Chapter 062. Principles of Human Genetics (Part 27) Genetic Linkage Genetic linkage refers to the fact that genes are physically connected, or linked, to one another along the chromosomes. Two fundamental principles are essential for understanding the concept of linkage: (1) when two genes are close together on a chromosome, they are usually transmitted together, unless a recombination event separates them (Figs. 62-3, 62-8); and (2) the odds of a crossover, or recombination event, between two linked genes is proportional to the distance that separates them. Thus, genes that are further apart are more likely to undergo a recombination event than genes that are very close together. The detection of chromosomal loci that segregate with a disease by linkage can be used to identify the gene responsible for the disease (positional cloning) and to predict the odds of disease gene transmission in genetic counseling. Polymorphisms are essential for linkage studies because they provide a means to distinguish the maternal and paternal chromosomes in an individual. On average, 1 out of every 1000 bp varies from one person to the next. Although this degree of variation seems low (99.9% identical), it means that >3 million sequence differences exist between any two unrelated individuals and the probability that the sequence at such loci will differ on the two homologous chromosomes is high (often >70–90%). These sequence variations include VNTRs, short tandem repeats (STRs), and SNPs. Most STRs, also called polymorphic microsatellite markers, consist of di-, tri-, or tetranucleotide repeats that can be measured readily using PCR (Fig. 62-12). Characterization of SNPs, using DNA chips, provides an important new tool for comprehensive analyses of genetic variation, linkage, and association studies. Although these sequence variations usually have no apparent functional consequences, they provide much of the basis for variation in genetic traits. Figure 62-12 CAG repeat length and linkage analysis in multiple endocrine neoplasia (MEN) type 1. Upper panel. Detection of different alleles using polymorphic microsatellite markers. The example depicts a CAG trinucleotide repeat. PCR with primers flanking the polymorphic region results in products of variable length, depending on the number of CAG repeats. After characterization of the alleles in the parents, transmission of the paternal and maternal alleles can be determined. Lower panel. Genotype analysis using microsatellite markers in a family with MEN-1. Two microsatellite markers, A and B, are located in close proximity to the MEN1 gene on chromosome 11q13. For each individual, the A and B alleles have been determined. Based on this analysis, the genotype A3,B4 is linked to the disease because it occurs in the two affected individuals I-1 and II-1 but not in unaffected siblings. Because the disease allele is linked to A3,B4 within the affected family, it is likely that the individual III-1 is a carrier of the mutated MEN1 gene. Although III-5 also has the A3,B4 genotype, she has inherited the allele from her unaffected father (II-4), who is not related to the original family. The A3,B4 genotype is only associated with MEN-1 in the original family, but not in the general population. Therefore, individual III-5 is not at risk for developing the disease. In order to identify a chromosomal locus that segregates with a disease, it is necessary to characterize polymorphic DNA markers from affected and unaffected individuals of one or several pedigrees. One can then assess whether certain marker alleles cosegregate with the disease. Markers that are closest to the disease gene are less likely to undergo recombination events and therefore receive a higher linkage score. Linkage is expressed as a lod (logarithm of odds) score—the ratio of the probability that the disease and marker loci are linked rather than unlinked. Lod scores of +3 (1000:1) are generally accepted as supporting linkage, whereas a score of –2 is consistent with the absence of linkage. An example of the use of linkage analysis is shown in Fig. 62-12. In this case, the gene for the autosomal dominant disorder MEN-1 is known to be located on chromosome 11q13. Using positional cloning, the MEN1 gene was identified and shown to encode menin, a tumor suppressor. Affected individuals inherit a mutant form of the MEN1 gene, predisposing them to certain types of tumors (parathyroid, pituitary, pancreatic islet) (Chap. 345). In the tissues that develop a tumor, a "second hit" occurs in the normal copy of the MEN1 gene. This somatic mutation may be a point mutation, a microdeletion, or loss of a chromosomal fragment (detected as loss of heterozygosity, LOH). Within a given family, linkage to the MEN1 gene locus can be assessed without necessarily knowing the specific mutation in the MEN1 gene. Using polymorphic STRs that are close to the MEN1 gene, one can assess transmission of the different MEN1 alleles and compare this pattern to development of the disorder to determine which allele is associated with risk of MEN-1. In the pedigree shown, the affected grandfather in generation I carries alleles 3 and 4 on the chromosome with the mutated MEN1 gene and alleles 2 and 2 on his other chromosome 11. Consistent with linkage of the 3/4 genotype to the MEN1 locus, his son in generation II is affected, whereas his daughter (who inherits the 2/2 genotype from her father) is unaffected. In the third generation, transmission of the 3/4 genotype indicates risk of developing MEN-1, assuming that no genetic recombination between the 3/4 alleles and the MEN1 gene has occurred. After a specific mutation in the MEN1 gene is identified within a family, it is possible to track transmission of the mutation itself, thereby eliminating uncertainty caused by recombination. . Chapter 062. Principles of Human Genetics (Part 27) Genetic Linkage Genetic linkage refers to the fact that genes are. polymorphic region results in products of variable length, depending on the number of CAG repeats. After characterization of the alleles in the parents, transmission of the paternal and maternal alleles. generally accepted as supporting linkage, whereas a score of –2 is consistent with the absence of linkage. An example of the use of linkage analysis is shown in Fig. 62-12. In this case, the