Debnath Mousumi, Prasad GBKS, Bisen PS. Molecular Diagnostics: Promises and Possibilities Dordrech Heidelberg London, Springer, 2010 pp 331-345. CHAPT...
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Debnath Mousumi, Prasad GBKS, Bisen PS. Molecular Diagnostics: Promises and Possibilities Dordrech Heidelberg London, Springer, 2010 pp 331-345. CHAPTER 20

DIAGNOSIS OF MUTATION AND GENETIC DISORDERS Abstract All diseases have a genetic component. However, the extent to which genes contribute to disease varies and much remains to be learned. Mutations may be inherited or developed in response to environmental stresses such as viruses or toxins. The ultimate goal is to use this information to treat, cure, or if possible, prevent the development of disease. Advances in understanding the genetic mechanisms behind these diseases enable the development of early diagnostic tests, new treatments, or interventions to prevent disease onset or minimize disease severity. Key words Single gene disorders, mutations, genetic disorders, genetic test, diagnostic test , DNA diagnosis , Cystic Fibrosis Transmembrane Conductance Regulator gene ,CFTR, the breast cancer genes , BRCA1 and BRCA2, retinoblastoma ,inherited disorder, monogenic disorders, pharmacogenetics, genetic

disease, cystic



muscular dystrophy,

haemophilia A and B, phenylketonuria, thalassaemia , Multiple endocrine neoplasia, factor V (Leiden) mutation, Hemochomatosis, colorectal cancer, Comparative genome hybridization

20.1 PROLOGUE The exponential increase in the discovery of genes over the past few years has transformed the DNA diagnosis of genetic disorders from a minor research-based activity to a major professional operation. For any genetic disease, once the defective gene is identified, knowledge of the pathogenic mutations is indispensable to offer DNA diagnosis. DNA diagnosis is offered at pre- and postnatal levels either by direct or indirect approaches. Direct mutational analysis and linkage studies with highly polymorphic intragenic markers are carried out depending on the feasibility of their detection. This is not as easy as it may sound, because many genes, including the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, the breast cancer genes (BRCA1 and BRCA2), and the retinoblastoma gene (RBI), lack mutational hot spots necessitating an exhaustive analysis of coding and flanking intronic and regulatory sequences.


20.2 CONCEPT The analysis of human DNA, RNA, chromosomes, proteins and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes or karyotypes for clinical purposes is essential. In the past decade, with the availability of technology and knowledge fueled by the investment and interest in the Human Genome Project, molecular diagnostics has recently enabled laboratories to offer diagnostic and predictive tests for inherited disorders (Harper et al., 2008). The technology will continue to become easier to apply and more affordable. Large amounts of genetic information can be determined at increasing efficiencies. Historically, genetic testing focused on single-gene disorders, where a disease is caused by a mutation in one gene. The classic example is sickle cell anemia. A single nucleic acid base change is responsible for the sickle cell trait, when inherited from one parent, or sickle cell disease when the nucleic acid base change is inherited from both parents. Although molecular diagnostics is available, sickle cell disease is most often diagnosed with hemoglobin electrophoresis. For single-gene disorders, mutation analysis is useful in diagnosis, confirmation of diagnosis, predisposition, prognosis and family planning. As with Huntington's disease, the identification of the gene and availability of testing opened another door to additional social and medical issues. In contrast, testing an individual with multiple endocrine neoplasia type-2 (MEN-2) for the RET proto-oncogene can avoid medullary carcinoma if the individual is positive and undergoes a prophylactic thyroidectomy. The pace of the molecular dissection of human disease can be measured by looking at the catalog of human genes and genetic disorders identified so far in Mendelian Inheritance in Man (Holzman and Waton ,2002) and in OMIM, its online version, which is updated daily (www.ncbi.nlm.nih.gov/omim). For 1100 genes, at least one disease-related mutation has been identified . Because different mutations in the same gene often result in more or less distinct disorders, the total number of diseases for which OMIM lists mutations approaches 1500 .The number of disease genes discovered so far is 1112. This number does not include at least 94 disease-related genes identified as translocation gene-fusion partners in neoplastic disorders. Numbers in parentheses indicate disease-related genes that are polymorphisms ("susceptibility genes").

