The Huntington's disease (HD) gene has been mapped in 4~16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4~16.3 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), repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4~16.3 haplotypes. The GAG), repeat appears to be located within the coding sequence of a predicted-346 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 4~16.3 gene to produce a dominant phenotype.
We have used RNA in situ hybridization to study the regional expression of the Huntington's disease gene (HD) and its rat homologue in brain and selected nonneural tissues. The HD transcript was expressed throughout the brain in both rat and human, especially in the neurons of the dentate gyrus and pyramidal neurons of the hippocampal formation, cerebellar granule cell layer, cerebellar Purkinje cells and pontine nuclei. Other brain areas expressed lower levels of the HD transcript without pronounced regional differences. Neuronal expression predominated over glial expression in all regions. HD mRNA was also expressed in colon, liver, pancreas and testes. The regional specificity of neuropathology in HD, which is most prominent in the basal ganglia, thus cannot be accounted for by the pattern of expression of HD.
The quest for the mutation responsible for Huntington's disease (HD) has required an exceptionally detailed analysis of a large part of 4p16.3 by molecular genetic techniques, making this stretch of 2.2 megabases one of the best characterized regions of the human genome. Here we describe the construction of a cosmid and P1 clone contig spanning the region containing the HD gene, and the establishment of a detailed, high resolution restriction map. This ordered clone library has allowed the identification of several genes from the region, and has played a vital role in the recent identification of the Huntington's disease gene. The restriction map provides the framework for the detailed analysis of a region extremely rich in coding sequences. This study also exemplifies many of the strategies to be used in the analysis of larger regions of the human genome.
We searched for mutations in the CCK gene in panic disorder with single-strand conformational polymorphism (SSCP) analysis of the three exons and promotor region of the gene. We found a C-->T transition at position -36 (CCK(-36C-->T)) in a GC box, a binding site for transcription factor Sp1, in the promotor region. The allele frequency was 0.168 (95% CI, 0.116-0.221) in 98 persons with panic disorder and 0.083 (95% CI, 0.059-0.107) in 247 geographically matched, unscreened controls. A transmission disequilibrium test based on panic disorder as the affected phenotype was nonsignificant (chi2 = 0.93), but when panic disorder or attacks were considered as affected, statistically significant transmission disequilibrium was detected (chi2 = 4.00, P < 0.05). Linkage analysis was uninformative. In exploratory analyses to search for clinical correlations, the "T" allele was found in 59% of 22 persons with panic attacks but not panic disorder, compared with 31% of those who met the criteria for panic disorder. An association between the CCK polymorphism and panic disorder cannot be considered established due to the inconsistencies in the results noted above, but if the provisional association can be replicated, the findings are consistent with CCK(-36C-->T) being a disease-susceptibility allele that alone is neither necessary nor sufficient to cause panic disorder but that increases vulnerability by acting epistatically.
We have used a combination of methods (exon amplification, direct selection, direct screening, evolutionary conservation, island rescue-PCR, and direct sequence analysis) to survey approximately 600 kb of genomic DNA surrounding the BRCA1 gene for transcribed sequences. We have cloned a set of fragments representing at least 26 genes. The DNA sequence of these clones reveals that 5 are previously cloned genes; the precise chromosomal location of 2 was previously unknown, and 3 have been cloned and mapped by others to this interval. Three other genes, including BRCA1 itself, have recently been mapped independently to this region. Sequences from 11 genes are similar but not identical matches to known genes; 5 of these appear to be the human homologues of genes cloned from other species. Another 7 genes have no similarity with known genes. In addition, 39 putative exons and 14 expressed sequence tags have been identified and mapped to individual cosmids. This transcript map provides a detailed description of gene organization for this region of the genome.
The Huntington's disease (HD) gene has been localized by recombination events to a region covering 2.2 megabases (Mb) DNA within chromosome 4p16.3. We have screened three yeast artificial chromosome (YAC) libraries in order to isolate and characterize 44 YAC clones mapping to this region. Approximately 50% of the YACs were chimaeric. Unstable YACs were identified across the whole region, but were particularly prevalent around the D4S183 and D4S43 loci. The YACs have been assembled into a contig extending from D4S126 to D4S98 covering roughly 2 Mb DNA, except for a gap of about 250 kilobases (kb). The establishment of a YAC contig which spans the region most likely to contain the HD mutation is an essential step in the isolation of the HD gene.
The identification of transcripts from large genomic regions cloned In yeast artificial chromosomes (YACs) or cosmids continues to be a critical and often rate-limiting step in positional cloning of human disease genes. We have developed a PCR-based method for rapid and efficient generation of probes from YACs or cosmids that can be used for cDNA library screening. The method, which we call island rescue PCR (IRP), is based upon the observation that the 5' ends of many genes are associated with (G+C)-rich regions called CpG islands. In IRP, the YAC of interest is digested with a restriction enzyme that recognizes sequences of high CpG content, and vectorette linkers are ligated to the cleaved ends. The PCR is used to amplify the region extending from the cleaved restriction enzyme site to the nearest SINE (Alu) repeat. In many cases this product contains sequences from the 5' end of the associated gene. cDNA clones isolated with these products are then verified by mapping them back to the original YAC.The method allows rapid screening of >500 kb of human genomic insert in one experiment, is tolerant of contaminating yeast sequences, and can also be applied to cosmid pools. In a control experiment, the method was able to identify cDNA clones for the neuroftbromatosis type 1 (NFI) gene using a probe generated from a YAC in the region. Application of IRP has yielded nine other genes from YACs isolated from chromosome locations 4pl6.3 and 17q21.An important step in the understanding and accurate diagnosis of human inherited diseases is isolation of the causal gene from the chromosomal region targeted by positional cloning studies. The limited resolution of genetic studies usually necessitates the cloning of large genomic regions. Once the candidate interval has been cloned in yeast artificial chromosomes (YACs) and/or cosmids, the challenge is to then identify coding regions within these largely uncharacterized genomic clones. A common conventional strategy involves screening small unique genomic fragments from the region for evolutionary conservation. More recent advances allow the isolation of exons by virtue of 5' and 3' splice acceptor sites (exon trapping) (1, 2), enrichment for coding sequences by magnetic bead capture (3-5), and direct screening of cDNA libraries by gel-purified YAC DNA or cosmids (6). However, all of these methods require DNA free of host sequences and are often difficult to apply to YACs.We have developed a method (Fig. 1A) that generates high-quality exon-enriched probes from genomic DNA derived directly from YAC inserts. CpG islands are targeted by the presence of an Eag I, Sac II, or BssHII site. Since, on the average, each of these enzymes is expected to cut 1.2 times within an island (7), most islands should be accessible. The procedure takes less than a day and yields probe suitable for cDNA library screening.CpG islands differ from bulk genomic DNA by being relatively (G+C)-rich (>60%) and having a high concentration (10-20 times above average) of the CpG dinucleotide (8).C...
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