Identification of crossovers in Wilson disease families as reference points for a genetic localization of the gene

Summary Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. A minimum recombinant analysis using D13S22, ESD, RB1, D13S31, D13S55, D13S26, D13S39, and D13S12, all localized at 13q14-q22, has been carried out in 20WD families of Northwest-European origin. No inconsistencies have been observed with respect to locus order or location of the WD locus (WND) compared with previous linkage studies. D13S31 was mapped as the closest marker proximal to WND, whereas D13S55 and D13S26 were mapped as the closest markers distal to WND. We have identified a crossover between WND and D13S31 in one family and a crossover between WND and D13S55 in another. These crossover sites can be used as reference points for new chromosome 13q14-q21 markers, and are therefore important for a more accurate mapping of the WD locus.     

Mapping the Wilson disease locus to a cluster of linked polymorphic markers on chromosome 13.

Linkage of both several chromosome 13 DNA markers and the locus for the red cell enzyme esterase D (ESD) to Wilson disease (WD), an autosomal recessive disorder affecting copper metabolism, was investigated in five Middle-Eastern kindreds. The single-copy probe 7D2, identifying the polymorphic region D13S10, was demonstrated to lie 7.5 centiMorgans (cM) from the locus, since a maximum lod score of 4.66 at a recombination frequency of .07 (7.5 cM) was found between the locus for WD (WND) and D13S10. Multipoint linkage analysis between several chromosome 13 markers and WND enables us to propose that the order of markers closely linked to WND is as follows: centromere-D13S10-ESD-WND.     

Polymorphic microsatellites and Wilson disease (WD)

Wilson disease (WD), an autosomal recessive disorder of copper metabolism, has been previously mapped to chromosome 13q. Highly informative PCR-based polymorphic microsatellites closely linked to the WD locus (WND) at 13q14.3, as well as sequence-tagged sites for closely linked loci, are described. Two polymorphic microsatellite markers at D13S118 and D13S119 lie within 3 cM of WND. Two others (D13S227 and D13S228) were derived from a yeast artificial chromosome containing D13S31. These were placed on a genetic linkage map of chromosome 13 and were typed in 74 multiplex WD families from a variety of geographic origins (166 affected members). Multipoint analysis provides very high odds that the location of WND is between D13S31/D13S227/D13S228 and D13S59. Previous odds with RFLP-based markers were only 7:1 more likely than any other location. Current odds are 5,000:1. Preclinical testing of three cases of WD by using the highly informative polymorphic microsatellite markers is described. The markers described here ensure that 95% of predictive tests using DNA from both parents and from at least one affected sib will have an accuracy >99%.     

Close linkage of the Wilsons's disease locus to D13S12 in the chromosomal region 13q21 and not to ESD in 13q14

Summary Wilson's disease (WD) is an autosomal recessive disorder resulting in copper accumulation notably in liver and brain tissue. Linkage of the WD locus (WND) to ESD at 13q14 was first shown by studies in families of Middle Eastern origin using the isozymic polymorphism of esterase D. Using RFLPs detected by the ESD cDNA we could not confirm this reported close linkage in an analysis of 17 WD families of northwest European origin. A tight linkage was detected, however, to the marker D13S12, located more distally at 13q21. No obligate cross-overs were detected in 63 gametes informative for this marker. Our data confirm an assignment of WND to 13q14-21. Its localization, however, seems to be more distal to ESD than previously reported. Although genetic heterogeneity cannot be excluded, the observed differences between the two populations are probably due to random variation.     

Esterase D and retinoblastoma gene loci are tightly linked to Wilson's disease in Chinese pedigrees from Taiwan

Summary Wilson's disease (WD) is a rare autosomal recessive disorder and has been mapped to the long arm of chromosome 13 (q14.1). We have analyzed the segregation of esterase D (ESD) and retinoblastoma (RB) gene loci in ten families of Chinese WD subjects living in Taiwan. The polymorphic information content (PIC) for ESD and RB was 0.18 and 0.31, respectively. We confirmed a tight linkage between these loci and WD with a lod score of 3.33 by multipoint linkage analysis. The data from this limited number of pedigrees also suggested the following order: centromere-WD-RB-ESD or centromere-ESD-RB-WD. ESD in conjunction with RB polymorphism would be useful in prenatal and presymptomatic diagnosis, as well as in carrier detection in informative pedigrees.     

Haplotype studies in Wilson disease.