Apart from the genes that cause monogenic disorders, the discovery of functional sequence variants that confer genetic susceptibility to common multifactorial disorders, such as cardiovascular disease, psychiatric disorders, autoimmune disorders and cancer, also creates demand for high throughput testing for clinically relevant polymorphisms. Single Nucleotide Polymorphisms (SNPs) are the most abundant type of DNA sequence variations in the human genome1, though only miniscule fraction causes significant changes in amino acid sequence. One area where SNPs may have an immediate impact on patient care, is the individual response to drug therapy, commonly referred to as pharmacogenetics. A genetic variability in N-acetyl transferase (NAT-2), for instance, is associated with a high incidence of peripheral neuropathy when taking isoniazid, an antituberculosis drug. A variant in the core promoter of the ALOX5 gene, on the other hand, is responsible for the failure of


some asthma patients to respond to treatment with ALOX5-pathway modifiers. The ultimate method for detection and definition of mutations is direct sequencing. But it comes at significant cost and labour, and the vast majority of sequencing reactions will only exclude the presence of a mutation. This has led to the development of physical, chemical and biological mutation screening methods that exclude the presence of mutations at a fraction of the cost of sequencing, but provide little, if any, information on the location and nature of sequence variation in mutated gene fragments. Most methods do well with regard to specificity, but fail miserably when it comes to sensitivity. They include such popular methods as single-strand conformation analysis, gel electrophoresis-based heteroduplex analysis, and denaturing gradient gel electrophoresis.

The diagnosis of a genetic disease requires a comprehensive clinical examination composed of three major elements: 1. A physical examination 2. A detailed medical family history 3. Clinical and laboratory testing if available. While primary care providers may not always be able to make a definitive diagnosis of a genetic disease, their role is critical in collecting a detailed family history, considering the possibility of a genetic disease in the differential diagnosis, ordering testing as indicated and when available, appropriately referring patients to genetic specialists.

20.3. FACTORS REGULATING A GENETIC DISEASE There are several factors that raise the possibility of a genetic disease in a differential diagnosis. One major factor is the occurrence of a condition among family members that is disclosed when the family history is obtained. The occurrence of the same condition in more than one family member (particularly first-degree relatives), multiple miscarriages, stillbirths, and childhood deaths are all suggestive of a genetic disease. Additionally, family history of common adult conditions (heart disease, cancer, and dementia) that occur in two or more relatives at relatively young ages may also suggest a genetic predisposition.

Other clinical symptoms that are suggestive of a genetic disease include developmental delay/mental retardation and congenital abnormalities. Dysmorphologies, often involving the heart and face, as well as growth problems are suggestive of a genetic disorder caused by an inherited mutation, a spontaneous mutation, a teratogen exposure, or unknown factors. While these clinical features may be caused by a number of factors, genetic conditions should also be considered as part of the differential diagnosis, particularly if the patient expresses several clinical features together that might be indicative of a syndrome (for example, mental retardation, distinct facies, and heart defect). Some physical features may appear unique or slightly different than the average such as wide-set or droopy eyes, flat face, short fingers, and tall stature. While these rare and seemingly mild features may not immediately be suggestive of a genetic disease to a primary care provider, an evaluation by a genetics specialist may be


helpful in ruling in/out a genetic disease.

While many genetic conditions appear during childhood, a genetic condition should not entirely be ruled out in adolescents or adults. Often a genetic disease can remain undetected for several years until an event such as puberty or pregnancy triggers the onset of symptoms or the accumulation of toxic metabolites manifests in disease. In these cases, a detailed family history and physical examination should be performed and a referral made to a genetics specialist if indicated.

