Molecular Detection of Inherited Diseases PDF

Chapter 12 Molecular Detection of Inherited Diseases Outline Objectives THE MOLECULAR BASIS OF INHERITED DISEASES 12.1 Describe Mendelian patterns of inheritance as CHROMOSOMAL ABNORMALITIES...

Chapter 12 Molecular Detection of Inherited Diseases Outline Objectives THE MOLECULAR BASIS OF INHERITED DISEASES 12.1 Describe Mendelian patterns of inheritance as CHROMOSOMAL ABNORMALITIES exhibited by family pedigrees. PATTERNS OF INHERITANCE IN SINGLE-GENE DISORDERS 12.2 Illustrate abnormalities in chromosome number MOLECULAR BASIS OF SINGLE-GENE DISORDERS and structure. Lysosomal Storage Diseases 12.3 Define penetrance and variable expressivity. Factor V Leiden 12.4 Relate disease syndromes with affected genes. Prothrombin 12.5 Give examples of laboratory methods designed to Methylenetetrahydrofolate Reductase detect single-gene disorders. Hemochromatosis 12.6 Discuss non-Mendelian inheritance, and give Cystic Fibrosis examples of these types of inheritance, such as Cytochrome P-450 mitochondrial disorders and nucleotide-repeat SINGLE-GENE DISORDERS WITH NONCLASSICAL PATTERNS expansion diseases. OF INHERITANCE 12.7 Show how genomic imprinting can affect disease Mutations in Mitochondrial Genes phenotype. Nucleotide-Repeat Expansion Disorders Fragile X Syndrome Huntington Disease Idiopathic Congenital Central Hypoventilation Syndrome Other Nucleotide Expansion Disorders Genomic Imprinting Multifactorial Inheritance LIMITATIONS OF MOLECULAR TESTING 344  Chapter 12 Molecular Detection of Inherited Diseases 345 Genetic and cytogenetic analyses are a critical com- ponent of diagnostic testing, especially for diseases Advanced Concepts that arise from known genetic events. The identifica- tion of a molecular or chromosomal abnormality is a According to the Lyon hypothesis (or Lyon’s direct observation of the source of some diseases. This hypothesis) first stated by Mary Lyon in 1961, chapter presents examples of clinical laboratory tests only one X chromosome remains genetically commonly performed in molecular genetics using these active in females.1,2 In humans, one X chromo- techniques. some is inactivated at random about the 16th day of embryonic development. The inactive X can be seen as a Barr body (X chromatin) in the inter- THE MOLECULAR BASIS phase nucleus. Not all X genes are shut off in the OF INHERITED DISEASES inactivated X chromosome. Furthermore, reactiva- tion of genes on the inactivated X occurs in germ Mutations are changes in DNA nucleotide sequences. cells before the first meiotic division for produc- These changes range from single base pair or point tion of eggs. mutations of various types to chromosomal. Not all mutations lead to disease. Polymorphisms are proportionately represented gen- otypes in a given population. Sequence polymorphisms can be located within genes or outside of genes. Benign CHROMOSOMAL ABNORMALITIES polymorphisms are useful for mapping disease genes, determining parentage, and identity testing. Balanced Genome mutations (abnormalities in chromosome polymorphisms can have offsetting phenotypes. number) can be detected by karyotyping, ploidy analysis Epigenetic alterations do not change the primary by flow cytometry, and fluorescent in situ hybridization DNA sequence. Epigenetic changes consist of three (FISH). Polyploidy (more than two of any autosome) different forms: DNA methylation, usually alterations in animals usually results in infertility and abnormal of cytosine in CpG islands; genomic imprinting; and appearance. Aneuploidy (gain or loss of any autosome) chromatin remodeling. DNA methylation mostly down- occurs with 0.5% frequency in term pregnancies and regulates RNA transcription. Genomic imprinting 50% in spontaneous abortions. Aneuploidy is caused selectively inactivates chromosomal regions (e.g., X chro- by erroneous separation of chromosomes during egg or mosome inactivation). Chromatin remodeling sequesters sperm production (chromosomal non-disjunction). Auto- large regions of chromosomal DNA through protein somal trisomy/monosomy (three copies/one copy of a binding and histone modification. Histone modification chromosome instead of two) results from fertilization controls the availability of DNA for RNA transcription. of gametes containing an extra chromosome or missing Mutations in germ cells result in inherited disease. a chromosome (n + 1 or n – 1 gametes, respectively). Mutations in somatic cells result in cancer and some Autosomal monosomy is generally, but not always, congenital malformations. Diseases with genetic compo- incompatible with life. Sex chromosome aneuploidy is nents are often referred to as congenital (“born with”) more frequently tolerated, although it is associated with diseases. Congenital disorders are not necessarily her- phenotypic abnormalities. itable, however. Congenital disorders are those present Mosaicism, two or more genetically distinct pop- in individuals at birth. Specifically, congenital disorders ulations of cells from one zygote in an individual (in result when some factor, such as a drug, a chemical, an contrast to chimerism: two or more genetically distinct infection, or an injury, upsets the developmental process. cell populations from different zygotes in an individual), Thus, a baby can have a heritable disease, such as hemo- results from mutation events affecting somatic or germ philia, that can be passed on to future generations or a cells. Early segregation errors during fertilized egg divi- congenital condition, such as spina bifida, that cannot be sion occasionally give rise to mosaicism. Mosaicism is passed to offspring. relatively common with sex chromosomes; for example, 346 Section III Techniques in the Clinical Laboratory TABLE 12.1 Examples of Genome Mutations Genetic Disorder Abnormality Incidence Clinical Features Down syndrome Trisomy 21, 47,XY,+21 1/700 live births Flat facial profile, mental retardation, cardiac problems, risk of acute leukemia, eventual neuropathological disorders, abnormal immune system Edward syndrome Trisomy 18, 47,XY,+18 1/3,000 live births Severe, clenched fist; survival less than 1 year Patau syndrome Trisomy 13, 47,XY,+13 1/5,000 live births Cleft palate, heart damage, mental retardation, survival usually less than 6 mo Klinefelter syndrome 47,XXY 1/850 live births Male hypogonadism, long legs, gynecomastia (male breast enlargement), low testosterone level XYY syndrome 47,XYY 1/1,000 live births Excessive height, acne, 1%–2% behavioral disorders Turner syndrome 45,X and variants 1/2,000 live births Bilateral neck webbing, heart disease, failure to develop secondary sex characteristics, hypothyroidism Multi X females 47,XXX; 48,XXXX 1/1,200 newborn Mental retardation increases with increasing X females 45,X/47,XXX (normal female chromosome complement somatic events (not inherited) and are most commonly is 46,XX). In this case, later nondisjunction will yield seen in cancer. Approximately 7.4% of conceptions additional