In 51 families with Wilson disease, we have studied DNA haplotypes of dinucleotide repeat polymorphisms (CA repeats) in the 13q14.3 region, to examine these markers for association with the Wilson disease gene (WND). In addition to a marker (D13S133) described elsewhere, we have developed three new highly polymorphic markers (D13S314, D13S315, and D13S316) close to the WND locus. We have examined the distribution of marker alleles at the loci studied and have found that D13S314, D13S133, and D13S316 each show nonrandom distribution on chromosomes carrying the WND mutation. We have studied haplotypes of these three markers and have found that there are highly significant differences between WND and normal haplotypes in northern European families. These findings have important implications for mutation detection and molecular diagnosis in families with Wilson disease.     

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.     

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.     

A centromere-based genetic map of the short arm of human chromosome 6

A genetic map of the short arm of chromosomes 6 (6p) has been constructed with 20 genetic markers that define 16 loci, including a locus at the centromere. The 40 CEPH families and, for 4 loci, 13 additional Utah families were genotyped. All 16 loci form a single linkage group extending from near the telomeric region to the centromere, covering 159 cM (Haldane) on the female map and 94 cM on the male map. Sex differences in recombination frequencies are noted for the 6p map, with an excess occurring in males at the distal end. The genetic order of loci is consistent with their physical localization on 6p. Proximal to the three most distal loci on the map, markers are especially dense, providing an extended region on 6p useful for localizing genes of interest.     

Autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly) is linked to chromosome 22q12-13

Macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly) is a rare autosomal dominant disorder characterized by thrombocytopenia, giant platelets, and D?hle body-like inclusions in leukocytes. To determine the genetic basis of this disorder, we performed a genome-wide screen for linkage in three families with May-Hegglin anomaly. For the pooled analysis of the three families, three markers on chromosome 22 had two-point logarithm-of-difference (lod) scores greater than 3, with a maximum lod score of 3.91 at a recombination fraction (theta) of 0.076 for marker D22S683. Within the largest family (MHA-1), the maximum lod score was 5.36 at theta=0 at marker D22S445. Fine mapping of recombination events using eight adjacent markers indicated that the minimal disease region of family MHA-1 alone is in the approximately 26 cM region from D22S683 to the telomere. The maximum lod score for the three families combined was 5.84 at theta=0 for marker IL2RB. With the assumption of locus homogeneity, haplotype analysis of family MHA-4 indicated the disease region is centromeric to marker D22S1045. These data best support a minimal disease region from D22S683 to D22S1045, a span of about 1 Mb of DNA that contains 17 known genes and 4 predicted genes. Further analysis of this region will identify the genetic basis of May-Hegglin anomaly, facilitating subsequent characterization of the biochemical role of the disease gene in platelet formation.     

Closely linked loci on the long arm of chromosome 13 flank a specific 2;13 translocation breakpoint in childhood rhabdomyosarcoma

A specific chromosomal translocation, t(2;13)(q35;q14), is present in tumor cells from about one-half of children with alveolar rhabdomyosarcoma, who generally have widely disseminated disease at diagnosis. Using a series of six DNA probes from five loci previously assigned to bands 13q12----q14, we have localized the translocation breakpoint on chromosome 13 by in situ hybridization. Each probe was used to examine metaphase spreads from two or more rhabdomyosarcoma cell lines that have the t(2;13), as well as from control lymphoblastoid cell metaphases. All six probes bound to chromosome 13q12----q14 in the control cell line, but showed no appreciable hybridization to other sites. With rhabdomyosarcoma metaphases, cDNA clones of the retinoblastoma susceptibility gene (RB1) and the esterase D gene (ESD), as well as the arbitrary genomic fragment 7D2 (D13S10), showed specific hybridization to the normal chromosome 13 and the der(2) marker, but not to the der(13). By contrast, the genomic fragments HU10 (D13S6) and 7F12 (D13S1) hybridized specifically to the normal chromosome 13 and the der(13), but not to the der(2). Thus, the breakpoint of this translocation lies distal to D13S6 and D13S1 and proximal to ESD, RB1, and D13S10. Our data indicate that the locus affected by the translocation breakpoint on chromosome 13, which we have termed RMS, is physically distinct from the RB1 locus and is, in fact, proximal to ESD, which others have placed at least 10(6) bp proximal to RB1. The consistent presence of the der(2) marker chromosome, coupled with occasional loss of the der(13), suggests that the RMS gene, or at least a critical component, moves to chromosome 2 in tumors with this translocation.     