20.4 GENETIC TESTING Genetic tests can be used for many different purposes (Holzman and Watson, 2002). Newborn screening is the most widespread use of genetic testing. Almost every newborn in the U.S. is screened for several genetic diseases. Early detection of these diseases can lead to interventions to prevent the onset of symptoms or minimize disease severity. Carrier testing can be used to help couples to learn if they carry and thus risk passing to their children—an allele for a recessive condition such as cystic fibrosis, sickle cell anaemia, and Tay-Sachs disease. This type of testing is typically offered to individuals who have a family history of a genetic disorder and to people in ethnic groups with an increased risk of specific genetic conditions. If both parents are tested, the test can provide information about a couple’s risk of having a child with a genetic condition. Prenatal diagnostic testing is used to detect changes in a foetus’s genes or chromosomes. This type of testing is offered to couples with an increased risk of having a baby with a genetic or chromosomal disorder.


Fig 20.1 Genetic tests may be used to confirm a diagnosis in a symptomatic individual or used to monitor prognosis of a disease or response to treatment.

A tissue sample for testing can be obtained through amniocentesis or chorionic villus sampling. Predictive or predispositional genetic testing can identify individuals at risk of getting a disease prior to the onset of symptoms. These tests are particularly useful if an individual has a family history of a specific disease and an intervention is available to prevent the onset of disease or minimize disease severity. Predictive testing can identify mutations that increase a person’s risk of developing disorders with a genetic basis, such as certain types of cancer.

Several different methods are currently used in genetic testing laboratories. The type of test will depend on the type of abnormality that is being measured. In general, three major types of genetic testing are available: cytogenetic, biochemical, and molecular testing.


Cytogenetics involves the examination of whole

chromosomes for abnormalities. Chromosomes of a dividing human cell can be clearly analyzed under a microscope. White blood cells, specifically T lymphocytes, are the most readily accessible cells for cytogenetic analysis since they are easily collected from blood and are capable of rapid division in cell culture. Cells from other tissues such as bone marrow (for leukemia), amniotic fluid (prenatal diagnosis), and other tissue biopsies can also be cultured for cytogenetic analysis Following several days of cell culture, chromosomes are fixed, spread on microscope slides, and then stained. The staining methods for routine analysis allow each of the chromosomes to be individually identified. The distinct bands of each chromosome revealed by staining allow for analysis of chromosome structure.

20.4.2 BIOCHEMICAL TESTING: The enormous numbers of biochemical reactions that routinely occur in cells require different types of proteins. Several classes of proteins exist to fulfill multiple functions, such as enzymes, transporters, structural proteins, regulatory proteins, receptors, and hormones. A mutation in any type of protein can result in disease if the mutation results in failure of the protein to correctly function . Clinical testing for a biochemical disease utilizes techniques that examine the protein instead of the gene. Tests can be developed to directly measure protein activity (enzymes), level of metabolites (indirect measurement of protein activity), and the size or quantity of protein (structural proteins). These tests require a tissue sample in which the protein is present, typically blood, urine, amniotic fluid, or cerebrospinal fluid. Because proteins are more unstable than DNA and can degrade quickly, the sample must be collected, stored properly, and shipped promptly according to the laboratory’s specifications.

20.4.3 MOLECULAR TESTING: For small DNA mutations, direct DNA testing may be the most effective method, particularly if the function of the protein is not known and a biochemical test cannot be developed. A DNA test can be performed on any tissue sample and requires very small amounts of sample. Some genetic diseases can be caused by many different mutations, making molecular testing challenging. For example, more than 1,000 mutations in the cystic fibrosis transmembrane conductance


regulator (CFTR) gene can cause cystic fibrosis (CF). It would be impractical to sequence the entire CFTR gene to identify the causative mutation since the gene is quite large. However, since the majority of CF cases are caused by approximately 30 mutations, this smaller group of mutations is first tested before more comprehensive testing is performed (Grody ,1999).