populations. Rarely, autosomal haploids will have chromosome mutations. Chromosome mutations be lost with the retention of the triploid lineage (e.g., are observed in 50% of spontaneous abortions and 5% 45,XY,-21, 46,XY/47,XY,+21 → 46,XY/47,XY,+21). of stillbirths. Examples of diseases arising from inher- Examples of genome mutations are shown in Table 12.1 ited chromosome structure abnormalities are shown in Chromosome mutations (abnormalities in chromosome Table 12.2. structure) larger than 4 million base pairs (bp) can be seen by karyotyping; smaller irregularities can be seen with the higher resolution of FISH or microarray tech- PATTERNS OF INHERITANCE nology. Structural alterations include translocations IN SINGLE-GENE DISORDERS (reciprocal, nonreciprocal), inversions (paracentric, peri- centric), deletions (terminal, interstitial, ring), duplica- Most phenotypes result from the interaction of multiple tions (isochromosomes), marker chromosomes, and genetic and environmental factors. Some phenotypes, derivative chromosomes. however, are caused by alteration of a single gene. If the Structural mutations require breakage and reunion of phenotype occurs as predicted by Mendelian genetics, DNA. Chromosomal breakage is caused by chemicals patterns of inheritance can be established. Patterns of and radiation. Chromosomal breakage also results from inheritance (transmission patterns) are determined by chromosome breakage syndromes (e.g., Fanconi anemia, examination of family histories. A pedigree is a diagram Bloom syndrome, and ataxia telangiectasia). Some aber- of the inheritance pattern of a phenotype of family rations have no immediate phenotypic effect (recip- members (Fig 12.1). There are three main Mendelian rocal translocations, inversions, some deletions, some transmission patterns: autosomal dominant, autosomal insertions). Others can be deleterious to cells includ- recessive, and X-linked or sex-linked recessive. These ing lethality. Chromosome translocations are usually patterns refer to the disease phenotype.  Chapter 12 Molecular Detection of Inherited Diseases 347 TABLE 12.2 Examples of Chromosomal Mutations Genetic Disorder Abnormality Incidence Clinical Features DiGeorge syndrome and del(22q) 1/4,000 live births CATCH 22 (cardiac abnormality/abnormal facies, velocardiofacial syndrome T-cell deficit, cleft palate, hypercalcemia) Cri du chat syndrome del(5p) 1/20,000–1/50,000 Growth deficiency, catlike cry in infancy, small live births head, mental retardation Contiguous gene syndrome; Wilms’ del(11p) 1/15,000 live births Aniridia (absence of iris), hemihypertrophy (one tumor, aniridia, genitourinary side of the body seems to grow faster than the anomalies, mental retardation other), and other congenital anomalies syndrome FIGURE 12.2 In an autosomal-dominant transmission pattern, heterozygous individuals express the affected pheno- type (filled symbols). Male Female Deceased male Deceased female that Mendelian inheritance is still present and that the Affected male Affected female partial dominance is a manifestation of how the genes are expressed. Codominant offspring simultaneously FIGURE 12.1 A pedigree is a diagram of family phenotype demonstrate the phenotype of both parents. A familiar or genotype. The pedigree will display the transmission pattern example of codominance is the ABO blood types. Dom- of a disease. Phenotypic traits are followed by coloring or pat- inant-negative phenotypes are seen in cases of multim- terns in the symbols. Lack of the trait is indicated by open eric proteins such as the tumor-suppressor tetramer, p53 symbols with no coloring. (Fig. 12.3). Even though only one allele is mutated, the mutated protein can interfere with the function of the In autosomal-dominant transmission, a child of an tetramer, producing an abnormal phenotype. affected individual and an unaffected mate has a 50% to The phenotype of a loss-of-function mutation is 100% risk or likelihood of expressing the disease pheno- usually recessive, but it depends on the type of protein type (Fig. 12.2). Gain-of-function mutations are usually affected. Complex metabolic pathways are suscepti- dominant because the mutated allele produces sufficient ble to loss-of-function mutations because of extensive abnormal factor to bring about the affected condition. interactions between and among proteins. Key structural In complete dominance, the heterozygous phenotype proteins, especially multimeric complexes, risk domi- of the offspring is the same as that of the homozygous nant negative phenotypes. Gain-of-function mutations parent. In partial dominance, the offspring phenotype are less common than loss-of-function mutations. Gain- is variably intermediate between the homozygous and of-function mutations include gene-expression/stability heterozygous parental phenotypes. The parental phe- defects that generate gene products at inappropriate sites notypes can reappear in the next generation, showing or times. 348 Section III Techniques in the Clinical Laboratory Autosomal recessive is the largest category of Men- mutations) such as type 2 diabetes may also be inborn delian disorders. The recurrence risk is 25% if siblings errors of metabolism.3 are affected, indicating the presence of the recessive mutation in both of the parents. The “margin of safety,” Advanced Concepts that is, having two copies of every gene, requires the loss of the normal allele through somatic events (loss of Autosomal-dominant mutations can originate from heterozygosity) or homozygosity for the manifestation gonadal mosaicism of new mutations in germ of the recessive disease phenotype. Autosomal-recessive cells, that is, DNA changes that arise in cells that diseases are more often observed as a result of two indi- produce eggs or sperm. Establishment of a new viduals heterozygous for the same mutation producing mutation as a dominant mutation in a family or in offspring (Fig. 12.4). New mutations are rarely detected a population is influenced by its effect on repro- in autosomal-recessive transmission patterns. Inborn ductive fitness. errors of metabolism are usually autosomal recessive. Risk factors for neoplastic diseases also fall in this cat- egory. Polygenic disorders (caused by multiple gene Almost all sex-linked disorders are X-linked because relatively few genes are carried on the Y chromosome. Chromosomes X-linked mutations are almost always recessive, but there are X-linked dominant diseases (e.g., vitamin Monomers Tetramers D–resistant rickets). Even though one X chromosome is inactivated in females, the inactivation is reversible + + so that a second copy of X-linked genes is available. + Normal function In contrast, males are hemizygous for X-linked genes, + having only one copy on the X chromosome. Males, + + therefore, are more likely to manifest the disease phe- notype (Fig. 12.5). + + + Abnormal + – – FIGURE 12.4 Autosomal-recessive mutations are not FIGURE 12.3 Dominant negative mutations affect multim- expressed in heterozygotes. The phenotype is displayed only in eric proteins. In this illustration, a single mutant monomer a homozygous individual; in this illustration, produced by the affects the function of the assembled tetramer. inbreeding of two cousins (double horizontal line). FIGURE 12.5 X-linked recessive diseases are carried by females but manifested most often in males.  