Chapter 062. Principles of Human Genetics (Part 31)

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More discrete sequence alterations rely heavily on the use of the PCR, which allows rapid gene amplification and analysis. Moreover, PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells, buccal cells, or hair roots. Screening for point mutations can be performed by numerous methods (Table 62-9); most are based on the recognition of mismatches between nucleic acid duplexes, electrophoretic separation of single- or double-stranded DNA, or sequencing of DNA fragments amplified by PCR. DNA sequencing can be performed directly on PCR products or on...

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Chapter 062. Principles of Human Genetics (Part 31) More discrete sequence alterations rely heavily on the use of the PCR, which allows rapid gene amplification and analysis. Moreover, PCR makes it possible to perform genetic testing and mutational analysis with small amounts of DNA extracted from leukocytes or even from single cells, buccal cells, or hair roots. Screening for point mutations can be performed by numerous methods (Table 62-9); most are based on the recognition of mismatches between nucleic acid duplexes, electrophoretic separation of single- or double-stranded DNA, or sequencing of DNA fragments amplified by PCR. DNA sequencing can be performed directly on PCR products or on fragments cloned into plasmid vectors amplified in bacterial host cells.
RT-PCR may be useful to detect absent or reduced levels of mRNA expression due to a mutated allele. Protein truncation tests (PTT) can be used to detect the broad array of mutations that result in premature termination of a polypeptide during its synthesis. The isolated cDNA is transcribed and translated in vitro, and the proteins are analyzed by gel electrophoresis. Comparison of electrophoretic mobility with the wild-type protein allows detection of truncated mutants. The majority of traditional diagnostic methods are gel-based. Novel technologies for the analysis of mutations, genotyping, large-scale sequencing, and mRNA expression profiles are in rapid development. DNA chip technologies allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used clinically for mutational analysis of several human disease genes, as well as for the identification of viral sequence variations. Together with the knowledge gained from the HGP, these technologies provide the foundation to expand from a focus on single genes to analyses at the scale of the genome. Faster and cheaper sequencing technologies are under development, and it has been anticipated that sequencing the whole genome of an individual for a cost of ≤$1000 will become a reality within this decade. The availability of comprehensive individual sequence information is expected to have a significant impact on medical care and preventative strategies, but it also raises
ethical and legal concerns how such information may be used by insurers and employers. A general algorithm for the approach to mutational analysis is outlined in Fig. 62-14. The importance of a detailed clinical phenotype cannot be overemphasized. This is the step where one should also consider the possibility of genetic heterogeneity and phenocopies. If obvious candidate genes are suggested by the phenotype, they can be analyzed directly. After identification of a mutation, it is essential to demonstrate that it segregates with the phenotype. The functional characterization of novel mutations is labor intensive and may require analyses in vitro or in transgenic models in order to document the relevance of the genetic alteration. Prenatal diagnosis of numerous genetic diseases in instances with a high risk for certain disorders is now possible by direct DNA analysis. Amniocentesis involves the removal of a small amount of amniotic fluid, usually at 16 weeks of gestation. Cells can be collected and submitted for karyotype analyses, FISH, and mutational analysis of selected genes. The main indications for amniocentesis include advanced maternal age above age 35, abnormal serum triple marker test (α-fetoprotein, βhuman chorionic gonadotropin, pregnancy-associated plasma protein A, or unconjugated estriol), a family history of chromosomal abnormalities, or a Mendelian disorder amenable to genetic testing. Prenatal diagnosis can also be performed by chorionic villus sampling (CVS), in which a