20.5 CURRENT STATUS OF MOLECULAR DIAGNOSIS OF SOME COMMON GENETIC DISESES Beyond the complexity of genetic sequencing, with rigorous quality control and assurance, is the difficulty in interpreting the results. As with HIV genotyping, some mutations have no known clinical significance and others impart antiretroviral drug resistance. Sometimes, two mutations act to cancel out the impact of one another. Likewise, some mutations in the gene associated with Gaucher's disease cause neuronopathic disease and other mutations do not. As a final example, some mutations in the CFTR cause cystic fibrosis. Other mutations have low penetrance. That is, these genes seem to have no clinical significance in the majority of individuals, but when combined with other gene expressions, impart classical or nonclassical cystic fibrosis disease. The current state of molecular diagnosis of some common genetic diseases, including cystic fibrosis, Duchenne muscular dystrophy, haemophilia A and B, phenylketonuria, and thalassaemia, has been reported . Data on carrier detection and prenatal diagnosis are presented and some objective problems and obstacles hampering efficient molecular diagnosis are discussed. The necessity for molecular diagnosis of some other inherited diseases (for example, von Willebrand's disease, Martin-Bell syndrome, polycystic kidney disease, Huntington's disease, and myotonic dystrophy) is stressed. The need for establishing new diagnostic centres dealing with the most common diseases, as well as rare genetic diseases, is substantiated. Perspectives on the implementation of new molecular methods and new technical approaches (preimplantation embryo diagnosis, fetal cells selected from maternal blood) are briefly outlined. It is still too early to estimate the significance of our Human Genome Programme as a whole, but its important impact on medical genetic studies in this country is quite obvious and has been highly appreciated.

There are a myriad of technologies capable of detecting single mutations or SNPs. These platforms are capable of performing medium throughput testing with standard laboratory robotic liquid handlers and at a reasonable hardware cost. These methods include allele-specific oligonucleotide hybridization, PCR RFLP, allele-specific PCR, Line Probe Assays (reverse dot blots), Invader (Third Wave Technologies), ReadIT (Promega), Nanochips (Nanogen), Homogenous PGR (includes TaqMan and Molecular Beacon) and many others. Gene quantitation can be done by several techniques, including fluorescent in situ hybridization (FISH), usually performed in the cytogenetics laboratory with the use of a computerized fluorescent microscope, real-time PCR analysis and comparative genome hybridization.


20.5.1 CYSTIC FIBROSIS Cystic fibrosis (CF) is one of the most commonly inherited diseases in the in Caucasian populations( Bouchara et al., 2009). Those affected have high levels of sodium and chloride (salt) in their sweat. More importantly, a thick, sticky mucous in the lungs causes persistent coughing, wheezing and frequent lung infections, including pneumonia. In 1989, a research team led by Dr. Francis Collins at the University of Michigan and LapChee Tsui and John Riodan at Toronto's Hospital for Sick Children discovered the gene responsible for cystic fibrosis. The protein is the cystic fibrosis transmembrance conductance regulator (CFTR). Since the discovery of the most common mutation that causes cystic fibrosis, approximately 1,000 additional mutations have been identified. Given that approximately 10 million Americans are carriers for cystic fibrosis and 30,000 have the disorder, testing for cystic fibrosis carrier status prior to conception (or if necessary, early after conception) is currently recommended. While cystic fibrosis is not curable, there are some treatments that greatly increase the life span and quality of life for patients with CF. Today, cystic fibrosis mutational analysis is the most commonly performed molecular d diagnostic test for inheritable disorders. Patterns of allelic polymorphisms in CF have been analysed and studied. Large scale testing of the AF508 mutation has become possible after adopting the PCR technique for dried blood spots on filter paper. The other major mutations of the CFTR gene known to be quite common in western populations (G551D, R553X, R334W, W1282X, R551X, 1716+ 12T-*C) are detected only occasionally in Russian samples (1 to 3%).However, CFTR gene mutation 3732delA (exon 19), recently found in southern France, was detected in almost 7% of Russian CF chromosomes Moreover, 1677delTA, originally discovered turned out to be a major mutation inthe populations of the Black Sea Basin. Three more new CFTR gene mutations (E504Q exon 10, W1282R exon 19, and S 1196X) have been recently shown by SSCP analysis followed by direct sequencing. According to these data the search for new mutations of the CFTR gene in our CF patients might be highly productive. At least three different mutations, AF508, 1677delTA, and 3732delA, might be used both for carrier detection and prenatal diagnosis of CF in Russia. These data are of special practical value as most of the families requesting prenatal diagnosis of CF in this country do not have a living index child and thus cannot be subjected to RFLP analysis. Identification of new major CFTR gene mutations, specific to our native populations, as well as RFLP analysis of new intragenic polymorphisms, such as the recently discovered highly polymorphic minisatellite DNA sequences in introns 6, 8, and 17b, is helping in more efficient application of molecular analysis in CF patients (Grody, 1999).