Chapter 12 Molecular Detection of Inherited Diseases 349 Due to the multifactorial nature of most diseases, substrates. With the discovery of genes that code for the the same gene mutations are not always manifested in enzymes and their subunits, molecular testing has been a disease phenotype. Penetrance is the frequency of used to some extent. Mutations can be detected by direct expression of disease phenotype in individuals with a sequencing, usually after an initial biochemical screen- gene lesion. Complete penetrance is the expression of the ing test for loss of enzyme activity. disease phenotype in every individual with the mutated gene. Complete penetrance is common in homozygous Factor V Leiden recessive phenotypes. Variable expressivity is a range of phenotypes in individuals with the same gene lesion. Mutations that lead to abnormal but survival states can Variable expressivity also reflects the interaction of be relatively frequently encountered in a population. An other gene products and the environment on the disease example is the hypercoagulation phenotype resulting in phenotype. mutations in the factor V gene. Discovered in 1994, the Leiden mutation (1691 A→G, R506Q) in the coagula- tion factor V gene F5 (1q23) causes a hypercoagulable MOLECULAR BASIS (thrombophilic) phenotype. This genotype is present in OF SINGLE-GENE DISORDERS heterozygous form in 4% to 8% of the general popu- lation, and 0.06% to 0.25% of the population is homo- Single-gene disorders affect structural proteins, cell zygous for this mutation. A blood clot or deep venous surface receptor proteins, growth regulators, and thrombosis is treated with anticoagulants. The risk of enzymes. Examples of diseases resulting from such dis- thrombosis increases with contraceptive use in women orders are shown in Table 12.3. Examples of molecular (Table 12.5). methods that have been or could be used to detect these Several approaches have been taken to test for the gene lesions are also listed. Not all of these methods are Leiden mutation. Polymerase chain reaction (PCR) in common use in molecular diagnostics. Some diseases methods include the use of restriction fragment length are effectively analyzed by morphological studies or polymorphism (PCR-RFLP) or PCR with sequence- clinical chemistry. For instance, hemoglobin S is classi- specific primers (SSP-PCR; Figs. 12.7 and 12.8). cally detected by protein electrophoresis. Final diagnosis Nonamplification molecular methods, such as Invader requires physiological, morphological, and laboratory technology, have also been developed to test for this results. gene mutation. Clot-based methods may be used to directly demonstrate thrombophilia before genetic testing. Family history may also be considered. Lysosomal Storage Diseases Lysosomes are subcellular organelles in which prod- Prothrombin ucts of cellular ingestion are degraded by acid hydrolase enzymes (Fig. 12.6). These enzymes work in an acid Prothrombin is the precursor to thrombin in the coag- environment. Substrates come from intracellular turn- ulation cascade and is required for the conversion over (autophagy) or outside the cell through phagocy- of fibrinogen to fibrin. A mutation in the 3′ untrans- tosis or endocytosis (heterophagy). Lysosomal storage lated region of the gene that codes for prothrombin disorders result from incompletely digested macromol- or coagulation factor II, F2 (11p11-q12), results in an ecules due to loss of enzymatic degradation. Storage autosomal-dominant increased risk of thrombosis (see disorders include defects in proteins required for normal Table 12.5). Laboratories may test for both F2 and F5 lysosomal function, giving rise to physical abnormali- mutations. Both may be present in the same individ- ties. The organs affected depend on the location and ual, in which case the risk of thrombosis is greater than site of degradation of the substrate material. Examples with one of the mutations alone. An example of a multi- of storage diseases are shown in Table 12.4. These dis- plex PCR-RFLP method to simultaneously test for both orders are screened by gene product testing, that is, mutations was described previously in Chapter 8. In this measuring the ability of serum enzymes to digest test method, primers that amplify prothrombin and factor V 350 Section III Techniques in the Clinical Laboratory TABLE 12.3 Single-Gene Disorders and Molecular Methods Examples of Type of Type of Molecular Protein Type of Disease Example Gene (Location) Mutation Methods Structural Hemoglobinopathies Sickle cell anemia Hemoglobin beta Missense Sequencing, (11p15.5) PCR-RFLP Connective tissue Marfan syndrome Fibrillin (15q21.1) Missense Sequencing, disorders linkage analysis Cell membrane– Muscular dystrophy Dystrophin, DMD Deletion Southern blot associated protein (Xp12.2) RFLP,21 multiplex dysfunction PCR, linkage analysis Cell surface Hypercholesteremia Familial Low-density Deletions, point Probe receptor hypercholesteremia lipoprotein receptor mutations amplification, proteins (19p13.2) sequencing Nutritional disorders Vitamin D–resistant Vitamin D receptor Point mutations Southern blot rickets (12q12-q14) RFLP, sequencing Cell growth Fibromas Neurofibromatosis type Neurofibromin Missense, Sequencing, regulators 1 (von Recklinghausen tumor suppressor frameshift, splice linkage analysis disease) (17q11.2) site mutations Fibromas Neurofibromatosis type Merlin tumor Nonsense, Linkage analysis 2 (von Recklinghausen suppressor, NF-2 frameshift, splice disease) (22q12) site mutations Cancer Li-Fraumeni syndrome p53 tumor- Missense Sequencing, SSCP, predisposition (LFS)* suppressor gene, mutations DGGE TP53 (17p13) Enzymes Metabolic diseases Alkaptonuria Homogentisic acid Missense, cDNA sequencing, (ochronosis) oxidase (3q21–q23) frameshift, splice SSCP site mutations Phenylketonuria Phenylalanine Splice site, missense Ligase chain hydroxylase, PAH or mutations, reaction,25,26 PKU1 (12q24.1) deletions direct sequencing Immunodeficiencies Severe combined Adenosine Point mutations Direct sequencing, immunodeficiency deaminase capillary (20q13.11) electrophoresis *A significant proportion of LFS and LFL (Li-Fraumeni–like) kindred do not have demonstrable TP53 mutations. are designed to destroy or produce HindIII restriction each lane reveal the F2 and F5 normal or mutant geno- sites in the presence of the F5 or F2 mutation, respec- types for each specimen.4 tively. The sizes of the amplicons and their restriction Thrombin time, prothrombin time, platelet count, and fragments allow resolution of both simultaneously by complete blood count are phenotypic tests that may be agarose gel electrophoresis. The fragment patterns in performed in addition or prior to molecular analysis.  