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...
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Chapter 062. Principles of Human Genetics (Part 31) 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 itpossible to perform genetic testing and mutational analysis with small amounts ofDNA extracted from leukocytes or even from single cells, buccal cells, or hairroots. Screening for point mutations can be performed by numerous methods(Table 62-9); most are based on the recognition of mismatches between nucleicacid duplexes, electrophoretic separation of single- or double-stranded DNA, orsequencing of DNA fragments amplified by PCR. DNA sequencing can beperformed directly on PCR products or on fragments cloned into plasmid vectorsamplified in bacterial host cells. RT-PCR may be useful to detect absent or reduced levels of mRNAexpression due to a mutated allele. Protein truncation tests (PTT) can be used todetect the broad array of mutations that result in premature termination of apolypeptide during its synthesis. The isolated cDNA is transcribed and translatedin vitro, and the proteins are analyzed by gel electrophoresis. Comparison ofelectrophoretic mobility with the wild-type protein allows detection of truncatedmutants. The majority of traditional diagnostic methods are gel-based. Noveltechnologies for the analysis of mutations, genotyping, large-scale sequencing, andmRNA expression profiles are in rapid development. DNA chip technologiesallow hybridization of DNA or RNA to hundreds of thousands of probessimultaneously. Microarrays are being used clinically for mutational analysis ofseveral human disease genes, as well as for the identification of viral sequencevariations. Together with the knowledge gained from the HGP, these technologiesprovide the foundation to expand from a focus on single genes to analyses at thescale of the genome. Faster and cheaper sequencing technologies are underdevelopment, and it has been anticipated that sequencing the whole genome of anindividual for a cost of ≤$1000 will become a reality within this decade. Theavailability of comprehensive individual sequence information is expected to havea significant impact on medical care and preventative strategies, but it also raisesethical and legal concerns how such information may be used by insurers andemployers. A general algorithm for the approach to mutational analysis is outlined inFig. 62-14. The importance of a detailed clinical phenotype cannot beoveremphasized. This is the step where one should also consider the possibility ofgenetic heterogeneity and phenocopies. If obvious candidate genes are suggestedby the phenotype, they can be analyzed directly. After identification of a mutation,it is essential to demonstrate that it segregates with the phenotype. The functionalcharacterization of novel mutations is labor intensive and may require analyses invitro or in transgenic models in order to document the relevance of the geneticalteration. Prenatal diagnosis of numerous genetic diseases in instances with a highrisk for certain disorders is now possible by direct DNA analysis. Amniocentesisinvolves the removal of a small amount of amniotic fluid, usually at 16 weeks ofgestation. Cells can be collected and submitted for karyotype analyses, FISH, andmutational analysis of selected genes. The main indications for amniocentesisinclude advanced maternal age above age 35, abnormal serum triple marker test(α-fetoprotein, βhuman chorionic gonadotropin, pregnancy-associated plasmaprotein A, or unconjugated estriol), a family history of chromosomalabnormalities, or a Mendelian disorder amenable to genetic testing. Prenataldiagnosis can also be performed by chorionic villus sampling (CVS), in which asmall amount of the chorion is removed by a transcervical or transabdominalbiopsy. Chromosomes and DNA obtained from these cells can be submitted forcytogenetic and mutational analyses. CVS can be performed earlier in gestation(weeks 9–12) than amniocentesis, an aspect that may be of relevance whentermination of pregnancy is a consideration. Later in pregnancy, beginning atabout 18 weeks of gestation, percutaneous umbilical blood sampling (PUBS)permits collection of fetal blood for lymphocyte culture and analysis. Incombination with in vitro fertilization (IVF) techniques, it is even possible toperform genetic diagnoses in a single cell removed from the four- to eight-cellembryo or to analyze the first polar body from an oocyte. Preconceptual diagnosisthereby avoids therapeutic abortions but is extremely costly and labor intensive.Lastly, it has to be emphasized that excluding a specific disorder by any of theseapproaches is ...