20.5.2 DUCHENNE MUSCULAR DYSTROPHY Molecular diagnosis of Duchenne muscular dystrophy (DMD) were carried out with intragenic and flanking DNA probes .They were later supplemented with multiplex polymerase chain reaction (MPCR) for exon deletion detection in the dystrophin gene. Both carrier detection and prenatal diagnosis have been done. A relatively low deletion detection rate with the standard set of exons tested


by MPCR, a somewhat unusual pattern of deletion distribution along the DMD cDNA, and a significant proportion of extensive deletions extending through the major part of the gene. One of the possible approaches, not yet tried here so far, concerns RNA amplification supplemented with MPCR analysis of cDNA. 5 Of special diagnostic value for at least some male fetuses at risk might also be the application of western blotting to muscle biopsies or direct immunocytochemical studies of dystrophin in muscle fibres. This approach might be of great benefit for the otherwise uninformative DMD families requesting prenatal diagnosis during the second trimester of pregnancy.

20.5.3 HAEMOPHILIA A Molecular diagnosis of haemophilia A was carried out using DNA probes.Highly polymorphic flanking DNA (probe StI4/TaqI) and intragenic polymorphic sites HindIII (intron 19), BclI (intron 18), and XbaI (intron 22) were used both for population studies and diagnostic purposes. Some intragenic polymorphic sites were used for polymerase chain reaction detection.Several prenatal diagnoses by PCR have been carried out .Mutation identification by SSCP analysis followed by direct sequencing of altered exons and amplification-mismatch detection studies of the factor VIII gene are now in progress.

20.5.4 HAEMOPHILIA B Intragenic DNA probes available for RFLP analysis of the factor IX gene . Southern blot RFLP analysis with these probes was later replaced by PCR for detection of intragenic polymorphic sites TaqI, XmnI (both in intron C), and HinfI/DdeI (intron A). Polymorphism identification was done with original sets of oligoprimers . Thus, direct identification of mutations by means of SSCP analysis or by the amplification-mismatch detection technique is advisable.

20.5.5 PHENYLKETONURIA Large scale newborn screening programmes for phenylketonuria (PKU), either by the Guthrie test or by an automated fluorescent assay, According to already available data the frequency of PKU in newborns varies between 1 in 5000 to 1 in 8000 in different regions, with an average of around 1 in 6000 .Molecular analysis of PKU in the USSR was initiated with the cDNA probe of the PAH gene. These studies were substantially helped later by the PCR method .New, efficient detection of point mutations by the limited by this group in collaboration with Australian scientists.Allele specific hybridisation supplemented with direct sequencing of exon 12 of the PAH gene showed the mutation in codon 408 as well as the exon-intron splicing mutation

20.5.6 β THALASSAEMIA Molecular studies of β thalassaemia were started in 1975 and were initially confined to mRNA analysis and its application to deletion detection in the a globin gene (L Lymborskaya). RFLP analysis in 32 e thalassaemia patients with a severe form of the disease showed 11 different haplotypes, one of which was encountered in half of all affected subjects. This particular haplotype was actually in strong linkage disequilibrium with mutations in codon 8. Five different mutations of the globin gene were discovered in seven patients by PCR followed by direct sequencing of the amplification products.


One of these mutations (deletion of a G nucleotide between codons 82 and 83) was found for the first time.Oligoprimer sets suitable for allele specific amplification and thus for direct identification of globin gene mutations have been suggested.

