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.     

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.     

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 theta 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.     

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.     

Sequence-tagged sites (STSs) spanning 4p16.3 and the Huntington disease candidate region.

The generation of sequence-tagged sites (STSs) has been proposed as a unifying approach to correlating the disparate results generated by genetic and various physical techniques being used to map the human genome. We have developed an STS map to complement the existing physical and genetic maps of 4p16.3, the region containing the Huntington disease gene. A total of 18 STSs span over 4 Mb of 4p16.3, with an average spacing of about 250 kb. Eleven of the STSs are located within the primary candidate HD region of 2.5 Mb between D4S126 and D4S168. The availability of STSs makes the corresponding loci accessibility to the general community without the need for distribution of cloned DNA. These STSs should also provide the means to isolate yeast artificial chromosome clones spanning the HD candidate region.     

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.     

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 linkage group of five DNA markers on human chromosome 10

Five chromosome 10 DNA markers (D10S1, D10S3, D10S4, D10S5, and RBP3) were typed in five large pedigrees with multiple endocrine neoplasia type 2A (MEN-2A) and in five non-MEN-2A pedigrees. Linkage analyses showed that these loci and the locus for MEN-2A (MEN2A) are in one linkage group spanning at least 70 cM. The order of the marker loci is RBP3-D10S5-D10S3-D10S1-D10S4, with interlocus recombination frequencies of 7, 13-19, 19, and 19%, respectively, all on the same side of MEN2A. Analyses of sex-specific recombination frequencies indicated no significant differences between males and females for any of the map intervals studied. Previous localization of D10S5 and RBP3 to the proximal region of the long arm and the pericentric region, respectively, comparison of results with other studies, and our preliminary results with other chromosome 10 markers suggest that the D10S4 end of the map extends into the long arm. Our linkage map has been constructed using only two- and three-locus analyses. It will be possible to combine our results with those of other groups to construct a more detailed and accurate genetic map of chromosome 10.     

Eight closely linked loci place the Wilson disease locus within 13q14-q21

Wilson disease (WD) is an autosomal recessive disorder resulting in an accumulation of copper in the liver, brain, and other organs. The WD locus (WND) has previously been linked to esterase D (ESD) and localized to 13q14-22. With the large Centre d'Etude Polymorphisme Humain cohort, a refined map of DNA markers from this region was constructed, with the following locus order: D13S1-D13S21-D13S22-D13S10-ESD-RB-WND-D 13S26-D13S12-D13S2. A significant excess of male recombination was observed between D13S21 and D13S22. Intervals distal to D13S22 showed an excess of female recombination. When these markers were tested on 19 WD families from a variety of ethnic backgrounds, the two closest loci were shown to be RB and D13S26. The retinoblastoma gene locus (RB) was shown to be proximal to WND at a distance of 4.4 centimorgans (cM), and D13S26 was placed distal to WND at a distance of 4.0 cM. ESD was assigned proximally at a distance of 9.4 cM. In all families studied WND was linked to one or more of the loci ESD, RB, or D13S26.     

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.     

D19S51 is closely linked with and maps distal to the myotonic dystrophy locus on 19q.

Recent genetic linkage studies have mapped the myotonic dystrophy (DM) locus to 19q13.3. All closely linked DM markers identified to date have been located on the centromeric side of the disease locus, with a relatively large genetic interval (9 cM) observed between the nearest distal marker and DM. We show here that the recently described marker p134C is tightly linked to DM (peak lod score 35.8 at peak recombination fraction .006) and confirm the previous suggestion that the p134C locus, D19S51 maps distal to the disease locus. D19S51 and the closest proximal flanking loci, ERCC1 and D19S115 (pE0.8), define a small genetic interval of less than 2 cM that contains the DM locus.     

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.     

Genetic mapping of the dentinogenesis imperfecta type II locus.

Dentinogenesis imperfecta type II (DGI-II) is an autosomal dominant disorder of dentin formation, which has previously been mapped to chromosome 4q12-21. In the current study, six novel short tandem-repeat polymorphisms (STRPs) have been isolated, five of which show significant evidence of linkage to DGI-II. To determine the order of the STRPs and define the genetic distance between them, nine loci (including polymorphisms for two known genes) were mapped through the CEPH reference pedigrees. The resulting genetic map encompasses 16.3 cM on the sex-averaged map. To combine this map with a physical map of the region, all of the STRPs were mapped through a somatic cell hybrid panel. The most likely location for the DGI-II locus within the fixed marker map is in the D4S2691-D4S2692 interval of 6.6 cM. The presence of a marker that shows no recombination with the DGI-II phenotype between the flanking markers provides an important anchor point for the creation of physical continuity across the DGI-II candidate region.     

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.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Physical mapping of probes within 14q32, a subtelomeric region showing a high recombination frequency

The genetic linkage map of chromosome 14q32 contains 11 loci which span a distance of more than 60 cM. We have assigned 10 of these loci and the AKT1 proto-oncogene to segments of 14q32, using breakpoints derived from four independent chromosomal deletions or rearrangements. The most telomeric breakpoint was found in a proband (HSC 6) carrying a ring-14 chromosome. HSC 6 is monosomic for the distal part of 14q32, which contains the immunoglobulin heavy-chain locus (IGH), and random markers D14S20, D14S19, and D14S23. Two other chromosomal breakpoints, found in probands HSC 121 and HSC 981, could not be distinguished from each other using DNA probes, although the cytogenetic breakpoints appeared to be different at 14q32.32 and 14q32.31, respectively. The region between the breakpoints of HSC 6 and HSC 121 contains AKT1, D14S1, D14S17, and D14S16. The entire telomeric band 14q32 is assumed to contain about 10% of chromosome 14, or approximately 10 Mb. The 8 most telomeric loci, including D14S1, map to 14q32.32-qter, which measures only several megabases. However, these loci span a genetic distance of 23 cM. The high recombination frequency contrasts with the observation that two of the gamma genes in the IGH constant region show a high degree of linkage disequilibrium, though 180 kb apart. This finding suggests that a telomeric localization per se does not lead to a higher recombination frequency and favors the hypothesis that the higher recombination frequency at the telomeres may be due to specific "hot spots" for recombination.     

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.     

A gene for pyridoxine-dependent epilepsy maps to chromosome 5q31

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder characterized by generalized seizures in the first hours of life and responding only to pyridoxine hydrochloride. The pathogenesis of PDE is unknown, but an alteration in the binding of pyridoxal 5-phosphate to glutamic acid decarboxylase (GAD) has been postulated in patients with PDE. Results are reported for genetic linkage analyses in four families with consanguineous parents and in one family with nonconsanguineous parents. The GAD1 (2q31) and GAD2 genes (10p23) were tested and excluded. A genomewide search was subsequently performed, using microsatellite markers at an average distance of 10 cM, and the search revealed linkage of the disease-causing gene to markers on chromosome 5q31.2-q31.3 (maximum LOD score [Z(max)] 8.43 at recombination fraction [theta] 0 and Zmax=7.58 at straight theta=0 at loci D5S2017 and D5S1972, respectively). A recombination event, between loci D5S638 and D5S463, in one family defined the distal boundary, and a second recombination event between loci D5S2011 and D5S2017 in another family defined the proximal boundary of the genetic interval encompassing the PDE gene (5.1 cM). Ongoing studies may lead to the identification of the disease-causing gene.     

Regional localization of the selenocysteine tRNA gene (TRSP) on human chromosome 19.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.