A new DNA marker (D4S90) is located terminally on the short arm of chromosome 4, close to the Huntington disease gene

Genetic linkage studies have mapped Huntington's disease (HD) to the distal portion of the short arm of chromosome 4 (4p16.3), 4 cM distal to D4S10 (G8). To date, no definite flanking marker has been identified. A new DNA marker, D4S90 (D5), which maps to the distal region of 4p16.3, is described. The marker was used in a genetic linkage study in the CEPH reference families with seven other markers at 4p16. The study, together with knowledge of the physical map of the region, places D4S90 as the most distal marker, 6 cM from D4S10. A provisional linkage study with HD gave a maximum lod score of 2.14 at a θ of 0.00 and no evidence of linkage disequilibrium. As D4S90 appears to be located terminally, it should play an important role in the accurate mapping and cloning of the HD gene.     

Increased recombination adjacent to the Huntington disease-linked D4S10 marker.

Huntington disease (HD) is caused by a genetic defect distal to the anonymous DNA marker D4S10 in the terminal cytogenetic subband of the short arm of chromosome 4 (4p16.3). The effort to identify new markers linked to HD has concentrated on the use of somatic cell hybrid panels that split 4p16.3 into proximal and distal portions. Here we report two new polymorphic markers in the proximal portion of 4p16.3, distal to D4S10. Both loci, D4S126 and D4S127, are defined by cosmids isolated from a library enriched for sequences in the 4pter-4p15.1 region. Physical mapping by pulsed-field gel electrophoresis places D4S126 200 kb telomeric to D4S10, while D4S127 is located near the more distal marker D4S95. Typing of a reference pedigree for D4S126 and D4S127 and for the recently described VNTR marker D4S125 has firmly placed these loci on the existing linkage map of 4p16.3. This genetic analysis has revealed that the region immediately distal to D4S10 shows a dramatically higher rate of recombination than would be expected based on its physical size. D4S10-D4S126-D4S125 span 3.5 cM, but only 300-400 kb of DNA. Consequently, this small region accounts for most of the reported genetic distance between D4S10 and HD. By contrast, it was not possible to connect D4S127 to D4S125 by physical mapping, although they are only 0.3 cM apart. A more detailed analysis of recombination sites within the immediate vicinity of D4S10 could potentially reveal the molecular basis for this phenomenon; however, it is clear that the rate of recombination is not continuously increased with progress toward the telomere of 4p.     

Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90.

A DNA probe (D4S95) that detects a variable number of tandem repeats and a single-site-variation polymorphism after digestion with a single restriction enzyme, AccI, has previously been described. The order of this probe relative to the gene for Huntington disease (HD) and other previously described markers has not been established. Analysis of 24 affected families with HD has shown that D4S95 is in tight linkage with the gene causing HD, with a maximal Lod score of 12.489 at a theta of .03. D4S90 is a probe which maps to 4p16.3, telomeric to D4S95, and detects polymorphisms with HincII and other enzymes. In one affected person, recombination has occurred between D4S10 and HD, between D4S95 and HD, and in all likelihood also between D4S90 and HD, which strongly suggests that the gene for HD is telomeric to all these DNA probes. This suggests that the gene causing HD is located in the most distal region of the short arm of chromosome 4, flanked by D4S90 and the telomere, and supports the locus order D4S10-D4S95-D4S90-HD-telomere. D4S95 is a most useful DNA marker for predictive testing programs, while D4S90 will serve as a useful starting point for identifying DNA fragments closer to the gene for HD.     

Defined physical limits of the Huntington disease gene candidate region.

The Huntington disease (HD) gene has been mapped 4 cM distal to D4S10 within the telomeric chromosome band, 4p16.3. The published physical map of this region extends from D4S10 to the telomere but contains two gaps of unknown size. Recombination events have been used to position the HD mutation with respect to genetic markers within this region, and one such event places the gene proximal to D4S168, excluding the distal gap as a possible location for the defect. One previously published recombination event appeared to have excluded the proximal gap. We have reassessed this event and have moved the proximal boundary for the HD candidate region centromeric to the gap within a "hot spot" for recombination between D4S10 and D4S125. We have closed the proximal gap and report here the complete physical map spanning the HD candidate region from D4S10 to D4S168, the maximum size of which can now be placed accurately at 2.5 Mb.     

Complex patterns of linkage disequilibrium in the Huntington disease region.

