screening results are the third most frequent indication for prenatal chromosome analysis. There have been several versions of maternal serum screening offered over the past few decades. Currently, the “quad” screen analyzes levels of α fetoprotein (AFP), human chorionic gonadotropin (hCG), estriol, and inhibin-A. The values of these analytes are used to adjust the maternal age–predicted risk of a trisomy 21 or trisomy 18 fetus.


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InternalMed_Harrison. Postnatal indications for cytogenetic or cytogenomic analysis in neonates or children are varied, and the list has been growing with the increasing ability to diagnose smaller genomic alterations via array-based techniques. Common indications include multiple congenital anomalies, suspicion of a known cytogenetic or cytogenomic syndrome, intellectual disability or developmental delay both with and without accompanying dysmorphic features, autism, failure to thrive in infancy or short stature during childhood, and disorders of sexual development. The ability to detect smaller genomic alterations with involvement of fewer genes, sometimes as few as a single gene, suggests that a wider range of phenotypes could be investigated by cytogenomic analysis. Reasons for chromosome testing in adults include recurrent miscarriages or infertility, where balanced chromosome rearrangements such as reciprocal translocations may occur. Additionally, some adults with anomalies who were not diagnosed


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InternalMed_Harrison. include recurrent miscarriages or infertility, where balanced chromosome rearrangements such as reciprocal translocations may occur. Additionally, some adults with anomalies who were not diagnosed when they were children are referred for cytogenetic analysis, often when other members of their family want to understand any potential genetic implications, as they plan their own families.


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InternalMed_Harrison. Aneuploidy (extra or missing chromosomes) is the most common type of abnormality, occurring in 3/1000 newborns and at much higher frequency (about 35%) in spontaneously aborted fetuses. The only autosomal trisomies that are compatible with being live born in humans are trisomies 13, 18, and 21, although there are several chromosomes that can be trisomic in mosaic form. Trisomy 21 is associated with the relatively common disorder Down syndrome. Down syndrome has characteristic features including recognizable facial features, along with intellectual disability and abnormalities of multiple other organ systems including the heart. Both trisomy 13 and trisomy 18 are much more severe disorders than Down syndrome, with low frequency of patients surviving past 1 year of age. Trisomy 13 is characterized by low birth weight, postaxial polydactyly, microcephaly, ocular malformations such as anophthalmia or microphthalmia, cleft lip and palate, cardiac defects, and renal malformations. Trisomy


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InternalMed_Harrison. by low birth weight, postaxial polydactyly, microcephaly, ocular malformations such as anophthalmia or microphthalmia, cleft lip and palate, cardiac defects, and renal malformations. Trisomy 18 neonates have distinct facial characteristics at birth accompanied by an abnormal neurologic exam, underdeveloped genitalia, general lack of responsiveness, and structural birth defects such as congenital heart disease, esophageal atresia, and omphalocele.


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InternalMed_Harrison. Mosaicism refers to the presence of two or more populations of cells with distinct chromosome constitutions: for example, an individual with a normal female karyotype in some cells (46,XX) and trisomy 21 in other cells (47,XX,+21). In general, individuals who are mosaic for a chromosomal abnormality have less severe phenotypes than individuals with that same finding in every cell. The severity and presentation of phenotypes are related to the mosaic levels and the tissue distribution of the abnormal cells. There are a number of trisomies that have been reported in mosaic form including mosaic trisomies for chromosomes 8, 9, 14, 17, and 22. A number of trisomies have also been reported in spontaneous abortions (SABs) that have not been seen in live-born individuals, including trisomy 16, which is the most common trisomy in SABs. Monosomy for human chromosomes is very rare, with the single exception being monosomy for the X chromosome, associated with Turner syndrome (45,X). Monosomy


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InternalMed_Harrison. which is the most common trisomy in SABs. Monosomy for human chromosomes is very rare, with the single exception being monosomy for the X chromosome, associated with Turner syndrome (45,X). Monosomy for the X chromosome occurs in 1% of all conceptions, yet 98% of these conceptions do not go to term and result in SABs. Trisomies for the sex chromosomes also occur, with 47,XXX (trisomy X or triple X syndrome), 47,XXY (Klinefelter syndrome), and 47,XYY all reported in individuals with relatively mild phenotypes (Chap. 410). Klinefelter syndrome is the most common clinically recognized sex chromosome abnormality, and clinical features include gynecomastia, azoospermia, small testes, and hypogonadism. The 47,XYY karyotype is most often found in boys with developmental delay and or behavioral difficulties, but population-based studies have shown that intelligence for individuals with this karyotype is generally within the normal range, although slightly lower than that found in siblings.


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InternalMed_Harrison. Structural chromosome abnormalities include deletions, duplications, translocations, inversions, as well as other types of abnormalities, each relatively rare, but nonetheless contributing to clinical disease resulting from chromosome anomalies. These rare alterations include isochromosomes, ring chromosomes, dicentric chromosomes, and marker chromosomes (structurally abnormal chromosomes that cannot be identified based on cytogenetics alone). Both translocations and inversions can be completely balanced in some cases, such that there is no disruption of coding regions of the genome, with a completely normal clinical phenotype; however, carriers are at risk for unbalanced forms of these rearrangements in their offspring.


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InternalMed_Harrison. Reciprocal translocations are found in approximately 1/500–1/600 individuals in the general population and result from the exchange of chromosomal segments between at least two chromosomes. These usually occur between nonhomologous chromosomes and can be identified based on an altered banding pattern on G-banding. Balanced translocation carriers are at risk for abnormal chromosome segregation during meiosis and therefore have a higher risk for infertility, SAB, and live-born offspring with multiple congenital malformations. These phenotypes are observed when only one of the pairs of chromosomes involved in a translocation is inherited from a parent, resulting in an unbalanced genotype (Fig. 83e-3). Sometimes the exchanged segments are so small that they cannot be appreciated by banding (cryptic translocation), and these are sometimes recognized 83e-5 when a phenotypically affected child with an unbalanced form is born. Parental chromosomes can then be studied by FISH to determine if


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InternalMed_Harrison. translocation), and these are sometimes recognized 83e-5 when a phenotypically affected child with an unbalanced form is born. Parental chromosomes can then be studied by FISH to determine if the rearrangement is inherited from a parent with a balanced form of the translocation. The majority of reciprocal, apparently balanced translocations occur in phenotypically normal individuals. The risk for a clinical abnormality when a new reciprocal translocation is identified (usually during prenatal diagnostic studies) is about 7%. Analysis of cytogenetically reciprocal translocations using arrays has demonstrated that translocations in clinically normal individuals are more likely to show no deletions or duplications at the breakpoint, whereas translocations in clinically affected individuals are more likely to have breakpoint-associated deletions or duplications. Most reciprocal translocations occur uniquely, at apparently random positions throughout the genome; however, there are a few


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InternalMed_Harrison. are more likely to have breakpoint-associated deletions or duplications. Most reciprocal translocations occur uniquely, at apparently random positions throughout the genome; however, there are a few exceptions with multiple cases of recurrent translocations occurring. These recurrent translocations include t(11;22), which results in Emanuel syndrome in the unbalanced form, and several translocations involving a region on 4p, 8p, and 12p. These recurrent translocations occur in regions of the genome that contain specific types of AT-rich repeats, or other repeat sequences, that are prone to rearrangement. A special category of translocations is the Robertsonian translocations, which involve the acrocentric chromosomes. An acrocentric chromosome has unique genetic material only on the long arm of the chromosomes, whereas the short arm contains repetitive DNA. The acrocentric chromosomes are 13, 14, 15, 21, and 22. Robertsonian translocations occur when an entire long arm of an


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InternalMed_Harrison. the long arm of the chromosomes, whereas the short arm contains repetitive DNA. The acrocentric chromosomes are 13, 14, 15, 21, and 22. Robertsonian translocations occur when an entire long arm of an acrocentric chromosome is translocated onto the short arm of another acrocentric chromosome. Balanced carriers of a Robertsonian translocation contain only 45 chromosomes, with one chromosome consisting of two long arms of an acrocentric chromosome. Technically, this is an unbalanced translocation, as two short arms of the acrocentric chromosomes are missing; however, because the short arms are repetitive, there is no phenotypic consequence. Unbalanced Robertsonian carriers have 46 chromosomes, but have three copies of the long arm of an acrocentric chromosome. The most


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InternalMed_Harrison. FIGuRE 83e-3 Segregation of a balanced translocation in a mother, with inheritance of an unbalanced form in her child. Note that the mother has two rearranged chromosomes, but her child only received one of these, resulting in extra copies of a region of the blue chromosome, with loss of some material from the red chromosome. 14. Unbalanced Robertsonian translocations involving chromosomes 13 and 21 result in trisomy 13 and Down syndrome, respectively. Approximately 4% of patients with Down syndrome have a translocation, and because recurrence risks are different for families of these individuals, all patients with clinically identified Down syndrome should have a karyotype to look for translocations.


