Physical and genetic mapping of polymorphic loci in Xq28 (DXS15, DXS52, and DXS134): analysis of a cosmid clone and a yeast artificial chromosome.

Sequences corresponding to the Xq28 loci DXS15, DXS52, DXS134, and DXS130 were shown to be present in a 140-kb yeast artificial chromosome (YAC XY58, isolated by Little et al.). This YAC clone appears to contain a faithful copy of this genomic region, as shown by comparison with human DNA and with a cosmid clone that contains probes St14c (part of the DXS52 sequences) and cpX67 (DXS134). cpX67 and St14c are contained in 11 kb and detect the same MspI RFLP polymorphism. A comparison of the YAC restriction map and pulsed-field gel electrophoresis data leads us to propose the following order of loci: DXS52(VNTR)-DXS33-DXF22S3-DXS130-DXS134 -DXS52-DXS15-DXS52, this whole cluster being comprised within 575 kb. The physical proximity of the DXS15, DXS52, and DXS134 loci led us to reinvestigate recombination events that had been reported between these loci in families from the Centre d'Etude du Polymorphisme Humain. Our results do not support the assumption that this region shows increased recombination.     

Genetic mapping of four dinucleotide repeat loci, DXS453, DXS458, DXS454, and DXS424, on the X chromosome using multiplex polymerase chain reaction.

Dinucleotide CA repeat sequences in the human genome have been shown to be highly polymorphic due to variation in the length of the repeat-containing segment. Therefore, these markers can serve as anchor loci in the construction of a high-resolution genetic map of the human genome. In this study, we improved the efficiency of typing dinucleotide repeats using multiplex polymerase chain reaction (PCR). Dinucleotide repeat sequences of four previously identified markers (DXS453, DXS458, DXS454, and DXS424) on the long arm of the X chromosome were simultaneously amplified in a single PCR reaction. This multiplex PCR was applied to genotype individuals from the 40 CEPH reference families, and the genotypic data were used to determine the map position of the four loci with respect to eight reference markers in the Xq region by linkage analysis.     

Genetic mapping of two loci,DXS454 and DXS458, with respect to the X-linked agammaglobulinemia gene locus

The dinucleotide repeat sequences at the DXS454 and DXS458 loci have been mapped genetically to Xq22, to the interval between DXS3 and DXS17. We have now mapped them with respect to XLA and five other loci, to within the DXS3 to XLA interval. The more precise localisation of these polymorphic loci will be useful for the fine-mapping of disease loci on the long arm of the X chromosome and enable these probes to be used for prenatal diagnosis and carrier status determination in families with XLA.     

YAC contigs mapping the human COL4A5 and COL4A6 genes and DXS118 within Xq21.3–q22

Sequence-tagged sites (STSs) were developed for three loci of uncertain X chromosomal localization (DXS122, DXS137, and DXS174) and were used to seed YAC contigs. Two contigs now total about 3.3 Mb formatted with 34 STSs. One contains DXS122 and DXS174 within 250 kb on single YACs; it is placed in Xq21.3–q22.1 by FISH analysis, which is consistent with somatic cell hybrid panel analyses and with the inclusion of a probe that detects polymorphism at the DXS118 locus already assigned to that general region. The other contig, which contains DXS137, is in Xq22.2 by FISH, consistent with cell hybrid analyses and with the finding that it covers the human COL4A5 and COL4A6 genes known to be in that vicinity. In addition to extending the cloned coverage of this portion of the X chromosome, these materials should aid, for example, in the further analysis of Alport syndrome.     

Localization of the highly polymorphic microsatellite DXS456 on the genetic linkage map of the human X chromosome

The CA repeat microsatellite DXS456, with a heterozygosity of 77%, has been localized by multipoint linkage analysis in relation to 20 other genetic markers. DXS456 mapped to a 4.2-cM interval defined by the flanking markers DXS178 and DXS287. The maximum likelihood order of markers, cen-(DXYS1X/DXYS13X/DXYS2X/DXYS12X)-DXS366 -DXS178-DXS456-DXS287-DXS358-DXS267- qter, is favored by odds greater than 1000:1 over the subset of most likely alternative orders. Linkage of DXS456 can be inferred for at least six disease genes that are known to be linked to markers in the region Xq21.31-Xq25 and the marker will serve as an important index point for orienting these and other disease and marker loci in the region.     