The genetic defect causing Huntington disease (HD) has been mapped to 4p16.3 by linkage analysis using DNA markers. Two apparently contradictory classes of recombination events in HD kindreds preclude precise targeting of efforts to clone the disease gene. Here, we report a new recombination event that increases support for an internal candidate region of 2.5 Mb between D4S10 and D4S168. Analysis of 23 DNA polymorphisms in 4p16.3 revealed a complex pattern of association with the disease gene that failed to narrow the size of the candidate region. The degree of linkage disequilibrium did not show a continuous increase across the physical map, nor was a region of extreme disequilibrium identified. Markers displaying no association with the disorder were interspersed with and, in many cases, close to markers displaying significant disequilibrium. Comparison of closely spaced marker pairs on normal and HD chromosomes, as well as analysis of haplotypes across the HD region, suggest that simple recombination subsequent to a single original HD mutation cannot easily explain the pool of HD chromosomes seen today. A number of different mechanisms could contribute to the diversity of haplotypes observed on HD chromosomes, but it is likely that there has been more than one and possibly several independent origins of the HD mutation.     

Localization of the Huntington's disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of late onset, characterized by progressive motor disturbance, psychological manifestations, and intellectual deterioration. The HD gene has been genetically mapped by linkage to the DNA marker D4S10, but the exact physical location of the HD defect has remained uncertain. To delineate critical recombination events revealing the physical position of the HD gene, we have identified restriction fragment length polymorphisms for two recently mapped chromosome 4 loci, RAF2 and D4S62, and determined the pattern of segregation of these markers in both reference and HD pedigrees. Multipoint linkage analysis of the new markers with D4S10 and HD establishes that the HD gene is located in a very small physical region at the tip of the chromosome, bordered by D4S10 and the telomere. A crossover within the D4S10 locus orients this segment on the chromosome, providing the necessary information for efficient application of directional cloning strategies for progressing toward, and eventually isolating, the HD gene.     

Defining the Proximal Border of the Huntington Disease Candidate Region by Multipoint Recombination Analyses

The candidate region for the Huntington disease (HD) gene has been narrowed down to a 2.2-Mb region between D4S10 and D4S98 on the short arm of chromosome 4. To map the HD gene within this candidate region 65 Dutch HD families were studied. In total 338 informative meioses were analyzed and 11 multiple informative crossovers were detected. Assuming a minimum number of recombinations and no double recombinations, our multiple informative crossovers are consistent with one specific genetic order for 12 loci: D4S10-(D4S81, D4S126)-D4S125-(D4S127, D4S95)-D4S43-(D4S115, D4S96, D4S111, D4S90, D4S141). This is in agreement with the known data derived from similar and other methods. The loci between brackets could not be mapped relative to each other. In our family material, two informative three-point marker recombination events were detected in the proximal HD candidate region, which are also informative for HD. Both recombination events map the HD gene distal to D4S81 and most likely distal to D4S125, narrowing down the HD candidate region to a 1.7-Mb region between D4S125 and D4S98.     

Recombination events suggest potential sites for the Huntington's disease gene

The Huntington's disease gene (HD) maps distal to the D4S10 marker in the terminal 4p16.3 subband of chromosome 4. Directed cloning has provided several DNA segments that have been grouped into three clusters on a physical map of approximately 5 X 10(6) bp in 4p16.3. We have typed RFLPs in both reference and HD pedigrees to produce a fine-structure genetic map that establishes the relative order of the clusters and further narrows the target area containing the HD gene. Despite the large number of meiotic events examined, the HD gene cannot be positioned relative to the most distal cluster. One recombination event with HD suggests that the terminal-most markers flank the disease gene; two others favor a telomeric location for the defect. Efforts to isolate the HD gene must be divided between these two distinct intervals until additional genetic data resolve the apparent contradiction in localization.     

A Huntington disease-like neurodegenerative disorder maps to chromosome 20p.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor disturbance, cognitive loss, and psychiatric manifestations. The disease is associated with a CAG trinucleotide-repeat expansion in the Huntington gene (IT15) on chromosome 4p16.3. One family with a history of HD was referred to us initially for predictive testing using linkage analysis. However, the chromosome 4p region was completely excluded by polymorphic markers, and later no CAG-repeat expansion in the HD gene was detected. To map the disease trait segregating in this family, whole-genome screening with highly polymorphic dinucleotide-, trinucleotide-, and tetranucleotide-repeat DNA markers was performed. A positive LOD score of 3.01 was obtained for the marker D20S482 on chromosome 20p, by two-point LOD-score analysis with the MLINK program. Haplotype analysis indicated that the gene responsible for the disease is likely located in a 2.7-cM region between the markers D20S193 and D20S895. Candidate genes from the mapping region were screened for mutations.     

Isolation and characterization of new highly polymorphic DNA markers from the Huntington disease region.