InternalMed_Harrison

InternalMed_Harrison. Inversions are another type of chromosome abnormality involving rearranged segments, where there are two breaks within a chromosome, with the intervening chromosomal material inserted in an inverted orientation. As with reciprocal translocations, if a break occurs within a gene or control region for a gene, a clinical phenotype may result, but often there are no consequences for the inversion carrier; however, there is risk for abnormalities in the offspring of carriers, as recombinant chromosomes may result after crossing over between a normal chromosome and an inverted chromosome during meiosis.


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InternalMed_Harrison. Deletion refers to the loss of a chromosomal segment, which results in the presence of only a single copy of that region in an individual’s genome. A deletion can be at the end of a chromosome (terminal), or it can be within the chromosome (interstitial). Deletions that are visible at the microscopic level in standard cytogenetic analysis are generally greater than 5 Mb in size. Smaller deletions have been identified by FISH and by chromosomal microarray. The clinical consequences of a deletion depend on the number and function of genes in the deleted region. Genes that cause a phenotype when a single copy is deleted are known as haploinsufficient genes (one copy is not sufficient), and it is estimated that less than 10% of genes are haploinsufficient. Genes associated with disease that are not haploinsufficient include genes for known recessive disorders, such as cystic fibrosis or Tay-Sachs disease.


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InternalMed_Harrison. The first chromosome deletion syndromes were diagnosed clinically and were subsequently demonstrated to be caused by a chromosome deletion on cytogenetic analysis. Examples of these disorders include the Wolf-Hirschhorn syndrome, which is associated with deletions of a small region of the short arm of chromosome 4 (4p); the cri-du-chat syndrome, associated with deletion of a small region of the short arm of chromosome 5 (5p); Williams syndrome, which is associated with interstitial deletions of the long arm of chromosome 7 (7q11.23); and the DiGeorge/velocardiofacial syndromes, associated with interstitial deletions of the long arm of chromosome 22 (22q11.2). Initial cytogenetic studies were able to provide a rough localization of the deletions in different patients, but with the increased usage of arrays, precise mapping of the extent and gene content of these deletions has become much easier. In many cases, one or two genes that are critical for the phenotype associated with these


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InternalMed_Harrison. usage of arrays, precise mapping of the extent and gene content of these deletions has become much easier. In many cases, one or two genes that are critical for the phenotype associated with these deletions have been identified. In other cases, the phenotype stems from the deletion of multiple genes. The increased utilization of genomic testing by array, which can identify deletions that are much smaller than those detectable by standard cytogenetic analysis, has resulted in the discovery of several new cytogenomic disorders. These include the 1q21.1, 15q13.3, 16p11.2, and 17q21.31 microdeletion syndromes.


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InternalMed_Harrison. Duplication of genomic regions is better tolerated than deletion, as evidenced by the viability of several autosomal trisomies (whole chromosome duplications) but no autosomal monosomies (whole chromosome deletions). There are several duplication syndromes where the duplicated region of the genome is present as a supernumerary chromosome. Utilization of chromosome microarray analysis has made analysis of the origins of duplicated chromosome material straightforward (Fig. 83e-2). Recurrent syndromes associated with supernumerary chromosomes include the inverted duplication 15 (inv dup 15) syndrome, caused by the presence of a marker chromosome derived from chromosome 15, with two copies of proximal 15q resulting in tetrasomy (four copies) of this region. The inv dup 15 syndrome has a distinct phenotype and is associated with hypotonia, developmental delay, intellectual disability, epilepsy, and autistic behavior. Another syndrome is the cat eye syndrome, named for the “cat-eyelike”


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InternalMed_Harrison. phenotype and is associated with hypotonia, developmental delay, intellectual disability, epilepsy, and autistic behavior. Another syndrome is the cat eye syndrome, named for the “cat-eyelike” appearance of the pupil, resulting from a coloboma of the iris. This syndrome results from a supernumerary chromosome derived from a portion of chromosome 22, and the marker chromosomes can vary in size and are often mosaic. Consistent with expectations of a mosaic disorder, the phenotype of this syndrome is highly variable and includes renal malformations, urinary tract anomalies, congenital heart defects, anal atresia with fistula, imperforate anus, and mild to moderate intellectual disability. Another rare duplication syndrome is the Pallister-Killian syndrome (PKS), which illustrates the principle of tissue-specific mosaicism. Individuals with PKS have coarse facial features with pigmentary skin anomalies, localized alopecia, profound intellectual disability, and seizures. The disorder is


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InternalMed_Harrison. of tissue-specific mosaicism. Individuals with PKS have coarse facial features with pigmentary skin anomalies, localized alopecia, profound intellectual disability, and seizures. The disorder is caused by a supernumerary isochromosome for the short arm of chromosome 12 (isochromosome 12p). Isochromosomes consist of two copies of one chromosome arm (p or q), rather than one copy of each arm. This isochromosome is not generally seen in peripheral blood lymphocytes when they are analyzed by G-banding, but it is detected in fibroblasts. Array technology has been reported to detect the isochromosome in uncultured peripheral blood in some patients, and it has been hypothesized that a growth bias against cells with the isochromosome prevents their identification in cytogenetic studies.


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InternalMed_Harrison. Numerical abnormalities, translocations, and deletions are the most common chromosome alterations observed in the diagnostic laboratory, but in addition to inversions and duplications, several other types of abnormal chromosomes have been reported, including ring chromosomes, where the two ends of the chromosome fuse to form a circle, and insertions, where a piece of one chromosome is inserted into another chromosome or elsewhere into the same chromosome.


InternalMed_Harrison

InternalMed_Harrison. Uniparental disomy (UPD) is the inheritance of a pair of chromosomes (or part of a chromosome) from only one parent. This usually occurs as a result of nondisjunction during meiosis, with a gamete missing or having an extra copy of a chromosome. A resulting fertilized egg would then have only one parental contribution for a given chromosome pair, or a trisomy for a given chromosome. If the monosomy or trisomy is not compatible with life, the embryo may undergo a “rescue” to normal copy number. If a monosomy is rescued, the single chromosome may be duplicated, resulting in a cell with two identical chromosomes (monosomy rescue) (Fig. 83e-4). In the case of trisomies, a subsequent nondisjunction can result in cells where one of the extra chromosomes is lost (trisomy rescue) (Fig. 83e-4). For trisomy rescue, there is a one in three chance that the lost FIGuRE 83e-4 Mechanisms of formation of uniparental disomy.


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InternalMed_Harrison. FIGuRE 83e-4 Mechanisms of formation of uniparental disomy. Panel A demonstrates nondisjunction in one parent (mother, represented in red), with trisomy in the zygote. A subsequent nondisjunction, with loss of the paternal chromosome (represented in blue), restores the diploid karyotype but leaves two copies of the maternal chromosome (maternal uniparental disomy [UPD]). Panel B demonstrates nondisjunction in one parent (mother, indicated by red oval), resulting in only one copy of this chromosome in the zygote. Subsequent nondisjunction duplicates the single chromosome, rescuing the monosomy, but resulting in two copies of the paternal chromosome (represented in blue; paternal UPD).


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InternalMed_Harrison. chromosome will be the sole chromosome from one parent, resulting in a cell with two chromosomes from the same parent. UPD is sometimes associated with clinical abnormalities, and this can occur by two mechanisms. UPD can cause disease when there is an imprinted gene on the involved chromosome, resulting in altered gene expression. Imprinting is the chemical marking of the parental origin of a chromosome, and genes that are imprinted are only expressed from either the maternal or paternal chromosome (Chap. 82). Imprinting therefore results in the differential expression of affected genes, based on parent of origin. Imprinting usually occurs through differential modification of the chromosome from one of the parents, and methylation is one of several epigenetic mechanisms (others include histone acetylation, ubiquitylation, and phosphorylation). Imprinted chromosomes that are associated with phenotypes include paternal UPD6 (associated with neonatal diabetes), maternal UPD7 and UPD11


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InternalMed_Harrison. histone acetylation, ubiquitylation, and phosphorylation). Imprinted chromosomes that are associated with phenotypes include paternal UPD6 (associated with neonatal diabetes), maternal UPD7 and UPD11 (associated with Russell-Silver syndrome), paternal UPD11 (associated with Beckwith-Wiedemann syndrome), paternal UPD14, maternal UPD15 (Angelman syndrome), and paternal UPD15 (Prader-Willi syndrome). UPD can also result in disease if the two copies from the same parent are the same chromosome (uniparental isodisomy), and the chromosome contains an allele involving a pathogenic mutation associated with a recessive disorder. Two copies of the deleterious allele would result in the associated disease, even though only one parent is a disease carrier.