20.5.7 WILSON'S DISEASE (HEPATOLENTICULAR DEGENERATION) Identification and biochemical investigations of copper binding protein caeruloplasmin (CP) in Wilson's disease patients were later extended to CP mRNA and CP gene molecular analysis. However, quite unexpectedly for this group, these studies failed to disclose any mutations of the CP gene in patients and thus indicated that the CP gene is not involved by itself in Wilson's disease. The later assignment of WD to chromosome 13 but not to chromosome (where the CP gene is located). Curiously enough, our in situ gene mapping with an original fragment of rat CP-DNA as a hybridisation probe disclosed a positive hybridisation signal not only on chromosome 3 (3q23-25, close to the transferrin gene) but also on chromosome 13 (13q23-24), that is, somewhere very close to the still unknown gene responsible for Wilson's disease.25 Thus the problem of the molecular nature of Wilson's disease is still awaiting a solution. Meanwhile chromosome 13 is one of the genome units selected by our Human Genome Project for detailed molecular analysis. Identification of the gene for Wilson's disease is one of the urgent tasks.

20.5.8 α ANTITRYPSIN DEFICIENCY DNA analysis of α antitrypsin deficiency (AD) was carried out.RFLP analysis was carried out in 659 patients with chronic non-specific lung disease and a substantial preponderance of the abnormal Z allele was found. An unusual neutral mutation of the MaeIII site in exon 3 of the AT gene in 20 to 30% of normal subjects with the MI allele, as well as its linkage disequilibrium with the Z allele in AD patients, was discovered. An allele specific amplification system for direct detection of the common mutation in codon 342 of AD patients with the Z haplotype was elaborated and tested.

20.5.9 FAMILIAL HYPERCHOLESTEROLAEMIA AND OTHER LIPOPROTEIN DISORDERS Molecular analysis has been confined to 20 families with familial hypercholesterolemia. No major mutations or rearrangements in the low density lipoprotein receptor gene have been reported so far. RFLP analysis of different polymorphic sites of the receptor gene in normal and affected subjects is in progress. Correlation of particular genotypes of Apo CIII, Apo B 100, and Apo Al genes with blood cholesterol lipoprotein levels in patients with cardiac ischaemic disease and in the general population is being studied.

A simple approach based on PCR mediated site directed mutagenesis has been

suggested for the identification of the common mutation in codon 3500 of the apo BiO gene.


20.5.10 HUNTINGTON'S DISEASE GENE Dr. George Huntington, along with his father and grandfather, published their observations in 1872 on familial cases of chorea near their home on Long Island, NY. The genetic disorder they described is now known as "Huntington's disease" (HD). Nearly 1% of Americans has HD or is at risk of passing along the disease to a child. HD affects as many people as hemophilia, cystic fibrosis or muscular dystrophy combined. In 1993, the HD gene was isolated and, eventually, a direct genetic test was developed that can accurately determine carrier status for the HD gene. The HD gene was found to contain a specific section with a pattern of so-called "trinucleotide repeats" which is expanded in people with HD. In most cases, the repeated pattern occurs 30 times or less. In HD it occurs more than 40 times (Harper et al., 2008). The test cannot predict when symptoms will begin, and therapy is palliative. In the absence of effective treatment and a cure, most individuals at risk elect not to take the test. HD is one of many trinucleotide expansion diseases characterized by genetic anticipation. This phenomenon manifests when a genetic disease either presents earlier, or presents with more severe symptoms in subsequent generations. For example, myotonic dystrophy has a broad spectrum of presentation varying from congenital myotonia to the seventh decade of life. Because of genetic anticipation, early and accurate diagnosis is vital for parents who may have minimal or late onset symptoms but can have children who are more severely affected. The advent of pre-implantation genetics allows for the detection of embryos that are homozygous for inheritable recessive disorders. Through detection of a mutation of the dystrophin gene in a fertilized cell, a couple can avoid bringing to term a child with Duchenne's muscular dystrophy. Approximately 30 other diseases like Huntington's disease, TaySachs, cystic fibrosis and familial dysautonomia can be diagnosed on an embryo prior to implantation in the womb.