The defect causing Huntington disease (HD) has been mapped to 4p16.3, distal to the DNA marker D4S10. Subsequently, additional polymorphic markers closer to the HD gene have been isolated, which has led to the establishment of predictive testing programs for individuals at risk for HD. Approximately 17% of persons presenting to the Canadian collaborative study for predictive testing for HD have not received any modification of risk, in part because of limited informativeness of currently available DNA markers. Therefore, more highly polymorphic DNA markers are needed, which will further increase the accuracy and availability of predictive testing, specifically for families with complex or incomplete pedigree structures. In addition, new markers are urgently needed in order to refine the breakpoints in the few known recombinant HD chromosomes, which could allow a more accurate localization of the HD gene within 4p16.3 and, therefore, accelerate the cloning of the disease gene. In this study we present the identification and characterization of nine new polymorphic DNA markers, including three markers which detect highly informative multiallelic VNTR-like polymorphisms with PIC values of up to .84. These markers have been isolated from a cloned region of DNA which has been previously mapped approximately 1,000 kb from the 4p telomere.     

Mapping of D4S98/S114/S113 confines the Huntington''s defect to a reduced physical region at the telomere of chromosome 4.

The dominant gene defect in Huntington's disease (HD) is linked to the DNA marker D4S10, near the telomere of the chromosome 4 short arm. Two other markers, D4S43 and D4S95, are closer, but still proximal to the HD gene in 4p16.3. We have characterized a new locus, D4S114, identified by cloning the end of a NotI fragment resolved by pulsed-field gel electrophoresis. D4S114 was localized distal to D4S43 and D4S95 by both physical and genetic mapping techniques. The "end"-clone overlaps a previously isolated NotI "linking" clone, and is within 150 kb of a second "linking" clone defining D4S113. Restriction fragment length polymorphisms for D4S113 and D4S114, one of which is identical to a SacI polymorphism detected by the anonymous probe pBS731B-C (D4S98), were typed for key crossovers in HD and reference pedigrees. The data support the locus order D4S10-(D4S43, D4S95)-D4S98/S114/S113-HD-telomere. The D4S98/S114/S113 cluster therefore represents the nearest cloned sequences to HD, and provides a valuable new point for launching directional cloning strategies to isolate and characterize this disease gene.     

Susceptibility loci for preeclampsia on chromosomes 2p25 and 9p13 in Finnish families

Preeclampsia is a common, pregnancy-specific disorder characterized by reduced placental perfusion, endothelial dysfunction, elevated blood pressure, and proteinuria. The pathogenesis of this heterogeneous disorder is incompletely understood, but it has a familial component, which suggests that one or more common alleles may act as susceptibility genes. We hypothesized that, in a founder population, the genetic background of preeclampsia might also show reduced heterogeneity, and we have performed a genomewide scan in 15 multiplex families recruited predominantly in the Kainuu province in central eastern Finland. We found two loci that exceeded the threshold for significant linkage: chromosome 2p25, near marker D2S168 (nonparametric linkage [NPL] score 3.77; P=.000761) at 21.70 cM, and 9p13, near marker D9S169 (NPL score 3.74; P=.000821) at 38.90 cM. In addition, there was a locus showing suggestive linkage at chromosome 4q32 between D4S413 and D4S3046 (NPL score 3.13; P=.003238) at 163.00 cM. In the present study the susceptibility locus on chromosome 2p25 is clearly different (21.70 cM) from the locus at 2p12 found in an Icelandic study (94.05 cM) and the locus at 2q23 (144.7 cM) found in an Australian/New Zealand study. The locus at 9p13 has been shown to be a candidate region for type 2 diabetes in two recently published genomewide scans from Finland and China. The regions on chromosomes 2p25 and 9p13 may harbor susceptibility genes for preeclampsia.     

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase beta-subunit gene in mouse and human: tight linkage to the Huntington disease region (4p16.3).

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE beta-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 +/- 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.     

Fine mapping of the congenital chloride diarrhea gene by linkage disequilibrium.

Congenital chloride diarrhea is a recessively inherited intestinal disorder affecting electrolyte transportation. The clinical presentation is a life-threatening watery diarrhea with a high chloride content. Recently, the congenital chloride diarrhea gene (CLD) was assigned to chromosome 7 by linkage in eight Finnish families. In the present study, refined mapping of CLD was performed by studying linkage and linkage disequilibrium in 24 Finnish and 4 Swedish families. Recombination mapping assigned CLD to an approximately 10-cM region flanked by D7S515 and D7S799. Linkage disequilibrium was detected over this large genetic region, with the strongest allelic association at D7S496. Application of the Luria and Delbrück-derived analysis allowed for a further narrowing of the CLD region to approximately 0.37 cM from the marker D7S496. Haplotype analysis placed CLD unequivocally between D7S501 and D7S692, very close to D7S496 and most likely on the distal side of D7S496. This combined analytical approach allowed highly accurate mapping of CLD, each component adding complementary and consistent mapping information.     

