Molecular genetics of restriction fragment length polymorphisms linked to the Huntington disease locus

Abstract

New Zealand families segregating the Huntington disease (HD) phenotype were investigated for linkage of the HD locus to the anonymous DNA locus G8, from chromosome 4p16.3. Linkage of the two loci was indicated in the largest family assessed. The results from this family together with those from 10 other smaller families were consistent with the existence of a single locus (that is, genetic homogeneity for HD). One crossover between the HD locus and the G8 marker locus was detected. Overall, the results of the linkage analysis indicate a distance of 4cM separating HD from G8. The usefulness of G8 as a marker in predictive testing for HD was examined in the New Zealand families. In agreement with overseas findings, G8 and DNA contiguous with G8 are useful for predictive testing in some families. The main limitation for predictive testing is the family structure which often results in the unavailability of key individuals for testing and thus prevents prediction in others. Sequence analysis of three sites which produce restriction fragment length polymorphisms (RFLPs) detected by G8 revealed that single point mutations were responsible for the presence or absence of the polymorphic sites *H1, *H2 and *E. The sequences were highly conserved between individuals in the regions investigated. The conservation of sequence provided the potential for the use of the polymerase chain reaction (PCR) to amplify each polymorphic region for more rapid assessment of these RFLPs. The three regions were amplified successfully from genomic DNA. In addition, amplification of the three regions was possible with template DNA which was degraded or crude or isolated from tissue which had been fixed in formalin. However, the results of the subsequent analyses of the amplified products by restriction enzyme digestion showed that there are problems that can render the PCR unreliable. The presence of non-target sequences hindered detection of the genotype in the *H1 and *H2 regions and masked the true genotype of a person in the *E region. Thus, the potential of the PCR for presymptomatic diagnosis of HD remains to be realised. However, when the problems are overcome a very rapid analysis of RFLPs linked with HD using the PCR will be possible, as will be retrospective analyses.