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InternalMed_Harrison. Chromosome changes can occur during meiosis or mitosis and can occur at any point across the lifespan. Mosaicism for a developmental disorder is one consequence of mitotic chromosome abnormalities, and another consequence is cancer, when the chromosome change 83e-7 confers a growth or proliferation advantage on the cell. The types of chromosome abnormalities seen in cancer are similar to those seen in the developmental disorders (e.g., aneuploidy, deletion, duplication, translocation, isochromosomes, rings, inversion). Tumor cells often have multiple chromosome changes, some of which happen early in the development of a tumor, and may contribute to its selective advantage, whereas others are secondary effects of the deregulation that characterizes many tumors. Chromosome changes in cancer have been studied extensively and have been shown to provide important diagnostic, classification, and prognostic information. The identification of cancer type–specific translocation breakpoints has


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InternalMed_Harrison. have been studied extensively and have been shown to provide important diagnostic, classification, and prognostic information. The identification of cancer type–specific translocation breakpoints has led to the identification of a number of cancer-associated genes.�For example, the small abnormal chromosome found to be associated with chronic myelogenous leukemia (CML) in 1960 was shown to be the result of translocation between chromosomes 9 and 22 once techniques for analysis of banded chromosomes were introduced, and subsequently, the translocation breakpoint was cloned to reveal the c-abl oncogene on chromosome 9. This translocation produces a fusion protein, which has been targeted for treatment of CML. For detailed discussion of cancer genetics, see Chap. 101e.


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InternalMed_Harrison. 446 the practice of Genetics in Clinical medicine Susan M. Domchek, J. Larry Jameson, Susan Miesfeldt APPLICATIONS OF MOLECULAR GENETICS IN CLINICAL MEDICINE Genetic testing for inherited abnormalities associated with disease 84 risk is increasingly used in the practice of clinical medicine. Germline alterations include chromosomal abnormalities (Chap. 83e), specific gene mutations with autosomal dominant or recessive patterns of transmission (Chap. 82), and single nucleotide polymorphisms with small relative risks associated with disease. Germline alterations are responsible for disorders beyond classic Mendelian conditions with genetic susceptibility to common adult-onset diseases such as asthma, hypertension, diabetes mellitus, macular degeneration, and many forms of cancer. For many of these diseases, there is a complex interplay of genes (often multiple) and environmental factors that affect lifetime risk, age of onset, disease severity, and treatment options.


InternalMed_Harrison

InternalMed_Harrison. The expansion of knowledge related to genetics is changing our understanding of pathophysiology and influencing our classification of diseases. Awareness of genetic etiology can have an impact on clinical management, including prevention and screening for or treatment of a range of diseases. Primary care physicians are relied upon to help patients navigate testing and treatment options. Consequently, they must understand the genetic basis for a large number of genetically influenced diseases, incorporate personal and family history to determine the risk for a specific mutation, and be positioned to provide counseling. Even if patients are seen by genetic specialists who assess genetic risk and coordinate testing, primary care providers should provide information to their patients regarding the indications, limitations, risks, and benefits of genetic counseling and testing. They must also be prepared to offer risk-based management following genetic risk assessment. Given the pace of


InternalMed_Harrison

InternalMed_Harrison. the indications, limitations, risks, and benefits of genetic counseling and testing. They must also be prepared to offer risk-based management following genetic risk assessment. Given the pace of genetics, this is an increasingly difficult task. The field of clinical genetics is rapidly moving from single gene testing to multigene panel testing, with techniques such as wholeexome and -genome sequencing on the horizon, increasing the complexity of test selection and interpretation, as well as patient education and medical decision making.


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InternalMed_Harrison. Adult-onset hereditary diseases follow multiple patterns of inheritance. Some are autosomal dominant conditions. These include many common cancer susceptibility syndromes such as hereditary breast and ovarian cancer (due to germline BRCA1 and BRCA2 mutations) and Lynch syndrome (caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2). In both of these examples, inherited mutations are associated with a high penetrance (lifetime risk) of cancer, although risk is not 100%. In other conditions, although there is autosomal dominant transmission, there is lower penetrance, thereby making the disorders more difficult to recognize. For example, germline mutations in CHEK2 increase the risk of breast cancer, but with a moderate lifetime risk in the range of 20–40%, as opposed to 50–70% for mutations in BRCA1 or BRCA2. Other adult-onset hereditary diseases are transmitted in an autosomal recessive fashion where two mutant alleles are necessary to cause disease.


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InternalMed_Harrison. as opposed to 50–70% for mutations in BRCA1 or BRCA2. Other adult-onset hereditary diseases are transmitted in an autosomal recessive fashion where two mutant alleles are necessary to cause disease. Examples include hemochromatosis and MYH-associated colon cancer. There are more pediatric-onset autosomal recessive disorders, such as lysosomal storage diseases and cystic fibrosis.


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InternalMed_Harrison. The genetic risk for many adult-onset disorders is multifactorial. Risk can be conferred by genetic factors at a number of loci, which individually have very small effects (usually with relative risks of <1.5). These risk loci (generally single nucleotide polymorphisms [SNPs]) combine with other genes and environmental factors in ways that are not well understood. SNP panels are available to assess risk of disease, but the optimal way of using this information in the clinical setting remains uncertain.


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InternalMed_Harrison. Many diseases have multiple patterns of inheritance, adding to the complexity of evaluating patients and families for these conditions. For example, colon cancer can be associated with a single germline mutation in a mismatch repair gene (Lynch syndrome, autosomal dominant), biallelic mutations in MYH (autosomal recessive), or multiple SNPs (polygenic). Many more individuals will have SNP risk alleles than germline mutations in high-penetrance genes, but cumulative lifetime risk of colon cancer related to the former is modest, whereas the risk related to the latter is significant. Personal and family histories provide important insights into the possible mode of inheritance.


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InternalMed_Harrison. When two or more first-degree relatives are affected with asthma, cardiovascular disease, type 2 diabetes, breast cancer, colon cancer, or melanoma, the relative risk for disease among close relatives ranges from twoto fivefold, underscoring the importance of family history for these prevalent disorders. In most situations, the key to assessing the inherited risk for common adult-onset diseases is the collection and interpretation of a detailed personal and family medical history in conjunction with a directed physical examination.


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InternalMed_Harrison. Family history should be recorded in the form of a pedigree. Pedigrees should convey health-related data on firstand second-degree relatives. When such pedigrees suggest inherited disease, they should be expanded to include additional family members. The determination of risk for an asymptomatic individual will vary depending on the size of the pedigree, the number of unaffected relatives, the types of diagnoses, and the ages of disease onset. For example, a woman with two first-degree relatives with breast cancer is at greater risk for a specific Mendelian disorder if she has a total of 3 female first-degree relatives (with only 1 unaffected) than if she has a total of 10 female first-degree relatives (with 7 unaffected). Factors such as adoption and limited family structure (few women in a family) should to be taken into consideration in the interpretation of a pedigree. Additional considerations include young age of disease onset (e.g., a 30-year nonsmoking woman with a myocardial


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InternalMed_Harrison. a family) should to be taken into consideration in the interpretation of a pedigree. Additional considerations include young age of disease onset (e.g., a 30-year nonsmoking woman with a myocardial infarction), unusual diseases (e.g., male breast cancer or medullary thyroid cancer), and the finding of multiple potentially related diseases in an individual (e.g., a woman with a history of both colon and endometrial cancer). Some adult-onset diseases are more prevalent in certain ethnic groups. For instance, 2.5% of individuals of Ashkenazi Jewish ancestry carry one of three founder mutation in BRCA1 and BRCA2. Factor V Leiden mutations are much more common in Caucasians than in Africans or Asians.


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InternalMed_Harrison. Additional variables that should be documented are nonhereditary risk factors among those with disease (such as cigarette smoking and myocardial infarction; asbestos exposure and lung disease; and mantle radiation and breast cancer). Significant associated environmental exposures or lifestyle factors decrease the likelihood of a specific genetic disorder. In contrast, the absence of nonhereditary risk factors typically associated with a disease raises concern about a genetic association. A personal or family history of deep vein thrombosis in the absence of known environmental or medical risk factors suggests a hereditary thrombotic disorder. The physical examination may also provide important clues about the risk for a specific inherited disorder. A patient presenting with xanthomas at a young age should prompt consideration of familial hypercholesterolemia. The presence of trichilemmomas in a woman with breast cancer raises concern for Cowden syndrome, associated with PTEN


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InternalMed_Harrison. at a young age should prompt consideration of familial hypercholesterolemia. The presence of trichilemmomas in a woman with breast cancer raises concern for Cowden syndrome, associated with PTEN mutations.