Polymorphic markers for the arylsulfatase A gene reveal a greatly expanded meiotic map for the human 22q telomeric region

Two microsatellite markers, D22S1743 and D22S1744, were developed for the arylsulfatase A (ARSA) region of chromosome 22q. Linkage analysis for 171 families, using nine reference markers covering all of 22q, placed these new markers 2.0 Kosambi cM distal to D22S526, making them more distal than any microsatellite markers currently on the Généthon or Marshfield linkage maps. Recombination between proximal markers D22S270/D22S683 and D22S446/D22S311 exhibited increased rates of female meiotic recombination compared to male recombination (P < 0.01). In contrast, the region encompassing sJCW16, D22S526, D22S1743, and D22S1744 exhibited relatively greater recombination in males (1.1 cM for females and 7.5 cM for males; chi(2); P < 0.005). These four distal markers lie in a region of hyperrecombination having a sex-averaged recombination ratio of between 8.3 (D22S1843/D22S1744) and 12 cM (sJCW16/D22S526) per megabase.     

The Friedreich ataxia gene is assigned to chromosome 9q13-q21 by mapping of tightly linked markers and shows linkage disequilibrium with D9S15.

Chamberlain et al. have assigned the gene for Friedreich ataxia (FA), a recessive neurodegenerative disorder, to chromosome 9, and have proposed a regional localization in the proximal short arm (9p22-cen), on the basis of linkage to D9S15 and to interferon-beta (IFNB), the latter being localized in 9p22. We confirmed more recently the close linkage to D9S15 in another set of families but found much looser linkage to IFNB. We also reported another closely linked marker, D9S5. Additional families have now been studied, and our updated lod scores are z = 14.30 at theta = .00 for D9S15-FA linkage and z = 6.30 at theta = .00 for D9S5-FA linkage. Together with the recent data of Chamberlain et al., this shows that D9S15 is very likely within 1 cM of the FA locus. We have found very significant linkage disequilibrium (delta Std = .28, chi 2 = 9.71, P less than .01) between FA and the D9S15 MspI RFLP in French families, which further supports the very close proximity of these two loci. No recombination between D9S5 and D9S15 was found in the FA families or Centre d'Etude du Polymorphisme Humain families (z = 9.30 at theta = .00). Thus D9S5, D9S15, and FA define a cluster of tightly linked loci. We have mapped D9S5 by in situ hybridization to 9q13-q21, and, accordingly, we assign the D9S5, D9S15, and FA cluster to the proximal part of chromosome 9 long arm, close to the heterochromatic region.     

Evidence for the close linkage between lymphocyte cytosol polypeptide with molecular weight of 64,000 (LCP1) and esterase D.

The genetic linkage of the loci for lymphocyte cytosol polypeptide with molecular weight of 64,000 (LCP1) and esterase (ESD) were examined by two-dimensional gel electrophoresis using blood from four informative families. No recombinants were observed in the four families totaling 17 children, giving a summed lod score of 4.221 at recombination fraction 0. This result indicates that the gene for LCP1 is closely linked to the ESD locus, which is assigned to the chromosomal region 13q14.11. This finding also suggests that the LCP1 locus is linked to the retinoblastoma gene, which is assigned to the same chromosomal region and closely linked to the ESD locus.     

Clinical, cytogenetic, and molecular characterization of seven patients with deletions of chromosome 22q13.3.

We have studied seven patients who have chromosome 22q13.3 deletions as revealed by high-resolution cytogenetic analysis. Clinical evaluation of the patients revealed a common phenotype that includes generalized developmental delay, normal or accelerated growth, hypotonia, severe delays in expressive speech, and mild facial dysmorphic features. Dosage analysis using a series of genetically mapped probes showed that the proximal breakpoints of the deletions varied over approximately 13.8 cM, between loci D22S92 and D22S94. The most distally mapped locus, arylsulfatase A (ARSA), was deleted in all seven patients. Therefore, the smallest region of overlap (critical region) extends between locus D22S94 and a region distal to ARSA, a distance of > 25.5 cM.     

A contiguous linkage map of chromosome 13q with 39 distinct loci separated on average by 5.1 centimorgans.

A fine-structure linkage map of chromosome 13q is presented. This map contains 39 continuously linked loci defined by genotypes generated from the CEPH family DNAs with 56 probe and enzyme combinations. An alpha-satellite probe for sequences on chromosome 13 was included, resulting in a complete map of 13q with 39 distinct loci. The map spans 1.715 M in males and 2.099 M in females and the mean genetic distance between adjacent loci is 5.1 cM. Although there was generally a several-fold excess of female recombination in the interstitial portion of 13q, an excess of recombination in males was observed at both ends of this chromosomal arm. This map should be useful for the localization of any additional marker, gene, or disease locus of interest on chromosome 13q.     

Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization

We have recently assigned the facioscapulohumeral muscular dystrophy (FSHD) gene to chromome 4 by linkage to the microsatellite marker Mfd 22 (locus D4S171). We now report that D4S139, a VNTR locus, is much more closely linked to FSHD. Two-point linkage analysis between FSHD and D4S139 in nine informative families showed a maximum combined lod score (Zmax) of 17.28 at a recombination fraction theta of 0.027. Multipoint linkage analysis between FSHD and the loci D4S139 and D4S171 resulted in a peak lod score of 20.21 at 2.7 cM from D4S139. Due to the small number of recombinants found with D4S139, the position of the FSHD gene relative to that of D4S139 could not be established with certainty. D4S139 was mapped to chromosome 4q35-qter by in situ hybridization, thus firmly establishing the location of the FSHD gene in the subtelomeric region of chromosome 4q. One small family yielded a negative lod score for D4S139. In the other families no significant evidence for genetic heterogeneity was obtained. Studies of additional markers and new families will improve the map of the FSHD region, reveal possible genetic heterogeneity, and allow better diagnostic reliability.     

A genetic linkage map of 96 loci on the short arm of human chromosome 3.

We constructed a genetic map of 96 loci on the short arm of human chromosome 3 (3p) in 59 families provided by the Centre d'Etude du Polymorphisme Humaine (CEPH). Twenty-nine continuously linked loci were placed on the map with likelihood support of at least 1000:1; one locus, D3S213, was placed on the map with likelihood support of 871:1; D3Z1, an alpha satellite centromeric repeat probe, was placed on the map with likelihood support of 159:1; 65 loci were assigned regional locations. The average heterozygosity of the uniquely ordered markers was 49%. The map extends from 3p26, the terminal band of 3p, to the centromere (from D3S211 to D3Z1). Multipoint linkage analysis indicated that the male, female, and sex-averaged maps extend for 102, 147, and 116 cM, respectively. The mean genetic distance between uniquely ordered loci on the sex-averaged map was 4.0 cM. Probe density was greatest for the region of 3p between D3F15S2e and the telomere. The sex-averaged map contained two intervals greater than 10 cM. Seventeen probes were localized by fluorescence in situ hybridization. The loci described in this report will be useful in building an integrated genetic and physical map of this chromosome.     

An AFLP linkage map for Douglas-fir based upon multiple full-sib families

An amplified fragment length polymorphism (AFLP) linkage map for coastal Douglas-fir (Pseudotsuga menziesii) was constructed from eight full-sib families each consisting of 40 progeny. These families were part of the British Columbia Ministry of Forests second-generation progeny test program and represent typical family sizes used in progeny trials. For map construction, ten primer pairs using EcoRI+3 and MseI+4 were employed to identify and assay AFLP loci that segregated in backcross configurations. A new technique was used to obtain a single recombination rate for each pair of marker loci: for each locus pair, a recombination rate and log-odd value were estimated across all segregating families using a joint maximum likelihood function that considered the full dataset of segregating genotypes. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 19 linkage groups spanning 938.6 cM at an average distance of 9.3 cM between markers. The simultaneous integration of data from multiple families may provide an effective way to construct a linkage map, using the genetic resources inherent in most tree improvement programs, where progeny tests of small size are conducted. The statistical property of number of families used is briefly discussed. For our data, at least three to four families greatly increased the chance of obtaining an informative locus in at least one family. Families as small as ten are adequate for closely linked loci (<10 cM), while the size used in our study (40) is adequate for loci within 30 cM.     

Homozygosity mapping of a Weill-Marchesani syndrome locus to chromosome 19p13.3-p13.2

Weill-Marchesani syndrome (WMS) is a rare disease characterized by short stature, brachydactyly, joint stiffness, and characteristic eye abnormalities, including microspherophakia, ectopia lentis, and glaucoma. Both autosomal recessive and autosomal dominant modes of inheritance have been described in association with WMS. We have performed a genome-wide search in two large consanguineous families of Lebanese and Saudian origin consistent with an autosomal recessive mode of inheritance. Here, we report the linkage of the disease gene to chromosome 19p13.3-p13.2 (Zmax=5.99 at theta=0 at locus D19S906). A recombination event between loci D19S905 and D19S901 defines the distal boundary, and a second recombination event between loci D19S221 and D19S840 defines the proximal boundary of the genetic interval encompassing the WMS gene (12.4 cM). We hope that our ongoing studies will lead to the identification of the disease-causing gene.