20.5.11 MULTIPLE ENDOCRINE NEOPLASIA Multiple endocrine neoplasia, type 2 (MEN 2) presents as two syndromes, 2A and 2B. In MEN 2A, medullary thyroid carcinoma (MTC) involving the thyroid interstitial C-cells is ultimately found in some 90% to 95% of affected individuals, pheochromocytoma in 25% to 50% and hyperparathyroidism in 15% to 20%. MEN 2B is characterized by MTC, pheochromocytoma, mucosal neuromata and marfanoid habitus. Classical linkage studies initiated in the early 1980s led to mapping of the MEN 2 gene to a centromeric chromosome 10 locus in 1987. Mutations of the RET proto-oncogene causative for MEN 2 were identified in 1993. Clinical laboratory testing became available a few years later. (Hoff et al., 2001)


The discovery of molecular abnormalities of the RET proto-oncogene in MEN 2 and FMTC has significantly improved clinical management of these disorders. The identified mutations are responsible for 90% to 95% of all hereditary MTC. Six to seven percent of patients with apparent sporadic MTC have been found to have germline mutations of RET indicative of hereditary MTC. Application of these tests in the management of MTC has improved diagnostic accuracy, lessened the likelihood that hereditary disease will be missed in the context of apparent sporadic MTC and improved the clinical management of this disease. Many of the common disorders that plague humans are multifactorial gene disorders, including cardiovascular disease, diabetes and most types of cancer. Some genes associated with these disorders may indicate increased predisposition, just as total cholesterol and LDL cholesterol serve as important but not exclusive predictors of cardiovascular disease. With additional studies, more genes are being identified with behaviors. These genes may become more important in the future as we develop a fuller understanding of the impact of genetic factors on behavioral disorders and characteristics. Common disorders for which intensive research is underway include cardiovascular disease, diabetes, cancer, neurological diseases (including Alzheimer's), bipolar disease, osteoporosis and behavioral disorders. The diagnostic tests that will be developed will complement current and future tests, including those involving proteomics and metabolic analysis. Many of these diagnostic tests may not live up to initial expectations and others may exceed them. Academic centers and large reference laboratories will be on the forefront of this frontier. The prospectors and pioneers will be followed by a growing group of laboratories as molecular diagnostic tests are more widely accepted and adopted. This is the same pattern that was observed with testing for HIV.

20.5.12 FACTOR V (LEIDEN) MUTATION The factor V (Leiden) mutation (1691G>A) occurs primarily in the Caucasian population and is a major risk factor for venous thrombosis (a lifetime risk of 12% to 30% in affected individuals) and a lesser risk factor for arterial thrombosis (cardiovascular disease). Additionally, factor V (Leiden) mutation is associated with arterial thrombosis (especially in smokers), complications of pregnancy (including fetal loss) and increased levels of factor VIII. The factor V (Leiden) mutation leads to the laboratory finding of activated protein C resistance (APCR) and a sevenfold increase in venous thromboembolic events in heterozygous individuals and an eightyfold increase in homozygous subjects. Due to a synergistic increase in venous thrombosis risk, individuals heterozygous for the factor V mutation are at greater risk when taking oral contraceptives. When a heterozygous mutation is coupled with oral contraceptive use, risk increases synergistically to thirtyfold (Grody et al., 2001). Since laboratory tests for APCR are highly sensitive, specific and simpler to perform, APCR is usually the test of choice; however, factor V mutation analysis is recommended to confirm positive APCR tests.


It is also recommended in place of APCR for patients with lupus anticoagulant, since such patients often have a false-positive APCR test. Although this test is highly specific, identification of a mutation may occur in the absence of APCR in rare cases. Sensitivity of this test for APCR is 94%; thus, a negative result does not rule out APCR or an increased risk of venous thrombosis. More than half of thromboembolic events associated with factor V (Leiden) mutation occur in the presence of additional risk factors, such as surgery and use of oral contraceptives. Thus, factor V (Leiden) mutation is a risk factor and not an indication of thromboembolic disease.

20.5.13 HEMOCHOMATOSIS GENE, HFE Hemochomatosis is an excess accumulation of iron that causes damage to organs, leading to such diseases as cirrhosis, cardiomyopathy, diabetes and arthritis. Two mutations in the HEE gene -- C282Y and H63D -- are associated with hemochromatosis. (Hanson et al., 2001) Disease develops in less than 1% of individuals with these genotypes. These mutations have low penetrance. Other factors, such as diet, hepatotoxins and likely other genes, are important factors that lead to hemochromarosis. The role of testing may be limited to family members of individuals with hemochromatosis. Even for these individuals, biochemical testing remains the cornerstone for diagnosis. Many molecular diagnostic tests may be similar to the HFE gene tests that have a limited role and must be interpreted in the context of family, clinical history, and other risk factors and laboratory tests.