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InternalMed_Harrison. Recall of family history is often inaccurate. This is especially so when the history is remote and families lose contact or separate geographically. It can be helpful to ask patients to fill out family history forms before or after their visits, because this provides them with an opportunity to contact relatives. Ideally, this information should be embedded in electronic health records and updated intermittently. Attempts should be made to confirm the illnesses reported in the family history before making important and, in certain circumstances, irreversible management decisions. This process is often labor


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InternalMed_Harrison. FIGURE 84-1 A 36-year-old woman (arrow) seeks consultation because of her family history of cancer. The patient expresses concern that the multiple cancers in her relatives imply an inherited predisposition to develop cancer. The family history is recorded, and records of the patient’s relatives confirm the reported diagnoses. Symbol key Breast cancer 52 Breast ca 44 46 Ovarian ca 43 Ovarian cancer 2 40 Ovarian ca 38 42 Breast ca 38 24 Pneumonia 56 36 62 69 Breast ca 44 55 Ovarian ca 54 6210 Accident 6 40 5 2 2 intensive and ideally involves interviews of additional family members or reviewing medical records, autopsy reports, and death certificates.


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InternalMed_Harrison. Although many inherited disorders will be suggested by the clustering of relatives with the same or related conditions, it is important to note that disease penetrance is incomplete for most genetic disorders. As a result, the pedigree obtained in such families may not exhibit a clear Mendelian inheritance pattern, because not all family members carrying the disease-associated alleles will manifest clinical evidence of the condition. Furthermore, genes associated with some of these disorders often exhibit variable disease expression. For example, the breast cancer–associated gene BRCA2 can predispose to several different malignancies in the same family, including cancers of the breast, ovary, pancreas, skin, and prostate. For common diseases such as breast cancer, some family members without the susceptibility allele (or genotype) may develop breast cancer (or phenotype) sporadically. Such phenocopies represent another confounding variable in the pedigree analysis.


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InternalMed_Harrison. Some of the aforementioned features of the family history are illustrated in Fig. 84-1. In this example, the proband, a 36-year-old woman (IV-1), has a strong history of breast and ovarian cancer on the paternal side of her family. The early age of onset and the co-occurrence of breast and ovarian cancer in this family suggest the possibility of an inherited mutation in BRCA1 or BRCA2. It is unclear however, without genetic testing, whether her father harbors such a mutation and transmitted it to her. After appropriate genetic counseling of the pro-band and her family, the most informative and cost-effective approach to DNA analysis in this family is to test the cancer-affected 42-year-old living cousin for the presence of a BRCA1 or BRCA2 mutation. If a mutation is found, then it is possible to test for this particular alteration in other family members, if they so desire. In the example shown, if the proband’s father has a BRCA1 mutation, there is a 50:50 probability that the


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InternalMed_Harrison. possible to test for this particular alteration in other family members, if they so desire. In the example shown, if the proband’s father has a BRCA1 mutation, there is a 50:50 probability that the mutation was transmitted to her, and genetic testing can be used to establish the absence or presence of this alteration. In this same example, if a mutation is not detected in the cancer-affected cousin, testing would not be indicated for cancer-unaffected relatives.


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InternalMed_Harrison. A critical first step before initiating genetic testing is to ensure that the correct clinical diagnosis has been made, whether it is based on family history, characteristic physical findings, pathology, or biochemical testing. Such careful clinical assessment can define the phenotype. In the traditional model of genetic testing, testing is directed initially toward the most probable genes (determined by the phenotype), which 447 prevents unnecessary testing. Many disorders exhibit the feature of locus heterogeneity, which refers to the fact that mutations in different genes can cause phenotypically similar disorders. For example, osteogenesis imperfecta (Chap. 427), long QT syndrome (Chap. 277), muscular dystrophy (Chap. 462e), and hereditary predisposition to breast (Chap. 108) or colon (Chap. 110) cancer can each be caused by mutations in a number of distinct genes. The patterns of disease transmission, disease risk, clinical course, and treatment may differ significantly depending


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InternalMed_Harrison. (Chap. 110) cancer can each be caused by mutations in a number of distinct genes. The patterns of disease transmission, disease risk, clinical course, and treatment may differ significantly depending on the specific gene affected. Historically, the choice of which gene to test has been determined by unique clinical and family history features and the relative prevalence of candidate genetic disorders. However, rapid changes in genetic testing techniques, as discussed below, may impact this paradigm. It is now technically and financially feasible to sequence many genes (or even the whole exome) at one time. The incorporation of multiplex testing for germline mutations is rapidly evolving.


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InternalMed_Harrison. Genetic testing is regulated and performed in much the same way as other specialized laboratory tests. In the United States, genetic testing laboratories are Clinical Laboratory Improvement Amendments (CLIA) approved to ensure that they meet quality and proficiency standards. A useful information source for various genetic tests is www.genetests.org. It should be noted that many tests need to be ordered through specialized laboratories.


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InternalMed_Harrison. Genetic testing is performed largely by DNA sequence analysis for mutations, although genotype can also be deduced through the study of RNA or protein (e.g., apolipoprotein E, hemoglobin S, and immunohistochemistry). For example, universal screening for Lynch syndrome via immunohistochemical analysis of colorectal cancers for absence of expression of mismatch repair proteins is under way at multiple hospitals throughout the United States. The determination of DNA sequence alterations relies heavily on the use of polymerase chain reaction (PCR), which allows rapid amplification and analysis of the gene of interest. In addition, PCR enables genetic testing on minimal amounts of DNA extracted from a wide range of tissue sources including leukocytes, mucosal epithelial cells (obtained via saliva or buccal swabs), and archival tissues. Amplified DNA can be analyzed directly by DNA sequencing, or it can be hybridized to DNA chips or blots to detect the presence of normal and altered DNA


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InternalMed_Harrison. saliva or buccal swabs), and archival tissues. Amplified DNA can be analyzed directly by DNA sequencing, or it can be hybridized to DNA chips or blots to detect the presence of normal and altered DNA sequences. Direct DNA sequencing is frequently used for determination of hereditary disease susceptibility and prenatal diagnosis. Analyses of large alterations of the genome are possible using cytogenetics, fluorescent in situ hybridization (FISH), Southern blotting, or multiplex ligation-dependent probe amplification (MLPA) (Chap. 83e).


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InternalMed_Harrison. Massively parallel sequencing (also called next-generation sequencing) is significantly altering the approach to genetic testing for adult-onset hereditary susceptibility disorder. This technology encompasses several high-throughput approaches to DNA sequencing, all of which can reliably sequence many genes at one time. Technically, this involves the use of amplified DNA templates in a flow cell, a very different process than traditional Sanger sequencing which is time-consuming and expensive.


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InternalMed_Harrison. Multiplex panels for inherited susceptibility are commercially available and include testing of a number of genes that have been associated with the condition of interest. For example, panels are available for Brugada syndrome, hypertrophic cardiomyopathy, and Charcot-Marie-Tooth neuropathy. For many syndromes, this type of panel testing may make sense. However, in other situations, the utility of panel testing is less certain. Currently available breast cancer susceptibility panels contain six genes or more. Many of the genes included in the larger panels are associated with only a modest risk of breast cancer, and the clinical application is uncertain. An additional problem of sequencing many genes (rather than the genes for which there is most suspicion) is the identification of one or more variants of uncertain significance (VUS), discussed below. Whole-exome sequencing (WES) is also now commercially available, although largely used in individuals with syndromes unexplained by


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InternalMed_Harrison. Whole-exome sequencing (WES) is also now commercially available, although largely used in individuals with syndromes unexplained by Chapter 84 The Practice of Genetics in Clinical Medicine traditional genetic testing. As cost declines, WES may be more widely used. Whole-genome sequencing is also commercially available. Although it may be quite feasible to sequence the entire genome, there are many issues in doing so, including the daunting task of analyzing the vast amount of data generated. Other issues include: (1) the optimal way in which to obtain informed consent, (2) interpretation of frequent sequence variation of uncertain significance, (3) interpretation of alterations in genes with unclear relevance to specific human pathology, and (4) management of unexpected but clinically significant genetic findings.


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InternalMed_Harrison. Testing strategies are evolving as a result of these new genetic testing platforms. As the cost of multiple gene panels and WES continue to fall, and as interpretation of such test results improve, there may be a shift from sequential single-gene (or a few genes) testing to multigene testing. For example, presently, a 30-year-old woman with breast cancer but no family history of cancer and no syndromic features would undergo BRCA1/2 testing. If negative, she would subsequently be offered TP53 testing. Notably, a reasonable number of individuals offered TP53 testing for Li-Fraumeni syndrome decline because mutations are associated with extremely high cancer risks (including childhood cancers) in multiple organs and there are no proven interventions to mitigate risk. Without features consistent with Cowden syndrome, the woman would not be routinely offered PTEN testing or testing for CHEK2, ATM, BRIP, BARD, NBN, and PALB2. However, it is now possible to synchronously analyze all of the


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InternalMed_Harrison. with Cowden syndrome, the woman would not be routinely offered PTEN testing or testing for CHEK2, ATM, BRIP, BARD, NBN, and PALB2. However, it is now possible to synchronously analyze all of the aforementioned genes, for a nominally higher cost than BRCA1/2 testing alone. Concerns about such panels include appropriate consent strategies related to unexpected findings, VUS, and unclear clinical utility of testing moderate-penetrance genes. Thus, changes from the traditional model of single-gene genetic testing should be done with caution (Fig. 84-2).