Chapter 12 Molecular Detection of Inherited Diseases 351 Phagocytosis Food particles Phagosome Lysosome Products of Golgi digestion apparatus Undigested material Autophagy Nucleus Cellular material Recycled material FIGURE 12.6 The lysosome is a depository for cell debris. The lysosome contains enzymes that are active in its acid environ- ment to digest proteins delivered from phagocytosis of foreign bodies, endocytosis, and autophagy of internal cellular components such as mitochondria. TABLE 12.4 Storage Diseases TABLE 12.5 Risk of Thrombosis Relative to Normal (1) Under the Indicated Genetic Substrate Accumulated Disease (F5, Prothrombin) and Environmental (OCP) Influences Sphingolipids Tay–Sachs disease Glycogen Von Gierke, McArdle, and Status Risk of Thrombosis Pompe disease Normal 1 Mucopolysaccharides Hurler, Sheie (MPS I), Hunter (MPS II), Sanfilippo (MPS III), Oral contraceptive (OCP) use 4 Morquio (MPS IV), Maroteaux– Lamy (MPS VI), Sly (MPS VII) Prothrombin mutation, 3 heterozygous Mucolipids Pseudo-Hurler polydystrophy Prothrombin mutation + OCP 16 Sulfatides Niemann–Pick disease R506Q heterozygous 5–7 Glucocerebrosides Gaucher disease R506Q heterozygous + OCP 30–35 R506Q homozygous 80 R506Q homozygous + OCP 100+ 352 Section III Techniques in the Clinical Laboratory Exon 10 Exon 10 F5 gene F5 gene Mnl I site MnlI site Mutant …A… Normal …G… Normal …G… Mnl I site Mnl I site destroyed Mutant …A… +/+ +/m m/m 148 bp +/+ +/m m/m 123 bp 153 bp 116 bp FIGURE 12.8 In sequence-specific PCR, a primer with thy- midine as its final 3′ base will yield a product only if the 67 bp adenine nucleotide is present. The resulting 148 bp PCR product reflects the presence of the mutation. By designing a primer slightly shorter than but complementary to the normal 37 bp (G) in the template, a distinct, shorter 123-bp normal product is amplified. In a heterozygous individual, both products will FIGURE 12.7 PCR-RFLP for the factor V Leiden mutation. appear. The R506Q amino acid substitution is caused by a G to A change in exon 10 of the F5 gene. This DNA mutation destroys an MnlI restriction enzyme site. An amplicon including the site co-substrate for conversion of homocysteine to methi- of the mutation, when cut with MnlI, will yield three fragments onine (Fig. 12.9). Two (of more than a dozen) genetic in normal DNA (+/+) and two products in homozygous mutant polymorphisms, 677C>T (p.A222V) and 1298 A>C DNA (m/m). A heterozygous specimen (+/m) will yield a com- bination of the normal and mutant pattern. (p.E429A), are associated with deficiencies in folate metabolism. These variants are detectable by standard or multiplex PCR with RFLP using restriction enzymes Automated systems that measure changes in light trans- HinfI and MboII or sequencing. Multiplex qPCR and mittance during clot formation generating a curve for high-resolution melt-curve methods have also been mathematical analysis replace some of the manual developed.5,6 methods. To further identify the genetic cause of abnor- mal coagulation, sequencing of factors IX and XIII is Hemochromatosis performed in addition to factor V and II analysis. Hemochromatosis is an autosomal-recessive condition that causes overabsorption of iron from food. Iron accu- Methylenetetrahydrofolate Reductase mulation subsequently causes pancreas, liver, and skin Deficiency of the 5,10-methylenetetrahydrofolate reduc- damage; heart disease; and diabetes. Classically, diagno- tase (MTHFR) gene product is an autosomal-recessive sis is made through measurement of blood iron levels, disorder that results in increased homocysteine levels transferrin saturation, or liver biopsy. (hyperhomocysteinemia), causing a predisposition to At the molecular level, hemochromatosis is caused venous and arterial thrombosis. by dysfunction of the hemochromatosis type I HFE or MTHFR catalyzes the conversion of 5,10- HLA-H gene product. HFE (6p21.3) codes for a mem- methylenetetrahydrofolate to 5-methyltetrahydrofolate, a brane-bound protein that binds with β2 microglobulin  Chapter 12 Molecular Detection of Inherited Diseases 353 Methylene THF Outside of cell H63D and S65C mutations MTHFR 5-methylTHF α1 heavy chain S S α2 Methionine Methionine synthase S-adenyl- Homocysteine NH2 NH2 methionine β2 -microglobulin S S S α3 S Methylation reactions Cell membrane HOOC C282Y mutation FIGURE 12.9 MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine-mediated remethylation to methionine. Mutations in this gene are associated with throm- bophilia and methylenetetrahydrofolate reductase deficiency. Cytosol HOOC and transferrin on the membrane of cells in the small intestine and also on the placenta. The protein directs iron absorption based on cellular iron loads. In the FIGURE 12.10 The HFE protein is associated with absence of HFE function, intestinal cells do not sense β2-microglobulin in the cell membrane. The location of the fre- iron stores, and iron absorption continues into overload. quently occurring mutations is shown. The most frequently observed mutation in hemo- chromatosis is C282Y, found in approximately 10% CF is caused by loss of function of the CF transmem- of the Caucasian population, with a disease frequency brane conductance regulator, the CFTR gene (7q31.2). of 2 to 3 per 1,000 people. Other mutations most fre- The gene codes for a chloride channel membrane protein quently detected in the HFE protein are H63D and S65C (Fig. 12.12). The first and most frequently observed (Fig. 12.10). Clinical symptoms and increased serum mutation in CFTR is a 3-bp deletion that removes a ferritin and transferrin-iron saturation are indications phenylalanine residue from position 508 of the protein for mutation testing. The C282Y mutation is detectable (F508del).7 More than 1,900 other mutations and varia- using PCR-RFLP (Fig. 12.11). tions have been reported in and around the CFTR gene in diverse populations. In a European survey of over 25,000 patients, the seven most frequent mutations were Cystic Fibrosis F508del, G542X, G551D, N1303K, R117H, W1282X, Cystic fibrosis (CF) is a life-threatening autosomal- and 1717-1G->A. These variants accounted for 75% recessive disorder that causes severe lung damage and of the alleles, with F508del present in 66.7% of the nutritional deficiencies. With earlier detection by genetic samples.8 A list of mutations, their locations, and refer- analysis and improved treatment strategies, people with ences is available through the Human Genome Variation CF can live more comfortably surviving beyond the Society at http://www.genet.sickkids.on.ca. fourth decade of life. CF affects the cells that produce Genetic testing for CF is important for diagnosis and mucus, sweat, saliva, and digestive juices. Normally, genetic counseling because early intervention is most these secretions are thin, but in CF, a defective gene effective