YAC clone contigs covering 5 Mb of a repeat sequence island on the mouse X Chromosome

We have initiated work towards the construction of YAC clone contigs across a repeat sequence island region on the mouse X Chromosome (Chr). The repeat sequence island region—the 141 island—located at band A3 contains 50 copies of a localized long complex repeat unit (LCRU). We have isolated 87 YAC clones from the 141 island and have used a dual faceted approach towards the construction of contigs across the repeat sequence island. First, we have identified YAC clones originating from the same region of the island by the identification of commonly held LCRU restriction site variants. Second, we have constructed rare cutter restriction maps of each YAC clone. Taken together, we have been able to assemble one large contig of 2.8 Mb and a number of smaller contigs. In total, contigs covering 5Mb of the island region have been identified. The island region would appear to represent a major component of the A3 Giemsa dark band on the mouse X Chr. Received: 1 September 1995 / Accepted: 11 December 1995     

Genetic and physical mapping around the properdin P gene.

A CA repeat has been found on the human X chromosome within 16 kb of the gene encoding properdin P factor (PFC) and has been shown to be a highly informative marker. Two more polymorphic CA repeats were found in a cosmid containing DXS228. The CA repeats, and other markers from proximal Xp, were mapped genetically in CEPH families and the likely order of markers was established as Xpter-(DXS7, MAO-A, DXS228)-(PFC, DXS426)-(TIMP, OATL1)-DXS255-Xcen. This places PFC in the region Xp11.3-Xp11.23, thus refining previous in situ hybridization data. Two yeast artificial chromosomes (YACs) (440 and 390 kb) contain both PFC and DXS426, and one of them (440 kb) also contains TIMP. This confirms the genetic order TIMP-(PFC, DXS426). PFC and TIMP are located on the same 100-kb SalI/PvuI fragment of the 440-kb YAC. Given the genetic orientation of TIMP and (PFC, DXS426), this YAC can now serve as a starting point for directional walking toward disease genes located in Xp11.3-Xp11.2 such as retinitis pigmentosa (RP2) and Wiskott-Aldrich syndrome.     

Dinucleotide polymorphism at the DXS1178 locus is tightly linked to PGK1 at Xq13

A polymorphic CA repeat (locus name DXS1178) was isolated from a 1-megabase YAC (OTCC) containing the OTC gene, located at Xp21.1. However, amplification in human-rodent hybrid cells and segregation analysis in three CEPH families mapped the DXS1178 locus at Xq13. The mapping ambiguity is apparently caused by the chimeric nature of the OTCC YAC clone.     

Mapping of locus for X-linked congenital stationary night blindness (CSNB1) proximal to DXS7.

A recombinant chromosome in a male affected with X-linked congenital stationary night blindness (CSNB1) provides new information on the location of the CSNB1 locus. A four-generation family with five males affected with X-linked CSNB was analyzed with five polymorphic markers for four X-chromosome loci spanning the region OTC (Xp21.1) to DXS255 (Xp11.22). Four of the males inherited the same X chromosome; one male inherited a chromosome that from OTC to DXS7, inclusive, was derived from the normal X chromosome of his unaffected grandfather and that from a location between DXS7 and DXS426 proximally was derived from the chromosome carrying the CSNB1 locus. This recombinant maps the CSNB1 locus in this family to a region on the short arm of the X chromosome proximal to the DXS7 locus.     

Assignment of the dystonia-parkinsonism syndrome locus, DYT3, to a small region within a 1.8-Mb YAC contig of Xq13.1.

A YAC contig was constructed of Xq13.1 in order to sublocalize the X-linked dystonia-parkinsonism (XDP) syndrome locus, DYT3. The contig spans a region of approximately 1.8 Mb and includes loci DXS453/DXS348/IL2R gamma/GJB1/CCG1/DXS559. For the construction of the contig, nine sequence-tagged sites and four short tandem repeat polymorphisms (STRPs) were isolated. The STRPs, designated as 4704#6 (DXS7113), 4704#7 (DXS7114), 67601 (DXS7117), and B4Pst (DXS7119) were assigned to a region flanked by DXS348 proximally and by DXS559 distally. Their order was DXS348/4704 #6/4704 #7/67601/B4Pst/DXS559. They were applied to the analysis of allelic association and of haplotypes in 47 not-obviously-related XDP patients and in 105 Filipino male controls. The same haplotype was found at loci 67601 (DXS7117) and B4Pst (DXS7119) in 42 of 47 patients. This percentage of common haplotypes decreased at the adjacent loci. The findings, together with the previous demonstration of DXS559 being the distal flanking marker of DYT3, assign the disease locus to a small region in Xq13.1 defined by loci 67601 (DXS7117) and B4Pst (DXS7119). The location of DYT3 was born out by the application of a newly developed likelihood method for the analysis of linkage disequilibrium.     

