Direct Mutation Analysis and Linkage Analysis

The ability to define mutations or gene regions associated with disease removes much of the art of risk assessment from evaluation of the pedigree and provides a more

Table 4-4. Factors Affecting Risk and Risk Assessment

Variable expressivity/pleiotropy

Age of onset




Gender-influenced expression (sex-limited vs. sex-influenced)

Family size/paucity of at-risk gender




New mutation

Mosaicism (somatic vs. germline) Modifying genes Environmental effects definitive answer in many cases. Currently, there are clinical or research tests being done for 1,269 different diseases (, accessed on June 14,2006),which continues to increase as definitive mutations are identified in newly described disease-associated genes. Methods of gene analysis vary among different laboratories (see chapter 2 and Reference 17). For large deletions and gene rearrangements, Southern blot analysis is used. Dosage analysis (determination of gene copy number utilizing densitome-try, multiplex ligation-dependent probe amplification [MLPA] or similar techniques) may be used in cases where an affected individual is not available for study and deletion is a common form of mutation, as is the case in Duchenne muscular dystrophy. Southern blot analysis also may be needed for sizing of large trinucleotide repeats, while smaller repeats can be identified by targeted polymerase chain reaction (PCR) analysis. PCR is also used in conjunction with allele-specific oligonucleotides (ASOs) for analysis of conditions with a single or few common mutations. In disorders where many unique, private mutations have been found, mutation-screening techniques may be utilized, including conformation sensitive gel electro-phoresis (CSGE), denaturing gradient gel electrophoresis (DGGE), denaturing high-performance liquid chromatography (DHPLC), two-dimensional gel scanning (TDGS), and single-strand conformation polymorphism (SSCP). Once gene segments with probable variants have been identified by these techniques, DNA sequencing is utilized to verify the presence of a mutation, polymorphism, or variant of unknown significance.

These direct methods of identifying mutations are invaluable when the disease-associated gene is known; however, historically and even today, for many conditions the causative gene has not been identified or is not characterized adequately to allow for mutation-specific testing. In these situations, it is possible to offer an indirect testing method, called linkage analysis, to clarify the risk status of family members if the responsible gene has been localized to a specific genomic map location. For some families, the most significant issue in linkage analysis is the need for specimens from a number of family members, both affected and unaffected, to ensure useful interpretation of results. Linkage analysis requires that the clinical status of the relatives and their relationships be accurately reported for accurate interpretation. Linkage analysis involves determination of "markers" for the disease gene, often variant forms of highly polymorphic short tandem repeats, within or near the genomic map location. It requires that a marker or markers near the genetic locus be informative; that is, key individuals in the family must be heterozygous, or have two different forms of the marker (alleles) at the locus in question. These markers are then tracked as they are passed from one individual to the next. It is thus essential to know which allele(s) is associated with the disease gene and which alleles track with the normal gene copy (setting phase). This typically involves analysis of DNA from a number of affected family members or a carefully selected group of affected and unaffected relatives. In addition, if the marker(s) is closely associated (linked) with the disease gene or is within the gene, presence of the disease-associated marker allele will correlate with presence of the disease gene. If the marker is genetically distant from the disease gene, it may become separated from the disease gene through recombination, and predictions about gene transmission may be inaccurate. Caution is required when doing linkage analysis of very large genes because a marker at one end of the gene may, through recombination, become unlinked from the (unknown) mutation if it resides at the other end of the gene. Accuracy of linkage analysis can be further increased by assessing more than one linked marker, preferably within or flanking opposite ends of the gene.

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