in relieving symptoms of the disease. Molec- causes the secretions to become thick and sticky. Respi- ular tests have been designed to detect a variety of ratory failure is the most dangerous consequence of CF. mutations that have been described in CF9 and include 354 Section III Techniques in the Clinical Laboratory Exon 4 HFE gene Outside of cell Carbohydrate Cell membrane Rsa1 site Normal …G… Rsa1 site Rsa1 site Mutant …A… Cytoplasm Nucleotide- binding Phosphate +/+ +/+ m/m +/m +/+ +/+ domain Regulatory 240 bp F508del domain 140 bp FIGURE 12.12 The CF transmembrane conductance regula- tor forms a channel in the cell membrane. The F508del and 110 bp other mutations that affect its function are responsible for the phenotype of CF. FIGURE 12.11 Detection of the C282Y mutation by PCR-RFLP. The G→A mutation in exon 4 of the HFE gene produces a site for the restriction enzyme, Rsa1. This region is first amplified using primers flanking exon 4 of the gene NADPH NADP! Bilirubin (arrows). In a normal specimen, the enzyme will produce two AH ! O2 fragments, 240 bp and 140 bp. If the mutation is present, the Heme FAD Heme AOH ! H2O 140-bp normal fragment is cut to a 110-bp and a 30-bp frag- oxygenase FMN e" ment (the 30-bp fragment is not shown). Heterozygous indi- P-450 viduals will have both the 140-bp and the 110-bp fragments. P-450 P-450 P-450 reductase RFLP, PCR-RFLP, heteroduplex analysis, temporal temperature-gradient gel electrophoresis, single-strand conformation polymorphism (SSCP), SSP-PCR, Cleav- ase, bead array technology, and direct sequencing. The American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists FIGURE 12.13 NADPH cytochrome P-450 reductase cata- (ACOG) have recommended a core panel of 23 muta- lyzes the reduction of NADPH and transfers electrons to flavin adenine dinucleotide (FAD). Electrons flow through FAD and tions that will identify 49% to 98% of carriers, depend- flavin mononucleotide (FMN) to the cytochromes. The cyto- ing on ethnic background. Sequencing panels include chromes then oxidize a variety of substrates (AH). NADPH these and more rare variants.10 Population differences cytochrome P-450 reductase also works with heme oxygenase and variable expressivity influence the choice of muta- to convert heme to biliverdin and eventually to bilirubin. tions to be covered. enzymes are mono-oxygenases; that is, they participate Cytochrome P-450 in enzymatic hydroxylation reactions and also transfer Cytochrome P-450 comprises a group of enzymes local- electrons to oxygen: ized to the endoplasmic reticulum (Fig. 12.13). These A− H + B− H 2 + O2 → A− OH + B + H 2 O  Chapter 12 Molecular Detection of Inherited Diseases 355 where A is the substrate and B is the hydrogen donor. these polymorphisms.11,12 CYP-450 polymorphisms may These enzymes influence steroid, amino acid, and drug also compound interactions of multiple drugs taken metabolism using NADH or NADPH as hydrogen simultaneously.13,14 donors. Oxygenation of lipophilic drugs renders them There are over 30 reported variations of CYP-450 more easily excreted. enzymes.15 Enzymes are classified according to families The cytochrome P-450 system is present in high con- and subfamilies. For example, CYP2A6 is cytochrome centrations in the liver and small intestine where the P-450, subfamily IIA, polypeptide 6. CYP1A2 and the enzymes metabolize and detoxify compounds taken in enzymes in the CYP2 and CYP3 families are consid- orally (Fig. 12.14). The P-450 system varies from one ered to be most important in drug metabolism. Some of person to another. This may in part account for differ- the enzymes reported to inhibit or induce drug metab- ent effects of drugs on different people. The metabo- olism include CYP1A2, CYP2A6, CYP2B6, CYP2C8, lism of hormones, caffeine, chemotherapeutic drugs, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, antidepressants, and oral contraceptives is affected by CYP3A4, and CYP3A5-7. The genes coding for these enzymes are located throughout the genome. Drug Genetic polymorphisms of cytochrome P-450 genes are unequally distributed geographically and in different ethnic populations. Testing for these polymorphisms is used to predict the response to drugs sensitive to metab- olism by this enzyme system. In the laboratory, testing CYP-450 for CYP-450 polymorphisms is performed by allele-spe- cific PCR for particular polymorphisms. Multiple P-450 genetic variants may be screened by microarray, bead array, or sequencing. Metabolite Conjugation (Kidney) SINGLE-GENE DISORDERS WITH NONCLASSICAL PATTERNS OF INHERITANCE (Urine) (Liver) Mitochondrial mutations, genomic imprinting, and Adduct gonadal mosaicism do not follow Mendelian rules of inheritance. Mitochondrial mutations are inherited (Intestine) maternally (Fig. 12.15). Genomic imprinting is responsi- ble for specific expression of genes in different cells and tissues. Imprinting is reset at meiosis and fertilization (Stool) and is different in egg and sperm production. FIGURE 12.14 The oxidation activities of cytochrome P-450 Gonadal mosaicism is the generation of new muta- proteins metabolize a variety of structurally diverse chemicals, tions in germline cells. The mutated cells give rise including therapeutic drugs. to eggs or sperm carrying the mutation, which then FIGURE 12.15 Mitochondrial mutations are maternally inherited. 356 Section III Techniques in the Clinical Laboratory FIGURE 12.16 Gonadal mosaicism arises as a result of a new mutation. In this example, a dominant disease phenotype has been inherited from two unaffected parents. The mutation is present only in the germ cells of the first-generation parents but is inherited in all cells of the offspring. becomes a heritable phenotype. Unusual pedigrees result HV 1 HV 2 (Fig. 12.16). Gonadal mosaicism is expected when (342 bp) (268 bp) phenotypically normal parents have more than one PH1 affected child (e.g., in osteogenesis imperfecta, an auto- PH2 somal-dominant phenotype in a child from unaffected PL LHON 14484T>C parents). MELA 3243A>G Deleted Advanced Concepts LHON areas 3460G>A An example of the nature of imprinting is illus- LHON trated by a comparison of mules and hinnies. A 11778G>A mule is the product of a male donkey and a female MERRF horse. A hinny is the product of a male horse and 8344A>G MERRF a female donkey. These animals are quite differ- 8393T>G ent in phenotype, even though they contain essen- FIGURE 12.17 Mutations and deletions throughout the mito- tially the same genotype. Another illustration of chondrial genome are associated with muscular and neurologi- the effects of imprinting is seen in animals cloned cal disorders. by nuclear transfer. Because this process bypasses the generation of eggs and sperm and fertilization, the most energy-demanding organs, the muscles and imprinting is not reset, and cloned animals display the nervous system.17 Mutations in several genes in the unexpected phenotypes, such as larger size or mitochondrial genome have been