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InternalMed_Harrison. Limitations to the accuracy and interpretation of genetic testing exist. In addition to technical errors, genetics tests are sometimes designed to detect only the most common mutations. In addition, genetic testing has evolved over time. For example, it was not possible to obtain commercially available comprehensive large genomic rearrangement testing for BRCA1 and BRCA2 until 2006. Therefore, a negative result must be qualified by the possibility that the individual may have a mutation that was not included in the test. In addition, a negative result does not mean that there is not a mutation in some other gene that causes a similar inherited disorder. A negative result, unless there is known mutation in the family, is typically classified as uninformative.


InternalMed_Harrison

InternalMed_Harrison. VUS are another limitation to genetic testing. A VUS (also termed unclassified variant) is a sequence variation in a gene where the effect of the alteration on the function of the protein is not known. Many of these variants are single nucleotide substitutions (also called missense mutations) that result in a single amino acid change. Although many VUSs will ultimately be reclassified as benign polymorphisms, some will prove to be functionally important. As more genes are sequenced (for example, in a multiplex panel or through WES), the percentage of individuals found to have a VUS increases significantly. The finding of a VUS is difficult for patients and providers alike and complicates decisions regarding medical management.


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InternalMed_Harrison. Clinical utility is an important consideration because genetic testing for susceptibility to chronic diseases is increasingly integrated into the practice of medicine. In some situations, there is clear clinical utility to genetic testing with significant evidence-based changes in medical management decisions based on results. However, in many cases, the discovery of disease-associated genes has outpaced studies that assess how such information should be used in the clinical management of the patient and family. This is particularly true for moderate-and low-penetrance gene mutations. Therefore, predictive genetic testing should be approached with caution and only offered to patients who have been adequately counseled and have provided informed consent. Predictive genetic testing falls into two distinct categories. Presymptomatic testing applies to diseases where a specific genetic alteration is associated with a near 100% likelihood of developing disease. In contrast, predisposition


InternalMed_Harrison

InternalMed_Harrison. two distinct categories. Presymptomatic testing applies to diseases where a specific genetic alteration is associated with a near 100% likelihood of developing disease. In contrast, predisposition testing predicts a risk for disease that is less than 100%. For example, presymptomatic testing is available for those at risk for Huntington’s disease; whereas, predisposition testing is considered for those at risk for hereditary colon cancer. It is important to note that for the majority of adult-onset disorders, testing is only predictive. Test results cannot reveal with confidence whether, when, or how the disease will manifest itself. For example, not everyone with the apolipoprotein


InternalMed_Harrison

InternalMed_Harrison. Traditional approach to genetic testing Genetic testing in the era of E4 allele will develop Alzheimer’s disease, and next-generation sequencing? individuals without this genetic marker can still develop the disorder. The optimal testing strategy for a family is to initiate testing in an affected family member first. Identification of a mutation can direct the testing of other at-risk family members (whether symptomatic or not). In the absence of additional familial or environmental risk factors, individuals who test negative for the mutation found in the affected family member can be informed that they are at general population risk for that particular disease. Furthermore, they can be reassured that they are not at risk for passing the mutation on to their children. On the other hand, asymptomatic family members who test positive for the known mutation must be informed that they are at increased risk for disease development and for transmitting the alteration to their children.


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InternalMed_Harrison. asymptomatic family members who test positive for the known mutation must be informed that they are at increased risk for disease development and for transmitting the alteration to their children. Pretest counseling and education are important, as is an assessment of the patient’s ability to understand and cope with test results. Genetic testing has implications for entire families, and thus individuals interested in pursuing genetic testing must consider how test results might impact their relationships with relatives, partners, spouses, and children. In families with a known genetic mutation, those who test posi-FIGURE 84-2 Approach to genetic testing. tive must consider the impact of their carrier status on their present and future lifestyles; those who test negative may manifest survivor guilt. Parents who are found to have a disease-associated mutation often express considerable anxiety and despair as they address the issue of risk to their children. In addition, some individuals


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InternalMed_Harrison. guilt. Parents who are found to have a disease-associated mutation often express considerable anxiety and despair as they address the issue of risk to their children. In addition, some individuals consider options such as preimplantation genetic diagnosis in their reproductive decision making.


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InternalMed_Harrison. When a condition does not manifest until adulthood, clinicians and parents are faced with the question of whether at-risk children should be offered genetic testing and, if so, at what age. Although the matter is debated, several professional organizations have cautioned that genetic testing for adult-onset disorders should not be offered to children. Many of these conditions have no known interventions in childhood to prevent disease; consequently, such information can pose significant psychosocial risk to the child. In addition, there is concern that testing during childhood violates a child’s right to make an informed decision regarding testing upon reaching adulthood. On the other hand, testing should be offered in childhood for disorders that may manifest early in life, especially when management options are available. For example, children with multiple endocrine neoplasia 2 (MEN 2) may develop medullary thyroid cancer early in life and should be considered for prophylactic


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InternalMed_Harrison. when management options are available. For example, children with multiple endocrine neoplasia 2 (MEN 2) may develop medullary thyroid cancer early in life and should be considered for prophylactic thyroidectomy (Chap. 408). Similarly, children with familial adenomatous polyposis (FAP) due to a mutation in APC may develop polyps in their teens with progression to invasive cancer in the twenties, and therefore, colonoscopy screening is started between the ages of 10 and 15 years (Chap. 110).


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InternalMed_Harrison. Informed consent for genetic testing begins with education and counseling. The patient should understand the risks, benefits, and limitations of genetic testing, as well as the potential implications of test results. Informed consent should include a written document, drafted clearly and concisely in a language and format that is understandable to the patient. Because molecular genetic testing of an asymptomatic individual often allows prediction of future risk, the patient should understand all potential long-term medical, psychological, and social implications of testing. There have long been concerns about the potential for genetic discrimination. The Genetic Information Nondiscrimination Act (GINA) was passed in 2008 and provides some protections related to job and health insurance discrimination. It is important to explore with patients the potential impact of genetic test results on future health as well as disability and life insurance coverage. Patients should understand that


InternalMed_Harrison

InternalMed_Harrison. It is important to explore with patients the potential impact of genetic test results on future health as well as disability and life insurance coverage. Patients should understand that alternatives remain available if they decide not to pursue genetic testing, including the option of delaying testing to a later date. The option of DNA banking should be presented so that samples are readily available for future use by family members, if needed.


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InternalMed_Harrison. Depending on the nature of the genetic disorder, posttest interventions may include: (1) cautious surveillance and awareness; (2) specific medical interventions such as enhanced screening, chemoprevention, or risk-reducing surgery; (3) risk avoidance; and (4) referral to support services. For example, patients with known deleterious mutations in BRCA1 or BRCA2 are strongly encouraged to undergo risk-reducing salpingo-oophorectomy and are offered intensive breast cancer screening as well as the option of risk-reducing mastectomy. In addition, such women may wish to take chemoprevention with tamoxifen, raloxifene, or exemestane. Those with more limited medical management and prevention options, such as patients with Huntington’s disease, should be offered continued follow-up and supportive services, including physical and occupational therapy and social services or support groups as indicated. Specific interventions will change as research continues to enhance our understanding of the


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InternalMed_Harrison. services, including physical and occupational therapy and social services or support groups as indicated. Specific interventions will change as research continues to enhance our understanding of the medical management of these genetic conditions and more is learned about the functions of the gene products involved.


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InternalMed_Harrison. Individuals who test negative for a mutation in a disease-associated gene identified in an affected family member must be reminded that they may still be at risk for the disease. This is of particular importance for common diseases such as diabetes mellitus, cancer, and coronary artery disease. For example, a woman who finds that she does not carry 449 the disease-associated mutation in BRCA2 previously discovered in the family should be reminded that she still requires the same breast cancer screening recommended for the general population.


