Zdenek Sedlacek

A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes

The Huntingtons disease (HD) gene has been mapped in 4p16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect. A new gene, IT15, isolated using cloned trapped exons from the target area contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4p16.3 haplotypes. The (CAG)n repeat appears to be located within the coding sequence of a predicted approximately 348 kd protein that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.

Physical maps of 4p16.3, the area expected to contain the Huntington disease mutation

The gene for Huntington disease, a neurodegenerative disorder with autosomal dominant inheritance, has been localized to the terminal portion of the short arm of human chromosome 4 (4p16.3) by linkage analysis. Since eventual isolation of the gene requires the application of high-resolution genetic analysis coupled with long-range DNA mapping and cloning techniques, we have constructed a physical map of the chromosomal region 4p16.3 using more than 20 independently derived probes. We have grouped these markers into three clusters which have been ordered and oriented by genetic and somatic cell genetic mapping information. The mapped region extends from D4S10 (G8) toward the telomere and covers minimally 5 Mb.

Mapping of cosmid clones in Huntington's disease region of chromosome 4

Huntingtons disease (HD) is tightly linked to genetic markers in 4p16.3. We have used a regional somatic cell hybrid mapping panel to isolate and map 25 cosmids to the proximal portion of 4p16.3 and 17 cosmids to the distal portion. The latter were positioned by long-range restriction mapping relative to previously mapped markers. One cosmid, L6 (D4S166), spans the critical breakpoint in the mapping panel that distinguishes proximal and distal 4p16.3. Four of the cosmids mapped distal toD4S90, the previous terminal marker on 4p, and stretched to within 75 kb of the telomere. Several of the cosmids that mapped between L6 andD4S90 were clustered near a number of previously isolated clones in a region with many NotI sites. Cosmid E4 (D4S168) was localized immediately proximal to the one remaining gap in the long-range restriction map of distal 4p16.3. Although pulsed field gel mapping with E4 failed to link the two segments of the map, the intervening gap was excluded as a potential site for theHD gene by genetic analysis.

The telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p.

A telomere YAC clone containing the most distal 115 kb of chromosome arm 4p has been previously isolated. This clone is of particular interest as it spans a potential candidate region for the Huntington disease gene. The YAC was subcloned into a phage vector, and a high-resolution restriction map extending to within 13 kb of the telomere was constructed. In situ hybridization of the YAC to human metaphase spreads gives a peak of hybridization on 4pter but also an increase in the number of signals close to several other telomeres. Where possible, these results were investigated further by the hybridization of probes from the YAC to somatic cell hybrids containing single human chromosomes. This analysis indicates that the most telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. The extent of this homology makes it less likely that the mutation for Huntingtons disease is located within the telomere YAC clone.

Evolutionary conservation and genomic organization of XAP-4, an Xq28 located gene coding for a human rab GDP-dissociation inhibitor (GDI)

After the development of efficient methods for the construction of transcription maps of defined genomic regions, the rate-limiting step in the analysis of the coding potentials of these regions is the elucidation of function of the novel genes and the examination of their possible involvement in hereditary diseases localized to the region. This can be greatly facilitated by the detection of sequence homology to a gene of known function. XAP-4 is one of the genes identified in the G6PD region of the human Xq28 by direct cDNA selection. The rapid assembly of this gene and the determination of its function was possible because of its sequence homology with the bovine smg p25A/rab3A GDP dissociation inhibitor (GDI). Sequence comparison with other GDIs in the databases has revealed that XAP-4 belongs to one of at least two distinct classes of mammalian rab GDIs. The rab GDIs, which play an important role in the regulation of cellular transport, are highly evolutionarily conserved, as are several other genes identified in the neighborhood of XAP-4. This genomic region is very gene dense, and all the cDNA clones from the approximately 2.5-kb-long transcript of XAP-4 map to a single 7.5-kb genomic EcoRI fragment. The genomic organization of XAP-4 has been examined to determine the distribution of the exonic sequences within this short segment of genomic DNA. It was found that, similar to several other genes from the region, XAP-4 is split into exons of average size, which are interrupted by very short introns.

Identification of tissue-specific expressed sequences in human band Xq28 with complex pig cDNA probes

As a part of the functional analysis of the region from the position of the fragile X mutation to the telomere of the long arm of the human X Chromosome (Chr), we have developed a number of different approaches to identify genes located in this area. We describe here a procedure allowing the rapid identification of expressed sequences based on the hybridization of radioactively labeled complex cDNA probes derived from different pig and human tissues to cosmid clones gridded onto nylon filters and to restriction fragments of these clones. This technique has allowed the identification of a number of differentially expressed sequences in cosmid clones covering most of the Xq27.3 to Xqter region. Using these sequences as hybridization probes, cDNA clones for new genes expressed in a tissue-specific manner were isolated. Applied to genomic regions defined by overlapping cosmid clones, this method will serve as a major component in our strategy to establish integrated physical and transcription maps.