Anderson Fabry disease,a close linkage with highly polymorphic DNA markers DXS17, DXS87 and DXS88

Summary Anderson Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A deficiency. Hemizygous males and some heterozygous females develop renal failure and cardiovacular complications in early adult life. We have investigated six large UK families to assess the possible linkage of five polymorphic DNA probes to the Anderson Fabry locus, previously localised to Xq21-24. No recombination was found between Anderson Fabry disease and DXS87, DXS88 and DXS17, which gave lod_max=6.4,6.4 and 5.8 respectively at θ=0.00, (upper confidence limit 0.10). DXS3 gave lod_max 2.9 at θ=0.10 (upper confidence limit 0.25). DXYS1 was excluded from linkage. The best fit map (DXYS1/DXS3) θ=0.192 (DXS17/DXS87/DXS88/Anderson Fabry locus) provided no information about the order of loci in parentheses due to the absence of recombinants. The close linkage of DXS17, DXS87 and DXS88, together with α-galactosidade A estimation, can be used for antenatal diagnosis and carrier detection until the application of a gene specific probe has been evaluated.     

Physical mapping of DXS134 close to the DXS52 locus

Summary The locus DXS134 (cpX67) has been physically linked to the cluster of polymorphic loci DXS52, DXS15, and DXS33. A comparison of physical and genetic distance indicates a high rate of recombination in this region.     

Definitive localization of Becker muscular dystrophy in Xp by linkage to a cluster of DNA polymorphisms (DXS43 and DXS9)

Summary A study of linkage between Becker muscular dystrophy and four X chromosome-specific DNA polymorphisms in 17 kindreds has indicated that this gene is located in Xp, as already anticipated by single pedigree analysis. In particular the DXS43 and DXS9 loci, identified by probes D2 and RC8, respectively, are closely linked to each other and are both located at approximately 15 cM from the Becker locus. These linkage data, together with the previously established linkage between Becker and the DXS7 locus identified by probe L 1.28, indicate that the Becker gene is located in the same region where Duchenne has been mapped and also yield information about relative genetic distances among different DNA polymorphisms of the X chromosome.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Linkage analysis of two cloned DNA sequences, DXS197 and DXS207, in hypophosphatemic rickets families

The human X-linked hypophosphatemic rickets gene locus (HYP, formerly HPDR) has been previously localized by linkage analysis to Xp22.31-Xp21.3 and the locus order Xpter-DXS43-HYP-DXS41-Xcen established. Recombination between HYP and these flanking markers is frequently observed and additional markers have been sought. The polymorphic loci DXS197 and DXS207 have been localized to Xpter-Xp11 and Xp22-Xp21, respectively. We have further localized DXS197 to Xpter-Xp21.3 by using a panel of rodent-human hybrid cells and have established the map positions of DXS197 and DXS207 in relation to HYP by linkage studies of hypophosphatemic rickets families. Linkage between DXS197 and the loci DXS43, DXS85, and DXS207 was established with peak lod score values of 6.19, 0 = 0.032; 4.14, 0 = 0.000; and 3.01, 0 = 0.000, respectively. Multilocus linkage analysis mapped the DXS197 and DXS207 loci distal to HYP and demonstrated the locus order Xpter-DXS85-(DXS207, DXS43, DXS197)-HYP-DXS41-Xcen. These additional genetic markers DXS197 and DXS207 will be useful as alternative markers in the genetic counseling of some families.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Genetic mapping of four DNA markers (DXS16, DXS43, DXS85, and DXS143) from the p22 region of the human X chromosome

Summary Linkage analysis of four polymorphic anonymous DNA markers from the Xp22 region was performed using families from the Centre d'Etude du Polymorphisme Humain. The loci DXS43 (pD2) and DXS16 (pXUT23) were found to be tightly linked ( = 0.02 at = 14.96) and proximal to both DXS85 (782) and DXS143 (dic56). Multipoint linkage analysis suggests the order:     

A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes for the DXS115 locus and two for the DXYS64 locus.

Two yeast artificial chromosome (YAC) libraries were screened for probes in Xq28, around the gene for coagulation factor VIII (F8). A set of 30 YACs were recovered and assembled into a contig spanning at least 1.6 Mb from the DXYS64 locus to the glucose 6-phosphate dehydrogenase gene (G6PD). Overlaps among the YACs were determined by several fingerprinting techniques and by additional probes generated from YAC inserts by using Alu-vector or ligation-mediated PCR. Analysis of more than 30 probes and sequence-tagged sites (STSs) made from the region revealed the presence of several homologous genomic segments. For example, a probe for the DXYS64 locus, which maps less than 500 kb 5' of F8, detects a similar but not identical locus between F8 and G6PD. Also, a probe for the DXS115 locus detects at least three identical copies in this region, one in intron 22 of F8 and at least two more, which are upstream of the 5' end of the gene. Comparisons of genomic and YAC DNA suggest that the multiple loci are not created artifactually during cloning but reflect the structure of uncloned human DNA. On the basis of these data, the most likely order for the loci analyzed is tel-DXYS61-DXYS64-(DXS115-3-DXS115-2)-5'F8-(D XS115-1)-3'F8-G6PD.