InternalMed_Harrison

InternalMed_Harrison. Genetic counseling should be distinguished from genetic testing and screening, although genetic counselors are often involved in issues related to testing. Genetic counseling refers to a communication process that deals with human problems associated with the occurrence of risk of a genetic disorder in a family. Genetic risk assessment is complex and often involves elements of uncertainty. Counseling, therefore, includes genetic education as well as psychosocial counseling. Genetic counseling can be useful in a wide range of situations (Table 84-1). The role of the genetic counselor includes the following: 1. Gather and document a detailed family history. 2. Educate patients about general genetic principles related to disease risk, both for themselves and for others in the family. 3. Assess and enhance the patient’s ability to cope with the genetic information offered. 4. Discuss how nongenetic factors may relate to the ultimate expression of disease. 5.


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InternalMed_Harrison. 3. Assess and enhance the patient’s ability to cope with the genetic information offered. 4. Discuss how nongenetic factors may relate to the ultimate expression of disease. 5. Address medical management issues. 6. Assist in determining the role of genetic testing for the individual and the family. 7. Ensure the patient is aware of the indications, process, risks, benefits, and limitations of the various genetic testing options. 8. Assist the patient, family, and referring physician in the interpretation of the test results. 9. Refer the patient and other at-risk family members for additional medical and support services, if necessary.


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InternalMed_Harrison. Genetic counseling is generally offered in a nondirective manner, wherein patients learn to understand how their values factor into a particular medical decision. Nondirective counseling is particularly appropriate when there are no data demonstrating a clear benefit associated with a particular intervention or when an intervention is considered experimental. For example, nondirective genetic counseling is used when a person is deciding whether to undergo genetic testing for Huntington’s disease. At this time, there is no clear benefit (in terms of medical outcome) to an at-risk individual undergoing genetic testing for this disease because its course cannot be altered by therapeutic interventions. However, testing can have an important impact on the individual’s perception of advanced care planning and his or her interpersonal relationships and plans for childbearing. Therefore, the decision to pursue testing rests on the individual’s belief system and values. On the other hand, a


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InternalMed_Harrison. care planning and his or her interpersonal relationships and plans for childbearing. Therefore, the decision to pursue testing rests on the individual’s belief system and values. On the other hand, a more directive approach is appropriate when a condition can be treated. In a family with FAP, colon cancer screening and prophylactic colectomy should be recommended for known APC mutation carriers. The counselor and clinician following this family must ensure that the at-risk family members have access to the resources necessary to adhere to these recommendations.


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InternalMed_Harrison. Genetic education is central to an individual’s ability to make an informed decision regarding testing options and treatment. An adequate knowledge of patterns of inheritance will allow patients to understand the probability of disease risk for themselves and other family members. It is also important to impart the concepts of disease penetrance and expression. For most complex adult-onset genetic Previous history of a child with birth defects or a genetic disorder Personal or family history suggestive of a genetic disorder


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InternalMed_Harrison. Previous history of a child with birth defects or a genetic disorder Personal or family history suggestive of a genetic disorder Chapter 84 The Practice of Genetics in Clinical Medicine disorders, asymptomatic patients should be advised that a positive test result does not always translate into future disease development. In addition, the role of nongenetic factors, such as environmental exposures and lifestyle, must be discussed in the context of multi-factorial disease risk and disease prevention. Finally, patients should understand the natural history of the disease as well as the potential options for intervention, including screening, prevention, and in certain circumstances, pharmacologic treatment or prophylactic surgery.


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InternalMed_Harrison. Specific treatments are available for a number of genetic disorders. Strategies for the development of therapeutic interventions have a long history in childhood metabolic diseases; however, these principles have been applied in the diagnosis and management of adult-onset diseases as well (Table 84-2). Hereditary hemochromatosis is usually caused by mutations in HFE (although other genes have been less commonly associated) and manifests as a syndrome of iron overload, which can lead to liver disease, skin pigmentation, diabetes mellitus, arthropathy, impotence in males, and cardiac issues (Chap. 428). When identified early, the disorder can be managed effectively with therapeutic phlebotomy. Therefore, when the diagnosis of hemochromatosis has been made in a proband, it is important to counsel and offer testing to other family members in order to minimize the impact of the disorder.


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InternalMed_Harrison. Preventative measures and therapeutic interventions are not restricted to metabolic disorders. Identification of familial forms of long QT syndrome, associated with ventricular arrhythmias, allows early electrocardiographic testing and the use of prophylactic antiarrhythmic therapy, overdrive pacemakers, or defibrillators. Individuals with familial hypertrophic cardiomyopathy can be screened by ultrasound, treated with beta blockers or other drugs, and counseled about the importance of avoiding strenuous exercise and dehydration. Those with Marfan’s syndrome can be treated with beta blockers or Abbreviations: AD, autosomal dominant; AR, autosomal recessive; HNPCC, hereditary nonpolyposis colorectal cancer; MRI, magnetic resonance imaging; XL, X-linked. angiotensin II receptor blockers and monitored for the development of aortic aneurysms.


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InternalMed_Harrison. angiotensin II receptor blockers and monitored for the development of aortic aneurysms. The field of pharmacogenetics identifies genes that alter drug metabolism or confer susceptibility to toxic drug reactions. Pharmacogenetics seeks to individualize drug therapy in an attempt to improve treatment outcomes and reduce toxicity. Examples include thiopurine methyltransferase (TPMT) deficiency, dihydropyrimidine dehydrogenase deficiency, malignant hyperthermia, and glucose-6-phosphate deficiency. Despite successes in this area, it is not always clear how to incorporate pharmacogenetics into clinical care. For example, although there is an association with CYP and VKORCgenotypes and warfarin dosing, there is no evidence that incorporating genotyping into clinical practice improves patient outcomes.


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InternalMed_Harrison. The identification of germline abnormalities that increase the risk of specific types of cancer is rapidly changing clinical management. Identifying family members with mutations that predispose to FAP or Lynch syndrome leads to recommendations of early cancer screening and prophylactic surgery, as well as consideration of chemoprevention and attention to healthy lifestyle habits. Similar principles apply to familial forms of melanoma as well as cancers of the breast, ovary, and thyroid. In addition to increased screening and prophylactic surgery, the identification of germline mutations associated with cancer may also lead to the development of targeted therapeutics, for example, the ongoing development of PARP inhibitors in those with BRCA-associated cancers.


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InternalMed_Harrison. Although the role of genetic testing in the clinical setting continues to evolve, such testing holds the promise of allowing early and more targeted interventions that can reduce morbidity and mortality. Rapid technologic advances are changing the ways in which genetic testing is performed. As genetic testing becomes less expensive and technically easier to perform, it is anticipated that there will be an expansion of its use. This will present challenges, but also opportunities. It is critical that physicians and other health care professionals keep current with advances in genetic medicine in order to facilitate appropriate referral for genetic counseling and judicious use of genetic testing, as well as to provide state-of-the-art, evidence-based care for affected or at-risk patients and their relatives. Chapter 84 The Practice of Genetics in Clinical Medicine


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InternalMed_Harrison. Mitochondrial DNa and heritable traits and Diseases Karl Skorecki, Doron Behar Mitochondria are cytoplasmic organelles whose major function is to generate ATP by the process of oxidative phosphorylation under aero-85e bic conditions. This process is mediated by the respiratory electron transport chain (ETC) multiprotein enzyme complexes I–V and the two electron carriers, coenzyme Q (CoQ) and cytochrome c. Other cellular processes to which mitochondria make a major contribution include apoptosis (programmed cell death) and additional cell type–specific functions (Table 85e-1). The efficiency of the mitochondrial ETC in ATP production is a major determinant of overall body energy balance and thermogenesis. In addition, mitochondria are the predominant source of reactive oxygen species (ROS), whose rate of production also relates to the coupling of ATP production to oxygen consumption. Given the centrality of oxidative phosphorylation to the normal activities of almost all cells, it is


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InternalMed_Harrison. whose rate of production also relates to the coupling of ATP production to oxygen consumption. Given the centrality of oxidative phosphorylation to the normal activities of almost all cells, it is not surprising that mitochondrial dysfunction can affect almost any organ system (Fig. 85e-1). Thus, physicians in many disciplines might encounter patients with mitochondrial diseases and should be aware of their existence and characteristics.


