Chapter 062. Principles of Human Genetics (Part 30) DNA testing is performed by mutational analysis or linkage studies in individuals at risk for a genetic disorder known to be present in a family. Mass screening programs require tests of high sensitivity and specificity to be cost- effective. Prerequisites for the success of genetic screening programs include the following: that the disorder is potentially serious; that it can be influenced at a presymptomatic stage by changes in behavior, diet, and/or pharmaceutical manipulations; and that the screening does not result in any harm or discrimination. Screening in Jewish populations for the autosomal recessive neurodegenerative storage disease Tay-Sachs has reduced the number of affected individuals. In contrast, screening for sickle cell trait/disease in African Americans has led to unanticipated problems of discrimination by health insurers and employers. Mass screening programs harbor additional potential problems. For example, screening for the most common genetic alteration in cystic fibrosis, the ∆F508 mutation with a frequency of ~70% in northern Europe, is feasible and seems to be effective. One has to keep in mind, however, that there is pronounced allelic heterogeneity and that the disease can be caused by >1400 other mutations. The search for these less common mutations would substantially increase costs but not the effectiveness of the screening program as a whole. Occupational screening programs aim to detect individuals with increased risk for certain professional activities (e.g., α 1 antitrypsin deficiency and smoke or dust exposure). Mutational Analyses DNA sequence analysis is increasingly used as a diagnostic tool and has significantly enhanced diagnostic accuracy. It is used for determining carrier status and for prenatal testing in monogenic disorders (Chap. 64). Numerous techniques are available for the detection of mutations (Table 62-9). In a very broad sense, one can distinguish between techniques that allow for screening the absence or presence of known mutations (screening mode) or techniques that definitively characterize mutations. Analyses of large alterations in the genome are possible using classic methods such as cytogenetics, fluorescent in situ hybridization (FISH), and Southern blotting (Chap. 63), as well as more sensitive novel techniques that search for multiple single exon deletions or duplications. Table 62-9 Methods Used for the Detection of Mutations Method Principle Type of Mutation Detected Commonly Used Techniques Cytogenetic analysis Unique visual appearance of various chromosomes Numerical or structural abnormalities in chromosomes Fluorescent in situ hybridization (FISH) Hybridization to chromosomes with fluorescently labeled probes Numerical or structural abnormali ties in chromosomes Southern blot Hybridization with genomic probe or cDNA probe after digestion of high molecular DNA Large deletion, insertion, rearrangement, expansions of triplet repeat, amplification Polymerase chain reaction (PCR) Amplification of DNA segment Expansion of triplet repeats, variable number of tandem repeats (VNTR), gene rearrangements, translocations; prepare DNA for other mutation methods Reverse transcriptase PCR (RT- PCR) Reverse transcription, amplification of DNA segment →absence or reduction of mRNA transcription Analyze expressed mRNA (cDNA) sequence; detect loss of expression Direct sequencing of PCR products DNA sequencing Sequencing of DNA Point mutations, small deletions and insertions segments cloned into plasmid vectors Restriction fragment polymorphism (RFLP) Detection of altered restriction pattern of genomic DNA (Southern blot) or PCR products Point mutations, small deletions and insertions Other Techniques Single-strand conformational polymorphism (SSCP) PCR of DNA segment: Mutations result in conformational change and altered mobility Point mutations, small deletions and insertions Denaturing gradient gel electrophoresis (DGGE) PCR of DNA segment: Mu tations result in conformational change and altered mobility Point mutations, small deletions and insertions RNAse cleavage Cleavage of mismatch Point mutations, between mutated and wild- type sequence small deletions and insertions Oligonucleotide specif ic hybridization (OSH) Hybridization of PCR products to wild- type or mutated oligonucleotides immobilized on chips or slides Point mutations, small deletions and insertions Point mutations, small deletions and insertions Microarrays Hybridization of PCR products to wild- type or mutated oligonucleotides Genotyping of SNPs Protein truncation test (PTT) Transcription/translation of cDNA isolated from tissue sample Mutations leading to premature truncations Pyrosequencing Clonal amplification of single DN A fragments on Sequencing of whole genomes of microparticles followed by massive parallel sequencing microorganisms, resequencing of amplicons Multiplex ligation-dependent probe amplification (MLPA) Quantification of PCR- generated amplicons reflecting the number of copies of a specific DNA sequence Copy number variations . Chapter 062. Principles of Human Genetics (Part 30) DNA testing is performed by mutational analysis or linkage studies. transcription, amplification of DNA segment →absence or reduction of mRNA transcription Analyze expressed mRNA (cDNA) sequence; detect loss of expression Direct sequencing of PCR products DNA. Clonal amplification of single DN A fragments on Sequencing of whole genomes of microparticles followed by massive parallel sequencing microorganisms, resequencing of amplicons Multiplex