defined. These muta- early onset of age-related diseases. tions result in a number of disease syndromes involving muscular and neurological disorders (Table 12.6). Mutations in Mitochondrial Genes Advanced Concepts Mitochondria are cellular organelles responsible for Disease severity depends on the number of mito- energy production. Mitochondria contain their own chondria affected. Mitochondria are actively genome, a circular DNA molecule 16,569 bp in length undergoing turnover in the cell, including fission (Fig. 12.17). The mitochondrial genome contains and fusion, which are part of the mitochondrial 37 genes, including a 12S and 16S rRNA, 22 tRNAs, network quality control. Alterations in mitochon- and 13 genes required for oxidative phosphorylation. In drial dynamics can cause neuropathies or optic addition, the mtDNA contains a 1000-nt control region atrophy.18 that encompasses transcription and replication regula- tory elements. A database of mitochondrial genes and mutations is available at http://www.MITOMAP.org.16 Mitochondrial mutations are easily detected by a variety Mutations in mitochondrial genes affect energy pro- of molecular methods. Southern blot is used for detect- duction and are therefore manifested as diseases in ing large deletions (Fig. 12.18). Point mutations are  Chapter 12 Molecular Detection of Inherited Diseases 357 TABLE 12.6 Diseases Resulting From Mutations in Mitochondrial Genes Disorder Gene Affected Molecular Methodology Kearns–Sayre syndrome 2–7 kb deletions Southern blot, PCR, PCR-RFLP Pearson syndrome Deletions Southern blot analysis of leukocytes, PCR-RFLP Pigmentary retinopathy, chronic progressive external tRNA (tyr) deletion, deletions PCR-RFLP, Southern blot analysis ophthalmoplegia of muscle biopsy Leber hereditary optic neuropathy Cyt6 and URF* point mutations PCR-RFLP Mitochondrial myopathy, encephalopathy, lactic tRNA (leu) point mutations PCR-RFLP, sequencing acidosis, and strokelike episodes Myoclonic epilepsy with ragged red fibers tRNA (lys) point mutations PCR-RFLP Deafness Neuropathy, ataxia, retinitis pigmentosa (NARP) ATPase VI point mutation PCR-RFLP Subacute necrotizing encephalomyelopathy with ATPase VI, NADH:ubiquinone PCR-RFLP neurogenic muscle weakness, ataxia, retinitis oxidoreductase subunit mutations pigmentosa (Leigh syndrome with NARP) Mitochondrial DNA depletion syndrome Thymidine kinase gene mutations PCR, sequencing *Unknown reading frame analyzed by PCR-RFLP (Fig. 12.19). Interpretation of with 3-bp repeating units) occur in coding and noncod- mutation analysis has long been complicated, however, ing sequences of genes. The most well-known examples by the extent of heteroplasmy (mutated mitochondria of triplet-repeat expansion diseases are fragile X syn- and normal mitochondria in the same cell) and the nature drome and Huntington disease. of the mutation.19 A range of phenotypes may be present, even in the same family. Advanced Concepts Genes that control mitochondrial functions are also found on the nuclear genome (Table 12.7). Unlike mito- In addition to family history, clinical tests includ- chondrial mutations that display maternal inheritance, ing electroencephalography, neuroimaging, cardiac these disorders have autosomal patterns of inheritance. electrocardiography, echocardiography, magnetic Although the causative gene mutation is located on a resonance spectroscopy, and exercise testing are nuclear gene, analysis of mitochondria may still show important for the accurate diagnosis of mito- deletions or other mutations caused by the loss of the chondrial disorders. High blood or cerebrospinal nuclear gene function. fluid lactate concentrations, as well as high blood glucose levels, are observed in patients with some mitochondrial diseases. More direct tests, such Nucleotide-Repeat Expansion Disorders as histological examination of muscle biopsies Nucleotide repeats include short tandem repeats (STRs) and respiratory chain complex studies on skeletal with 1 to 10-bp repeating units. During DNA replica- muscle and skin fibroblasts, are more specific for tion and meiosis, these STRs can expand (or contract) mitochondrial dysfunction. in length. Triplet-repeat mutations (expansions of STR 358 Section III Techniques in the Clinical Laboratory Fragile X Syndrome Fragile X syndrome is associated with a triplet-repeat (CGG) expansion in the noncoding region 5′ to the fragile X mental retardation gene, FMR-1 (Fig. 12.20). The expansion becomes so large in full fragile X syn- drome (more than 2,000 CGG repeats) that the region is microscopically visible (Fig. 12.21). The CGG repeat expansion 5′ to the FMR-1 gene also results in meth- ylation of the region and transcriptional shutdown of Spec. 1 2 3 Mspl U C U C U C 551 bp FIGURE 12.18 A mitochondrial deletion as revealed by 345 bp Southern blot. DNA was cut with PvuII, a restriction enzyme 206 bp that cuts once in the mitochondrial genome. The membrane was probed for mitochondrial sequences. Normal mitochondria (N) yield one band at 16.6 kb when cut with PvuII (C). Super- coiled, nicked, and a few linearized mitochondrial DNA circles can be seen in the uncut DNA (U). DNA from a patient with Kearns–Sayres syndrome (P) yields two mitochondrial popula- tions, one of which has about 5 kb of the mitochondrial FIGURE 12.19 Detection of the NARP mitochondrial point genome–deleted sequences. Because both normal and mutant mutation (ATPase VI 8993T>C or T>G) by PCR-RFLP. The mitochondria are present, this is a state of heteroplasmy. (Photo PCR product was digested with the enzyme MspI (C) or undi- courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical gested (U). If the mutation is present, the enzyme will cut the Center.) PCR product into two pieces, as seen is specimen 3. (Photo courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical Center.) TABLE 12.7 Some Disorders Caused by Nuclear Gene Mutations Disorder Gene Affected (Location) Molecular Methodology Mitochondrial DNA depletion syndrome Succinate-CoA ligase, ADP-forming, beta subunit, Southern blot SUCLA 2 (13q12.2-q13) Mitochondrial neurogastrointestinal Platelet-derived endothelial cell growth factor, Sequencing encephalomyopathy ECGF (22q13-qter) Progressive external ophthalmoplegia Chromosome 10 open reading frame 2, C10ORF2 Southern blot, SSCP, sequencing (10q24); polymerase, DNA, gamma, POLG (15q25); solute carrier family 25 (mitochondrial carrier), member 4, SLC25A4/ANT1 (4q25)  Chapter 12 Molecular Detection of Inherited Diseases 359 Normal 5′ CGG(CGG)5–55 FMR-1 3′ Amplification Premutation (carrier) 5′ CGGCGGCGG(CGG)56–200 FMR-1 3′ Amplification and methylation Full mutation (affected) 5′ CGGCGGCGGCGGCGGCGG(CGG)200–2000+ FMR-1 3′ Unaffected carrier FIGURE 12.20 Triplet-repeat (CGG) expansions in Learning disabled sequences 5′ to the FMR-1 gene are observed in fragile X car- FXS riers (up to 200 repeats) and fully affected individuals (more than 200 repeats). Normally there are fewer than 60 repeats. Expansion