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InternalMed_Harrison. The integrated activity of an estimated 1500 gene products is required for normal mitochondrial biogenesis, function, and integrity. Almost all of these are encoded by nuclear genes and thus follow the rules and patterns of nuclear genomic inheritance (Chap. 84). These nuclear-encoded proteins are synthesized in the cell cytoplasm and imported to their location of activity within the mitochondria through a complex biochemical process. In addition, the mitochondria contain their own small genome consisting of numerous copies (polyploidy) per mitochondrion of a circular, double-strand mitochondrial DNA (mtDNA) molecule comprising 16,569 nucleotides. This mtDNA sequence (also known as the “mitogenome”) might represent the remnants of endosymbiotic prokaryotes from which mitochondria are thought to have originated. The mtDNA sequence contains a total of 37 genes, of which 13 encode mitochondrial protein components of the ETC (Fig. 85e-2). The remaining 22 tRNAand 2 rRNA-encoding genes are


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InternalMed_Harrison. to have originated. The mtDNA sequence contains a total of 37 genes, of which 13 encode mitochondrial protein components of the ETC (Fig. 85e-2). The remaining 22 tRNAand 2 rRNA-encoding genes are dedicated to the process of translating the 13 mtDNAencoded proteins. This dual nuclear and mitochondrial genetic control of mitochondrial function results in unique and diagnostically challenging patterns of inheritance. The current chapter focuses on heritable traits and diseases related to the mtDNA component of the dual genetic control of mitochondrial function. The reader is referred to Chaps. 84 and 462e for consideration of mitochondrial disease originating from mutations in the nuclear genome. The latter include disorders due to mutations in nuclear genes directly encoding structural components or assembly factors of the oxidative phosphorylation complexes, (2) disorders due to mutations in nuclear genes encoding proteins indirectly related to oxidative phosphorylation, and of mtDNA


InternalMed_Harrison

InternalMed_Harrison. or assembly factors of the oxidative phosphorylation complexes, (2) disorders due to mutations in nuclear genes encoding proteins indirectly related to oxidative phosphorylation, and of mtDNA copy number in affected tissues without mutations or rear-85e-1 rangements in the mtDNA.


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InternalMed_Harrison. As a result of its circular structure and extranuclear location, the replication and transcription mechanisms of mtDNA differ from the corresponding mechanisms in the nuclear genome, whose nucleosomal packaging and structure are more complex. Because each cell contains many copies of mtDNA, and because the number of mitochondria can vary during the lifetime of each cell, mtDNA copy number is not directly coordinated with the cell cycle. Thus, vast differences in mtDNA copy number are observed between different cell types and tissues and during the lifetime of a cell. Another important feature of the mtDNA replication process is a reduced stringency of proofreading and replication error correction, leading to a greater degree of sequence variation compared to the nuclear genome. Some of these sequence variants are silent polymorphisms that do not have the potential for a phenotypic or pathogenic effect, whereas others may be considered pathogenic mutations.


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InternalMed_Harrison. With respect to transcription, initiation can occur on both strands and proceeds through the production of an intronless polycistronic precursor RNA, which is then processed to produce the 13 individual mRNA and 24 individual tRNA and rRNA products. The 37 mtDNA genes comprise fully 93% of the 16,569 nucleotides of the mtDNA in what is known as the coding region. The control region consists of ~1.1 kilobases (kb) of noncoding DNA, which is thought to have an important role in replication and transcription initiation.


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InternalMed_Harrison. In contrast to homologous pair recombination that takes place in the nucleus, mtDNA molecules do not undergo recombination, such that mutational events represent the only source of mtDNA genetic diversification. Moreover, with very rare exceptions, it is only the maternal DNA that is transmitted to the offspring. The fertilized oocyte degrades mtDNA carried from the sperm in a complex process involving the ubiquitin proteasome system. Thus, although mothers transmit their mtDNA to both their sons and daughters, only the daughters are able to transmit the inherited mtDNA to future generations. Accordingly, mtDNA sequence variation and associated phenotypic traits and diseases are inherited exclusively along maternal lines.


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InternalMed_Harrison. As noted below, because of the complex relationship between mtDNA mutations and disease expression, sometimes this maternal inheritance is difficult to recognize at the clinical or pedigree level. However, evidence of paternal transmission can almost certainly rule out an mtDNA genetic origin of phenotypic variation or disease; conversely, a disease affecting both sexes without evidence of paternal transmission strongly suggests a heritable mtDNA disorder (Fig. 85e-2). MULTIPLE COPY NUMBER (POLYPLOIDY), HIGH MUTATION RATE, HETEROPLASMY, AND MITOTIC SEGREGATION


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InternalMed_Harrison. Each aerobic cell in the body has multiple mitochondria, often numbering many hundreds or more in cells with extensive energy production requirements. Furthermore, the number of copies of mtDNA within each mitochondrion varies from several to hundreds; this is true of both somatic as well as germ cells, including oocytes in females. In the case of somatic cells, this means that the impact of most newly acquired somatic mutations is likely to be very small in terms of total cellular or organ system function; however, because of the manyfold higher mutation rate during mtDNA replication, numerous different mutations may accumulate with aging of the organism. It has been proposed that the total cumulative burden of acquired somatic mtDNA mutations with age may result in an overall perturbation of mitochondrial function, contributing to age-related reduction in the efficiency of oxidative phosphorylation and increased production of damaging ROS. The accumulation of such acquired somatic


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InternalMed_Harrison. of mitochondrial function, contributing to age-related reduction in the efficiency of oxidative phosphorylation and increased production of damaging ROS. The accumulation of such acquired somatic mtDNA mutations with aging may contribute to age-related diseases, such as metabolic syndrome and diabetes, cancer, and neurodegenerative and cardiovascular disease in any given individual. However, somatic mutations are not carried forward to the next generation, and the individual, play a pivotal role in the manifestation and severity of disease and are crucial to understanding the complexity of inheritance of mtDNA disorders. At the level of the oocyte, the percentage of mtDNA molecules bearing each version of the polymorphic sequence variant or mutation depends


InternalMed_Harrison

InternalMed_Harrison. Liver on stochastic events related to partition- Hepatopathy ing of mtDNA molecules during the process of oogenesis itself. Thus, oocytes differ from each other in the degree of ATP heteroplasmy for that sequence variant or mutation. In turn, the heteroplasmic state is carried forward to the zygote and to the organism as a whole, to varying Nuclear Subunits Oxidative degrees, depending on mitotic segrega-DNA phosphorylation tion of mtDNA molecules during organ Fanconi's syndrome system development and maintenance. Glomerulopathy For this reason, in vitro fertilization, Brain followed by preimplantation genetic diagnosis (PGD), is not as predictive of the genetic health of the offspring in Mitochondrial the case of mtDNA mutations as in the DNA case of the nuclear genome. Similarly, Dementia Pancreas Migraine Diabetes mellitus the impact of somatic mtDNA mutations acquired during development and subsequently also shows an enormous spectrum of variability.


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InternalMed_Harrison. Dementia Pancreas Migraine Diabetes mellitus the impact of somatic mtDNA mutations acquired during development and subsequently also shows an enormous spectrum of variability. Blood Mitotic segregation refers to the Pearson's syndrome unequal distribution of wild-type and mutant versions of mtDNA molecules Inner ear during all cell divisions that occur dur-Sensorineural ing prenatal development and subsehearing loss Colon quently throughout the lifetime of an Pseudo-obstruction individual. The phenotypic effect or FIGURE 85e-1 Dual genetic control and multiple organ system manifestations of mitochondrial disease impact will, thus, be a funcdisease. (Reproduced with permission from DR Johns: Mitochondrial DNA and disease. N Engl J Med tion not only of the inherent disruptive 333:638, 1995.) hereditary impact of mtDNA mutagenesis requires separate consideration of events in the female germline.


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InternalMed_Harrison. The multiple mtDNA copy number within each cell, including the maternal germ cells, results in the phenomenon of heteroplasmy, in contrast to much greater uniformity (homoplasy) of somatic nuclear DNA sequence. Heteroplasmy for a given mtDNA sequence variant or mutation arises as a result of the coexistence within a cell, tissue, or individual of mtDNA molecules bearing more than one version of the sequence variant (Fig. 85e-3). The importance of the heteroplasmy phenomena to the understanding of mtDNA-related mitochondrial diseases is critical. The coexistence of mutant and nonmutant mtDNA and the variation of the mutant load among individuals from the same maternal sibship, and across organs and tissues within the same FIGURE 85e-2 Maternal inheritance of mitochondrial DNA (mtDNA) disorders and heritable traits. Affected women (filled circles) transmit the trait to their children. Affected men (filled squares) do not transmit the trait to any of their offspring.


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InternalMed_Harrison. effect (pathogenicity) on the mtDNA tions) or integrity of the mtDNA molecule (control region mutations), but also of its distribution among the multiple copies of mtDNA in the various mitochondria, cells, and tissues of the affected individual. Thus, one consequence can be the generation of a bottleneck due to the marked decline in given sets of mtDNA variants, consequent to such mitotic segregation. Heterogeneity arises from differences in the degree of heteroplasmy among oocytes of the affected female, together with subsequent mitotic segregation of the pathogenic mutation during tissue and organ development, and throughout the lifetime of the individual offspring. The actual expression of disease might then depend on a threshold percentage of mitochondria whose function is disrupted by mtDNA mutations. This in turn confounds hereditary transmission patterns and hence genetic diagnosis of pathogenic heteroplasmic mutations. Generally, if the proportion of mutant mtDNA is less than


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InternalMed_Harrison. mtDNA mutations. This in turn confounds hereditary transmission patterns and hence genetic diagnosis of pathogenic heteroplasmic mutations. Generally, if the proportion of mutant mtDNA is less than 60%, the individual is unlikely to be affected, whereas proportions exceeding 90% cause clinical disease.