Fine mapping and SNP analysis of positional candidates at the preeclampsia susceptibility locus (PREG1) on chromosome 2

Genome scans in Icelandic, Australian and New Zealand, and Finnish families have localized putative susceptibility loci for preeclampsia/ eclampsia to chromosome 2. The locus mapped in the Australian and New Zealand study (designated PREG1) was thought to be the same locus as that identified in the Icelandic study. In both these studies, two distinct quantitative trait locus (QTL) regions were evident on chromosome 2. Here, we describe our fine mapping of the PREG1 locus and a genetic analysis of two positional candidate genes. Twenty-five additional microsatellite markers were genotyped within the 74-cM linkage region defined by the combined Icelandic and Australian and New Zealand genome scans. The overall position and shape of the localization evidence obtained using nonparametric multipoint analysis did not change from that seen previously in our 10-cM resolution genome scan; two peaks were displayed, one on chromosome 2p at marker D2S388 (107.46 cM) and the other on chromosome 2q at 151.5 cM at marker D2S2313. Using the robust two-point linkage analysis implemented in the Analyze program, all 25 markers gave positive LOD scores with significant evidence of linkage being seen at marker D2S2313 (151.5 cM), achieving a LOD score of 3.37 under a strict diagnostic model. Suggestive evidence of linkage was seen at marker D2S388 (107.46 cM) with a LOD score of 2.22 under the general diagnostic model. Two candidate genes beneath the peak on chromosome 2p were selected for further analysis using public single nucleotide polymorphisms (SNPs) within these genes. Maximum LOD scores were obtained for an SNP in TACR1 (LOD = 3.5) and for an SNP in TCF7L1 (LOD = 3.33), both achieving genome-wide significance. However, no evidence of association was seen with any of the markers tested. These data strongly support the presence of a susceptibility gene on chromosome 2p11-12 and substantiate the possibility of a second locus on chromosome 2q23.     

Mapping of recombinants near the Huntington disease locus by using G8 (D4S10) and newly isolated markers in the D4S10 region.

Genetic linkage between the marker G8 (D4S10) and Huntington disease (HD) was studied in six Dutch pedigrees. The informativeness of the D4S10 locus was increased by isolation of a cosmid, C5.5, with a G8 subclone used as probe. We present a restriction map of 70 kb in the D4S10 region. Two subclones of C5.5, H5.52 and F5.53, detect MspI and SinI RFLPs, respectively. These probes increase the informativeness of D4S10 in the Dutch HD population from 55% to 95%. Seven recombinations were found in 124 informative meioses in which multipoint segregation of D4S10 haplotypes and the HD locus was studied. Two of the recombinations occurred within the D4S10 region. The other five recombinations are highly valuable for the mapping of present and future markers relative to each other and to the HD gene. In addition, several recombinations between markers in meioses from unaffected parents were noted, which will also be useful in ordering new markers. On the basis of our three-point recombination data, the orientation of the D4S10 region relative to HD is HD-H5.52-G8-F5.53, which independently confirms the previously derived polarity for D4S10.     

Identification of multiple CpG islands and associated conserved sequences in a candidate region for the Huntington disease gene

The HD locus has been assigned to 4p16.3 distal to the DNA segment D4S10. However, the precise location of this gene is still unknown. At least three regions, together encompassing more than 3.5 Mb of DNA, can still be considered as candidate regions for the HD gene. Our efforts are directed toward the cloning and the complete characterization of one of these regions. Thus far we have cloned 460 kb of DNA in contiguously overlapping cosmids distal to D4S111 and have developed a detailed long-range restriction map orienting the contig within the terminal region of 4p16.3. We characterized 15 CpG-rich islands defined by tightly clustered rare cutter restriction sites for the enzymes NotI, BssHII, EagI, NruI, and SacII. In addition, we show that the sequences associated with the CpG-rich islands detect cross-species conservation. The detailed genetic analysis of the 460-kb contig provides a framework for the identification of genes, which can be assessed for the characteristics expected for the HD gene.     

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.     

Refining the localization of the PKD2 locus on chromosome 4q by linkage analysis in Spanish families with autosomal dominant polycystic kidney disease type 2.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder. At least two distinct forms of ADPKD are now well defined. In approximately 86% of affected European families, a gene defect localized to 16p13.3 was responsible for ADPKD, while a second locus has been recently localized to 4q13-q23 as candidate for the disease in the remaining families. We present confirmation of linkage to microsatellite markers on chromosome 4q in eight Spanish families with ADPKD, in which the disease was not linked to 16p13.3. By linkage analysis with marker D4S423, a maximum lod score of 9.03 at a recombination fraction of .00 was obtained. Multipoint linkage analysis, as well as a study of recombinant haplotypes, placed the PKD2 locus between D4S1542 and D4S1563, thereby defining a genetic interval of approximately 1 cM. The refined map will serve as a genetic framework for additional genetic and physical mapping of the region and will improve the accuracy of presymptomatic diagnosis of PKD2.