Genetic mapping of two new DNA markers in Xq26-q28 relative to the fragile-X syndrome locus.

We have characterized and genetically mapped two new DNA markers (DXS311 and DXS312) with respect to 10 existing loci in Xq26----Xq28 in a set of 15 families in which the fragile-X [fra(X)] syndrome was segregating. Two-point and multipoint linkage analyses were performed taking into account the incomplete penetrance of the fra(X) mutation. The most likely order on the basis of these data is centromere-DXS79-DXS10-DXS311-DXS86-(F9-DXS99 )-(DXS98-DXS312)-fra(X)-DXS52- DXS15-F8C-telomere. DXS98 and one of the new loci, DXS312, were found to be the proximal markers closest to the fra(X) locus. The order F9-(DXS98-DXS312)-fra(X) was found to be 5.9 x 10(4) times more likely than the order (DXS98-DXS312)-F9-fra(X).     

Multipoint genetic mapping of the Xq26-q28 region in families with fragile X mental retardation and in normal families reveals tight linkage of markers in q26–q27

Summary The q26–q28 region of the human X chromosome contains several important disease loci, including the locus for the fragile X mental retardation syndrome. We have characterized new polymorphic DNA markers useful for the genetic mapping of this region. They include a new BclI restriction fragment length polymorphism (RFLP) detected by the probe St14-1 (DXS52) and which may therefore be of diagnostic use in hemophilia A families. A linkage analysis was performed in fragile X families and in large normal families from the Centre d'Etude du Polymorphisme Humain (CEPH) by using seven polymorphic loci located in Xq26-q28. This multipoint linkage study allowed us to establish the order centromere-DXS100-DXS86-DXS144-DXS51-F9-FRAX-(DXS52-DXS15). Together with other studies, our results define a cluster of nine loci that are located in Xq26-q27 and map within a 10 to 15 centimorgan region. This contrasts with the paucity of markers (other than the fragile X locus) between the F9 gene in q27 and the G6PD cluster in q28, which are separated by about 30% recombination.     

Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile-X and hemophilia-B families.

We have characterized and genetically mapped new polymorphic DNA markers in the q27-q28 region of the X chromosome. New informative RFLPs have been found for DXS105, DXS115, and DXS152. In particular, heterozygosity at the DXS105 locus has been increased from 25% to 52%. We have shown that DXS105 and DXS152 are contained within a 40-kb region. A multipoint linkage analysis was performed in fragile-X families and in large normal families from the Centre d'Etudes du Polymorphisme Humain (CEPH). This has allowed us to establish the order centromere-DXS144-DXS51-DXS102-F9-DXS105-FRAX A-(F8, DXS15, DXS52, DXS115). DXS102 is close to the hemophilia-B locus (z[theta] = 13.6 at theta = .02) and might thus be used as an alternative probe for diagnosis in Hemophila-B families not informative for intragenic RFLPs. DXS105 is 8% recombination closer to the fragile-X locus than F9 (z[theta] = 14.6 at theta = .08 for the F9-DXS105 linkage) and should thus be a better marker for analysis of fragile-X families. However, the DXS105 locus appears to be still loosely linked to the fragile-X locus in some families. The multipoint estimation for recombination between DXS105 and FRAXA is .16 in our set of data. Our data indicate that the region responsible for the heterogeneity in recombination between F9 and the fragile-X locus is within the DXS105-FRAXA interval.     

Genetic mapping of new RFLPs at Xq27-q28.

The development of the human gene map in the region of the fragile X mutation (FRAXA) at Xq27 has been hampered by a lack of closely linked polymorphic loci. The polymorphic loci DXS369 (detected by probe RN1), DXS296 (VK21A, VK21C), and DXS304 (U6.2) have recently been mapped to within 5 cM of FRAXA. The order of loci near FRAXA has been defined on the basis of physical mapping studies as cen-F9-DXS105-DXS98-DXS369-DXS297-FRAXA-++ +DXS296-IDS-DXS304-DXS52-qter. The probe VK23B detected HindIII and XmnI restriction fragment length polymorphisms (RFLPs) at DXS297 with heterozygote frequencies of 0.34 and 0.49, respectively. An IDS cDNA probe, pc2S15, detected StuI and TaqI RFLPs at IDS with heterozygote frequencies of 0.50 and 0.08, respectively. Multipoint linkage analysis of these polymorphic loci in normal pedigrees indicated that the locus order was F9-(DXS105, DXS98)-(DXS369, DXS297)-(DXS293,IDS)-DXS304-DXS52. The recombination fractions between adjacent loci were F9-(0.058)-DXS105-(0.039)-DXS98-(0.123)-DXS369-(0.00)- DXS297-(0.057)-DXS296- (0.00)-IDS-(0.012)-DXS304-(0.120)-DXS52. This genetic map will provide the basis for further linkage studies of both the fragile X syndrome and other disorders mapped to Xq27-q28.     

Heterogeneity in X-linked recessive Charcot-Marie-Tooth neuropathy.