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InternalMed_Harrison. In contrast to classic mtDNA diseases, most of which begin in childhood and are the result of heteroplasmic mutations as noted above, during the course of human evolution, certain mtDNA sequence variants have drifted to a state of homoplasmy, wherein all of the mtDNA molecules in the organism contain the new sequence variant. This arises due to a “bottleneck” effect followed by genetic drift during the very process of oogenesis itself (Fig. 85e-3). In other words, during certain stages of oogenesis, the mtDNA copy number becomes so substantially reduced that the particular mtDNA species bearing the novel or derived sequence variant may become the increasingly predominant, mate because of the phenotypic heterogeneity that occurs as a function of heteroplasmy, the challenge of detecting and assessing heteroplasmy in different affected tissues, and the other unique features of mtDNA function and FIGURE 85e-3 Heteroplasmy and the mitochondrial genetic bottleneck.


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InternalMed_Harrison. FIGURE 85e-3 Heteroplasmy and the mitochondrial genetic bottleneck. inheritance described above. It is estimated that at least During the production of primary oocytes, a selected number of mitochondrial DNA (mtDNA) molecules are transferred into each oocyte. Oocyte maturation is mutation with the potential to causes disease, but that associated with the rapid replication of this mtDNA population. This restriction amplification event can lead to a random shift of mtDNA mutational load actually affect up to approximately 1 in 8500 individuals. between generations and is responsible for the variable levels of mutated mtDNA The true disease burden relating to mtDNA sequence observed in affected offspring from mothers with pathogenic mtDNA mutations. variation will only be known when the following capa-


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InternalMed_Harrison. The true disease burden relating to mtDNA sequence observed in affected offspring from mothers with pathogenic mtDNA mutations. variation will only be known when the following capa- Mitochondria that contain mutated mtDNA are shown in red, and those with bilities become available: (1) ability to distinguish a normal mtDNA are shown in green. (Reproduced with permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. Nat Rev Genetics 6:389, 2005.) and eventually exclusive, version of the mtDNA for that particular nucleotide site. All of the offspring of a woman bearing an mtDNA sequence variant or mutation that has become homoplasmic will also be homoplasmic for that variant and will transmit the sequence variant forward in subsequent generations.


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InternalMed_Harrison. Considerations of reproductive fitness limit the evolutionary or population emergence of homoplasmic mutations that are lethal or cause severe disease in infancy or childhood. Thus, with a number of notable exceptions (e.g., mtDNA mutations causing Leber’s hereditary optic neuropathy; see below), most homoplasmic mutations are considered to be neutral markers of human evolution, which are useful and interesting in the population genetics analysis of shared maternal ancestry but which have little significance in human phenotypic variation or disease predisposition.


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InternalMed_Harrison. More importantly is the understanding that this accumulation of homoplastic mutations occurs at a genetic locus that is transmitted only through the female germline and that lacks recombination. In turn, this enables reconstruction of the sequential topology and radiating phylogeny of mutations accumulated through the course of human evolution since the time of the most recent common mtDNA ancestor of all contemporary mtDNA sequences, some 200,000 years ago. The term haplogroup is usually used to define major branching points in the human mtDNA phylogeny, nested one within the other, which often demonstrate striking continental geographic ancestral partitioning. At the level of the complete mtDNA sequence, the term haplotype is usually used to describe the sum of mutations observed for a given mtDNA sequence and as compared to a reference sequence, such that all haplotypes falling within a given haplogroup share the total sum of mutations that have accumulated since the most recent


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InternalMed_Harrison. a given mtDNA sequence and as compared to a reference sequence, such that all haplotypes falling within a given haplogroup share the total sum of mutations that have accumulated since the most recent common ancestor and the bifurcation point they mark. The remaining observed variants are private to each haplotype. Consequentially, human mtDNA sequence is an almost perfect molecular prototype for a nonrecombining locus, and its variation has been extensively used in phylogenetic studies. Moreover, the mtDNA mutation rate is considerably higher than the rate observed for the nuclear genome, especially in the control region, which contains the displacement, or


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InternalMed_Harrison. Oocyte maturation D-loop, in turn comprising two adjacent hypervariable 85e-3 and mtDNA amplification Fertilization regions (HVR-I and HVR-II). Together with the absence of recombination, this amplifies drift to high frequencies of novel haplotypes. As a result, mtDNA haplotypes are more highly partitioned across geographically defined High level of mutation (affected offspring) populations than sequence variants in other parts of the genome. Despite extensive research, it has not been well established that such haplotype-based partitioning has a significant influence on human health conditions. However, mtDNA-based phylogenetic analysis can be used both as a quality assurance tool and as a filter of mutation (mildly affected offspring) in distinguishing neutral mtDNA variants comprising human mtDNA phylogeny from potentially deleterious mutations.


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InternalMed_Harrison. Low level of mutation (unaffected offspring) The true prevalence of mtDNA disease is difficult to esti notype-modifying or pathogenic mutation, (2) accurate assessment of heteroplasmy that can be determined with fidelity, and (3) a systems biology approach (Chap. 87e) to determine the network of epistatic interactions of mtDNA sequence variations with mutations in the nuclear genome.


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InternalMed_Harrison. Given the vital roles of mitochondria in all nucleated cells, it is not surprising that mtDNA mutations can affect numerous tissues with pleiotropic effects. More than 200 different disease-causing, mostly heteroplasmic mtDNA mutations have been described affecting ETC function. Figure 85e-4 provides a partial mtDNA map of some of the better characterized of these disorders. A number of clinical clues can increase the index of suspicion for a heteroplasmic mtDNA mutation as an etiology of a heritable trait or disease, including (1) familial clustering with absence of paternal transmission; (2) adherence to one of the classic syndromes (see below) or paradigmatic combinations of disease phenotypes involving several organ systems that normally do not fit together within a single nuclear genomic mutation category; (3) a complex of laboratory and pathologic abnormalities that reflect disruption in cellular energetics (e.g., lactic acidosis and neurodegenerative and myodegenerative


InternalMed_Harrison

InternalMed_Harrison. genomic mutation category; (3) a complex of laboratory and pathologic abnormalities that reflect disruption in cellular energetics (e.g., lactic acidosis and neurodegenerative and myodegenerative symptoms with the finding of ragged red fibers, reflecting the accumulation of abnormal mitochondria under the muscle sarcolemmal membrane); or (4) a mosaic pattern reflecting a heteroplasmic state.


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InternalMed_Harrison. Heteroplasmy can sometimes be elegantly demonstrated at the tissue level using histochemical staining for enzymes in the oxidative phosphorylation pathway, with a mosaic pattern indicating heterogeneity of the genotype for the coding region for the mtDNA-encoded enzyme. Complex II, CoQ, and cytochrome c are exclusively encoded by nuclear DNA. In contrast, complexes I, III, IV, and V contain at least some subunits encoded by mtDNA. Just 3 of the 13 subunits of the ETC complex IV enzyme, cytochrome c oxidase, are encoded by mtDNA, and, therefore, this enzyme has the lowest threshold for dysfunction when a threshold level of mutated mtDNA is reached. Histochemical staining for cytochrome c oxidase activity in tissues of patients affected with heteroplasmic inherited mtDNA mutations Parkinsonism, LS, MELAS, PEO, LHON, MELAS, myopathy, cardiomyopathy, diabetes and deafness Myopathy, LHON cardiomyopathy, PEO Myopathy, MELAS Myopathy, lymphoma


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InternalMed_Harrison. Parkinsonism, LS, MELAS, PEO, LHON, MELAS, myopathy, cardiomyopathy, diabetes and deafness Myopathy, LHON cardiomyopathy, PEO Myopathy, MELAS Myopathy, lymphoma LS, ataxia, chorea, myopathy PEO Myopathy, PEO Myoglobinuria, motor neuron disease, PPK, deafness, Myopathy, multisystem disease, NARP, MILS, Myopathy, PEO Cardiomyopathy ECM ECM, LHON, myopathy, cardiomyopathy, MELAS and parkinsonism LHON, MELAS, diabetes, LHON and dystonia ND5 LS, MELAS Cardiomyopathy, ECM PEO, myopathy, LHON, myopathy, LHON and dystonia Progressive myoclonus, epilepsy, and optic atrophy Cardiomyopathy, encephalomyopathy FBSN SIDS, ECM Cardiomyopathy, LS, ECM, PEO, MERRF, myoglobinuria MELAS, deafness FIGURE 85e-4 Mutations in the human mitochondrial genome known to cause disease.