20.5.14 COLON CANCER GENE, APC Dr. Bert Vogelstein generated enormous excitement 15 years ago when he and his colleagues at Johns Hopkins described a series of genetic alterations leading to colorectal cancer, (Vogelstein et al., 1998) that occurs in the different phases of cancer development, starting from normal epithelium and moving through adenomatous polyps to cancer. This suggested a genetic pathway in tumor development. Unfortunately, the number and nature of the genetic alterations may vary in a population. There may be a spectrum of routes that describes the genetic alterations leading to cancer. The interaction with environmental factors, including diet and other genetic factors, is open for exploration. Today, genetic testing for colorectal cancer is quite limited.

20.6.MICROARRAY ANALYSIS FOR DETECTION OF COMPLEX PATTERN OF GENES Comparative genome hybridization technique used in microarrays hold enormous hope in the field of oncology. Many solid and hematologic malignancies have gene duplications and/or gene deletions associated with them. Amplification of the protooncogene Her2/neu has been demonstrated to correlate with a higher grade of malignancy and with tumor response to the chemotherapeutic agent Herceptin. Comparative genome hybridization microarrays have the ability to scan the entire genome for such


insertions and deletions and may revolutionize drug development and prognostic testing. Multiple SNP analysis is currently necessary for cystic fibrosis carrier detection, which requires 25 mutations and six polymorphisms to be analyzed simultaneously. A low-density microarray has been developed for this purpose. Suggestions have been made to screen patients for multiple drug sensitivity pharmacogenetic SNPs using a single chip. This approach suffers from HIPAA and compliance issues, as genetic testing would be performed without a definite indication. Microarrays will likely be used in one of four ways: expression arrays, resequencing microarrays, multiple genotyping arrays and comparative genome hybridization arrays. Expression arrays consist of DNA probes immobilized on chips that are used to detect mRNA, primarily in solid tumors. Promising data suggests these expression arrays can be used to predict malignant potential in stage 1 breast cancer and other malignancies.Resequencing microarrays are being developed to replace expensive DNA sequencing assays for large genes, such as BRCA1 and BRCA2. These chips may reduce the cost of BRCA1 sequencing. If this can be accomplished, the indications for BRCA1 and BRCA2 testing may be broadened. Genetic testing, as all clinical laboratory testing, must always be interpreted in the light of clinical findings. Molecular testing for hemochromatosis, alone, is not capable of making the diagnosis of hemochromatosis. Less than 5% of patients homozygous for C282Y in the HFe gene will ever develop symptoms of hemochromatosis. Molecular genotyping tests are extremely sensitive and specific for the mutations they are designed to detect, but may be less so for disorders associated with those diseases. These complex tests may need to be interpreted with additional clinical and routine test results. Regulatory oversight and ethical debate will strive to keep pace with the rapid advances and applications.

20.7 CONCLUSION Molecular diagnosis of inherited diseases will expand rapidly into clinical labs in the coming years. Laboratorians must develop internal capabilities to perform and interpret selective tests that are appropriate for their setting, understand the clinical application of these tests, examine the technical issues with performing these tests and maintain the expertise to interpret the test results in the full context of the patient and the family being tested. The future will certainly include more demand for information that will improve the lives of individuals, including testing of embryo and foetus. Technology will make it easier for laboratories to perform molecular diagnostics of common disorders. The application of microarray will aid in the discovery of complex patterns of genes, whose functions may not be understood, that define prognostic patterns and lead to therapeutic recommendations. From Mendel's rudimentary study of peapods a century and a half ago, to the sophisticated clinical laboratories of today, the rapid growth of molecular genetics provides unsurpassed diagnostic insights into complicated hereditary diseases that affect the lives of human beings worldwide. From these


insights, medical science can contribute solutions to the families who struggle to resolve hereditary issues. After all, life should be as simple as "she has your smile and my eyes."



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