results from amplification of the triplet sequences FIGURE 12.22 The symptoms of fragile X syndrome (FXS) become more severe with each generation. The fragile X chro- during meiotic recombination events. The very large repeats mosome cannot be transmitted from fathers to sons but can be (more than 200 repeats) are methylated on the C residues. This transmitted from grandfathers to grandsons through their methylation turns off FMR-1 transcription. daughters. primary ovarian insufficiency (FXPOI), with amenor- rhea, menopausal follicle-stimulating hormone (FSH) levels, and possible estrogen deficiency.20 Another con- dition associated with PM status is fragile X tremor and ataxia syndrome (FXTAS), with declining overall cogni- tive abilities with age.21 In addition to the fragile X chromosome observed by karyotyping, the state of the repeat expansion is also analyzed using PCR and by Southern blot (Fig. 12.23). Premutations in fragile X carriers are easily detected by PCR, with full mutations detectable by triple-repeat- primed PCR. AGG interruptions can be detected with Fragile X chromosome AGG-triplet-repeat primers.22 Southern blot can reveal (in metaphase) cases of mosaicism where both premutations and full FIGURE 12.21 The fragile X chromosome is characterized fragile X chromosomes are present in separate cell pop- by a threadlike process just at the telomere of the long arm ulations from the same patient. (arrow). This is the site of disorganization of chromatin struc- Capillary electrophoresis is an increasingly popular ture by the GC-rich repeat expansions. option for identifying expanded FMR alleles, replacing the gel procedures (Fig. 12.24). Peak patterns indicate FMR-1. CGG repeats can be interrupted by AGG, which the presence of normal, premutation, and full fragile dampens the methylation and silencing of FMR-1. X mutation. The presence of AGG interrupters of the Symptoms of fragile X syndrome include learn- CGG repeats shows as gaps in the series of peaks. AGG ing disorders and mental retardation (IQ ~20), long promotes stability or slower expansion of the repeat face, large ears, and macroorchidism (large genitalia). region. The capillary electrophoresis method is faster and Symptoms are more apparent at puberty. Symptoms technically less demanding than Southern blot; however, increase in severity with each generation in a fragile the latter procedure may still be required to identify the X family (Fig. 12.22). Approximately 20% of women presence of deletions within the FMR-1 gene or the 5′ with FMR-1 premutation (PM) will develop fragile X repeats in a percentage of the cells (mosaicism). 360 Section III Techniques in the Clinical Laboratory PCR Southern blot 50–90 20–40 Inactive X Premutations Full mutations FIGURE 12.23 Detection of premutations by PCR (left) and full fragile X mutations by Southern blot (right). Primers (one of which is labeled with 32P) flanking the repeat region are used to generate labeled PCR products. Premutations appear as large amplicons in the 50- to 90-repeat range on the autoradiogram at left. Normal samples fall in the 20- to 40-repeat range. Full fragile X repeats are too large and GC rich to detect by standard PCR. Southern blots reveal full mutations in three of the samples shown. The inactive (methylated) X chromosome in four female patients is detected by cutting the DNA with a methylation-specific restriction enzyme. (Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical Center.) FU A 150 200 250 300 350 400 FU B 150 200 250 300 350 400 FU C 150 200 250 300 350 400 FIGURE 12.24 Fragile X premutations and full mutations appear as altered peak patterns in an electropherogram. PCR products of the FMR-1 CGG repeat region in the normal X chromosome (A) are detected as peaks of less than 250 bases (A), whereas the premutation (B) and full mutation (C) are visible as regular peaks extending up to 400 bases.  Chapter 12 Molecular Detection of Inherited Diseases 361 Huntingtin 80–170 bp FIGURE 12.25 The huntingtin repeat expansion P occurs within the coding region of the huntingtin #40 repeats gene. The expansion is detected directly by PCR using primers flanking the expanded region (top). A 32 P-labeled primer is used, and the bands are detected by autoradiography of the polyacrylamide gel (bottom). In this example, PCR products from the 10–29 repeats patient (P) fall within the normal range with the neg- ative control (–). The positive control (+) displays the band sizes expected in Huntington disease. (Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush Uni- versity Medical Center.) Huntington Disease In contrast to fragile X, where the repeat expansion takes place 5′ to the coding sequences, the Huntington Huntington disease, first described by George Hunting- expansion occurs within the coding region of the gene. ton in 1872, is associated with expansion within the The triplet expansion inserts multiple glutamine residues huntingtin structural gene (4p16.3). In this repeat expan- in the 5′ end of the huntingtin protein. This causes the sion, the sequence CAG expands from 9 to 37 repeats to protein to aggregate in plaques, especially in nervous 38 to 86 in the huntingtin gene. The clinical symp- tissue, causing the neurological symptoms seen in this toms of Huntington disease include impaired judgment, disease. The expansion does not reach the size of the slurred speech, difficulty in swallowing, abnormal body fragile X expansion and is detectable by standard PCR movements (chorea), personality changes, depression, methods and capillary electrophoresis (Fig. 12.25). mood swings, unsteady gait, and an intoxicated appear- ance. With onset in the 30s or 40s, these symptoms do Idiopathic Congenital Central not become obvious until the fourth or fifth decade of Hypoventilation Syndrome life, usually after family planning. The child of a person with Huntington disease has a 50% chance of inheriting Idiopathic congenital central hypoventilation syndrome the disorder. Genetic counseling, therefore, is important (CCHS) is a rare pediatric disorder characterized by for younger persons with family histories of this disease, inadequate breathing while asleep. More-affected chil- especially with regard to having children. dren may also experience hypoventilation while awake. CCHS occurs in association with an intestinal disorder called Hirschsprung disease and symptoms of diffuse autonomic nervous system dysregulation/dysfunction. Advanced Concepts A number of gene mutations have been observed in CCHS, including a polyalanine expansion of the paired- The FMR protein (FMRP) binds RNA and is asso- like homeobox (PHOX2b) gene (4p12)13. The PHOX2b ciated with polysomes. FMRP regulates translation protein is a transcription factor containing a domain of its bound mRNAs through alternative mRNA (homeobox) similar to other proteins that bind DNA. splicing, mRNA stability, and mRNA trafficking In CCHS, a triplet-repeat expansion occurs inside of from the nucleus