small amount of the chorion is removed by a transcervical or transabdominal biopsy. Chromosomes and DNA obtained from these cells can be submitted for cytogenetic and mutational analyses. CVS can be performed earlier in gestation (weeks 9–12) than amniocentesis, an aspect that may be of relevance when termination of pregnancy is a consideration. Later in pregnancy, beginning at about 18 weeks of gestation, percutaneous umbilical blood sampling (PUBS) permits collection of fetal blood for lymphocyte culture and analysis. In combination with in vitro fertilization (IVF) techniques, it is even possible to perform genetic diagnoses in a single cell removed from the four- to eight-cell embryo or to analyze the first polar body from an oocyte. Preconceptual diagnosis thereby avoids therapeutic abortions but is extremely costly and labor intensive. Lastly, it has to be emphasized that excluding a specific disorder by any of these approaches is never equivalent to the assurance of having a normal child. Mutations in certain cancer susceptibility genes, such as BRCA1 and BRCA2, may identify individuals with an increased risk for the development of malignancies and result in risk-reducing interventions. The detection of mutations is an important diagnostic and prognostic tool in leukemias and lymphomas. The demonstration of the presence or absence of mutations and polymorphisms is also relevant for the rapidly evolving field of pharmacogenomics, including the identification of differences in drug treatment response or metabolism as a function of genetic background. For example, the thiopurine drugs 6-
mercaptopurine and azathioprine are commonly used cytotoxic and immunosuppressive agents. They are metabolized by thiopurine methyltransferase (TPMT), an enzyme with variable activity associated with genetic polymorphisms in 10% of Caucasians and complete deficiency in about 1/300 individuals. Patients with intermediate or deficient TPMT activity are at risk for excessive toxicity, including fatal myelosuppression. Characterization of these polymorphisms allows mercaptopurine doses to be modified based on TPMT genotype. Pharmacogenomics may increasingly permit individualized drug therapy, improve drug effectiveness, reduce adverse side effects, and provide cost-effective pharmaceutical care. Further Readings Altshuler D et al: for The International HapMap Consortium: A haplotype map of the human genome. Nature 437:1299, 2005 Guttmacher AE, Collins FS: Realizing the promise of genomics in biomedical research. JAMA 294:1399, 2005 [PMID: 16174701] ——— et al: The family history—more important than ever. N Engl J Med 351:2333, 2004
Rockman MV, Kruglyak L: Genetics of global gene expression. Nat Rev Genet 7:862, 2006 [PMID: 17047685] Roden DM et al: Pharmacogenomics: Challenges and opportunities. Ann Intern Med 145:749, 2006 [PMID: 17116919] Service RF: Gene sequencing. The race for the $1000 genome. Science 311:1544, 2006 [PMID: 16543431] Wolfsberg TG et al: A user's guide to the human genome. Nat Genet 35(Suppl 1): 2003 Bibliography Alberts B et al (eds): Molecular Biology of the Cell, 5th ed. New York, Garland, 2007 American Society of Human Genetics Board of Directors, American College of Medical Genetics Board of Directors: Points to consider: Ethical, legal, and psychosocial implications of genetic testing in children and adolescents. Am J
Hum Genet 57:1233, 1995 Balmain A et al: The genetics and genomics of cancer. Nat Genet 33(Suppl):238, 2003 Clayton EW: Ethical, legal, and social implications of genomic medicine. N Engl J Med 349:562, 2003 [PMID: 12904522] Egger G et al: Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:457, 2004 [PMID: 15164071] Feuk L et al: Structural variation in the human genome. Nat Rev Genet 7:85, 2006 [PMID: 16418744] Florez JC et al: The inherited basis of diabetes mellitus: Implications for the genetic analysis of complex traits. Annu Rev Genomics Hum Genet 4:257, 2003 [PMID: 14527304] Garrigan D, Hammer MF: Reconstructing human origins in the genomic era. Nat Rev Genet 7,669, 2006
Gelehrter TD et al (eds): Principles of Medical Genetics, 2d ed. Philadelphia, Lippincott, 1998 Haga SB, Willard HF: Science and society: Defining the spectrum of genome policy. Nat Rev Genet 7:966, 2006 [PMID: 17139328] Harper PS: Practical Genetic Counseling, 6th ed. Stoneham, MA, Butterworth-Heinemann, 2004 Jaenisch R, Bird A: Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat Genet 33(Suppl):245, 2003 Lanpher B et al: Inborn errors of metabolism: The flux from Mendelian to complex diseases. Nat Rev Genet 7:449, 2006 [PMID: 16708072] Lewin B: Genes VIII. Oxford, Oxford University Press, 2004 Mayeux R: Mapping the new frontier: Complex genetic disorders. J Clin Invest 115:1404, 2005 [PMID: 15931374]
Nussbaum RL et al (eds): Thompson and Thompson Genetics in Medicine, 7th ed. New York, Elsevier Science, 2007 Ott J: Analysis of Human Genetic Linkage. 3d ed. Baltimore, Johns Hopkins, 1999 Pfeifer A, Verma IM: Gene therapy: Promises and problems. Ann Rev Genom Hum Genet 2:177, 2001 [PMID: 11701648] Rimoin DL et al (eds): Emery and Rimoin's Principles and Practice of Medical Genetics, 5th ed. New York, Churchill Livingstone, 2006 Scriver CR et al (eds): The Metabolic and Molecular Bases of Inherited Disease, 8th ed. New York, McGraw-Hill, 2001 Tyers M, Mann M: From genomics to proteomics. Nature 422:193, 2003 [PMID: 12634792] Wilkins JF, Haig D: What good is genomic imprinting: The function of parent-specific gene expression. Nat Rev Genet 4: 359, 2003 [PMID: 12728278]