to the cytoplasm. FMRP may be the PHOX2b gene, resulting in the insertion of multi- associated with the miRNA pathway as well, pre- ple alanine residues into the protein. The severity of the venting helicase-mediated miRNA suppression.23,24 disease increases with increasing numbers of repeats. The expansion is detected by PCR (Fig. 12.26).25 362 Section III Techniques in the Clinical Laboratory PHOX2b exon 3 PAGE (Normal) (Normal) Agarose (rapid test) FIGURE 12.26 The triplet-repeat expansion of PHOX2b includes triplets that code for alanine (top). The expansion is detected by PCR with a 32P-labeled primer and polyacrylamide gel electrophoresis (center) or by standard PCR and agarose gel electropho- resis (bottom). Normal specimens yield a single PCR product. CCHS specimens yield another larger product in addition to the normal product. The standard PCR test can rapidly show the presence of the expansion, and the PAGE test allows determination of the exact number of alanine codons that are present in the expansion. (Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical Center.) CCHS is usually apparent at birth. In some children, The difference is exhibited in genetic disorders in which late onset of the disease occurs at 2 to 4 years of age. An one or the other allele of a gene is lost. estimated 62% to 98% of patients with CCHS have the A uniparental disomy/deletion demonstrates the PHOX2b gene mutation.26 nature of imprinting on chromosome 15. A deletion in the paternal chromosome 15, del(15)(q11q13), causes Prader–Willi syndrome. Symptoms of this disorder Other Nucleotide Expansion Disorders include mental disability, short stature, obesity, and Fragile X, Huntington disease, and CCHS are three of hypogonadism. Loss of the same region from the mater- a group of diseases caused by trinucleotide-repeat dis- nal chromosome 15 results in Angelman syndrome, a orders. This category of diseases is subclassified into disorder with very different symptoms, including ataxia, polyglutamine expansion disorders, which includes seizures, and inappropriate laughter. Both syndromes Huntington disease, and non–polyglutamine expansion can occur in four ways: a deletion on the paternal or disorders. Larger repeat units may also be involved in maternal chromosome 15, a mutation on the paternal or expansion mutations. Expansion of a hexanucleotide maternal chromosome 15, a translocation with loss of repeat unit is found in cases of amyotrophic lateral scle- the critical region from one chromosome, and maternal rosis (ALS).27 Examples of repeat expansion diseases or paternal uniparental disomy in which both chromo- are listed in Table 12.8. somes 15 are inherited from the mother and none from the father or vice versa. Cytogenetic methods are used to test for these genetic Genomic Imprinting lesions. Translocations and some deletions are detect- Genomic imprinting is transcriptional silencing through able by standard karyotyping. High-resolution karyotyp- histone or DNA modification. Imprinting occurs during ing can detect smaller deletions; however, other cases egg and sperm production and is different in DNA are not detectable microscopically. FISH with labeled brought in by the egg or the sperm upon fertilization. probes to the deleted region can detect over 99% of  Chapter 12 Molecular Detection of Inherited Diseases 363 TABLE 12.8 Examples of Nonpolyglutamine Expansion Disorders Expansion, (Normal)— Disorder Gene Repeat (Symptomatic)* Fragile XE Fragile X mental retardation 2 (Xq28) GCC (6–35) – (over 200) Friedreich ataxia Frataxin, FRDA or X25 (9q13) GAA (7–34) – (over 100) Myotonic dystrophy Dystrophia myotonica protein kinase (9q13.2–13.3) CTG (5–37) – (over 50) Spinocerebellar ataxia type 8 Spinocerebellar ataxia type 8 (13q21) CTG (16–37) – (110–250) Spinocerebellar ataxia type 12† Spinocerebellar ataxia type 12 (5q31–33) CAG (7–28) – (66–78) Amyotrophic lateral sclerosis, C9 open reading frame 72 (9p21.1) GGGGCC (2–20) – (over 100) frontotemporal disorder *The phenotypic effects of intermediate numbers of repeats is not known. †Although CAG codes for glutamine, this expansion occurs outside of the coding region of this gene and is not translated. cases. PCR of RFLP or STR analysis has also been used combinations of genetic (and somatic) variants. Further to demonstrate uniparental disomy. Because imprinting annotation of demographics, such as ethnicity or gender, (DNA methylation) is different on maternal and paternal and lifestyles, such as smoking or diet, will further add chromosomes, methylation-specific PCR and Southern to the prognostic and diagnostic value of gene mutation blot using methylation-specific restriction enzymes can analysis in these cases. aid in the diagnosis of these disorders. Assays developed for the detection of copy-number variants, including FISH, array-based comparative genomic hybridization LIMITATIONS OF MOLECULAR TESTING (aCGH), and next-generation sequencing (NGS), have been used for the detection of genome-wide uniparental Although molecular testing for inherited diseases is disomy (UPD) associated with constitutional and neo- extremely useful for early diagnosis and genetic counsel- plastic disorders.27 ing, there are circumstances in which genetic testing may not be the optimal methodology. To date, most therapeutic targets are phenotypic so that treatment is better directed Multifactorial Inheritance to the phenotype. In genes with variable expressivity, Most disorders (and normal conditions) are controlled finding a gene mutation may not predict the severity of by multiple genetic and environmental factors. Multifac- the phenotype. For instance, clotting time and transferrin torial inheritance is displayed as a continuous variation saturation are better guides for anticoagulant treatment in populations, with a normal distribution, rather than than the demonstration of the causative gene mutations. as a specific inheritance pattern. Nutritional or chemi- Molecular testing may discover genetic lesions in the cal exposures alter this distribution. The range may be absence of symptoms. This raises a possible problem as discontinuous, with a threshold of manifestation. The to whether treatment is indicated. This is increasingly phenotypic expression is conditioned by the number of possible with the use of large array or sequencing panels controlling genes inherited. The chance of a first-degree targeting hundreds of genes. Several genetic lesions may relative having a similar phenotype is 2% to 7%. be present or polymorphic states of other normal genes High-resolution array methods and next-genera- may influence the disease state. Research methods and tion sequencing have further defined the genetic com- clinical trials using array technology and sequencing ponents of multifactorial illnesses. Large databases methods designed to scan at the genomic level may con- such as ClinVar and dbSNP aid in the interpretation of tribute to better diagnosis of complex diseases.