Three families presenting with X-linked recessive Charcot-Marie-Tooth neuropathies (CMT) were studied both clinically and genetically. The disease phenotype in family 1 was typical of CMT type 1, except for an infantile onset; two of five affected individuals were mentally retarded, and obligate-carrier females were unaffected. Families 2 and 3 showed distal atrophy with weakness, juvenile onset, and normal intelligence. Motor-nerve conduction velocities were significantly slowed, and electromyography data were consistent with denervation in affected CMT males in all three families. Thirty X-linked RFLPs were tested for linkage studies against the CMT disease loci. Family 1 showed tight linkage (recombination fraction [theta] = 0) to Xp22.2 markers DXS16, DXS143, and DXS43, with peak lod scores of 1.75, 1.78, and 2.04, respectively. A maximum lod score of 3.48 at DXS16 (theta = 0) was obtained by multipoint linkage analysis of the map DXS143-DXS16-DXS43. In families 2 and 3 there was suggestion of tight linkage (theta = 0) to Xq26 markers DXS86, DXS144, and DXS105, with peak lod scores of 2.29, 1.33, and 2.32, respectively. The combined maximum multipoint lod score of 1.81 at DXS144 (theta = 0) for these two families occurred in the map DXS10-DXS144-DXS51-DXS105-DXS15-DXS52++ +. A joint homogeneity analysis including both regions (Xp22.2 and Xq26-28) provided evidence against homogeneity (chi 2 = 9.12, P less than .005). No linkage to Xp11.12-q22 markers was observed, as was reported for X-linked dominant CMT and the Cowchock CMT variant. Also, the chromosomes 1 and 17 CMT loci were excluded by pairwise linkage analysis in all three families.     

Genetic mapping of nine DNA markers in the q11----q22 region of the human X chromosome

We have ordered nine polymorphic DNA markers within detailed map of the proximal part of the human X chromosome long arm, extending from band q11 to q22, by use of both physical mapping with a panel of rodent-human somatic hybrids and multipoint linkage analysis. Analysis of 44 families (including 17 families from the Centre d'Etude du Polymorphisme Humain) provided highly significant linkage data for both order and estimation of map distances between loci. We have obtained the following order: DXS1-DXS159-DXYS1-DXYS12-DXS3-(DXS94 , DXS178)-DXYS17. The most probable location of DXYS2 is between DXS159 and DXS3, close to DXYS1 and DXYS12. The high density of markers (nine loci within 30 recombination units) and the improvement in the estimation of recombination frequencies should be very useful for multipoint mapping of disease loci in this region and for diagnostic applications.     

Physical mapping of the factor VIII gene proximal to two polymorphic DNA probes in human chromosome band Xq28: implications for factor VIII gene segregation analysis

Genomic DNA segments for the coagulation factor VIIIc gene (F8C), which exhibits only limited restriction length polymorphism, map to the proximal region of band Xq28 by somatic cell hybridization analysis and in situ hybridization. Using somatic cell hybrids, we have obtained data which place probes DX13 (used to detect locus DXS15) and St14 (used to detect DXS52) distal to F8C, within band Xq28. Previous studies have mapped the factor IX gene (F9) and probe 52A (used to detect DXS51) proximal to F8C, in Xq26----q27 and Xq27, respectively (Camerino et al., 1984; Drayna et al., 1984; Mattei et al., 1985). Thus, the relative order of genetic marker loci in the Xq27----qter region is most likely cen-F9-DXS51-F8C-(DXS15, DXS52)-Xqter. The collection of these molecular probes is thus potentially useful in three-factor crosses of factor VIII gene segregation.     

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.     

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.     

Linkage studies in a large fragile X family.

We have analyzed the segregation of five loci in the region Xq27/28 in a large family affected by the fragile X syndrome. The marker DXS115 (767) is shown to be polymorphic with the enzyme PstI, as well as with BstXI. This marker will be useful in the analysis of both fragile X and haemophilia A families. The data presented here are consistent with the following order of loci: Xcen-F9-DXS105(cX55.7,55E)-DXS98(4D-8)- FRAXA-DXS52(St14)-DXS115(767)-qter.     

Linkage analysis using multiple DNA polymorphic markers in normal families and in families with fragile X syndrome

Summary Linkage data, using the polymorphic markers 52A (DXS51), F9, 4D-8(DXS98), and St14(DXS52), are presented from 14 fragile X pedigrees and from 7 normal pedigrees derived from the collection of the Centre d'Étude du Polymorphisme Humaine. A multipoint linkage analysis indicates that the most probable order of these four loci in normal families is DXS51-F9-DXS98-DXS52. Recombination frequencies ( ) corresponding to maximum LOD scores ( ) were obtained by two-point linkage analysis for a nuber of linkage groups, including: DXS51-F9 ( =5.94, =0.03), F9-DXS98 ( =0.51, =0.26), F9-DXS52 ( =0.84, =0.27), and DXS98-DXS52 ( =0.32, =0.20). A multipoint linkage analysis of these loci, including the fragile X locus, was also performed for the fragile X population and the data support the relative order (DSX51, F9, DXS98)-FRAXA-DXS52. Recombination frequencies and maximum LOD scores, which again were derived from two-point linkage analyses, were obtained for the linkage groups DXS51-F9 ( =9.96, =0) and F9-DXS52 ( =0.07, =0.45), as well as for the groups DXS51-FRAXA ( =2.42, =0.15), F9-FRAXA ( =1.30, =0.18), DXS98-FRAXA ( =0.05 =0.36), and DXS52-FRAXA ( =2.42 =0.15). The linkage data was further tested for the presence of genetic heterogeneity both within and between the fragile X and normal families for the intervals DXS51-F9, F9-DXS52, F9-FRAXA, and DXS52-FRAXA using a modification of the A test. Except for the interval F9-FRAXA (P<0.10) there was no evidence of genetic heterogeneity for each of the various linkage groups examined. The heterogeneity detected for the interval F9-FRAXA, however, was most likely due to one family (Fx-28) that displayed very tight linkage between these two loci.     

A new polymorphic marker very closely linked to DXS52 in the q28 region of the human X chromosome

Summary We have isolated an X chromosome probe, St35.691 (DXS305), which detects two RFLPs with TaqI and PstI, whose combined heterozygosity is about 60%. This probe has been assigned to Xq28 by physical and genetic mapping and is very closely linked to DXS52, DXS15, and the coagulation factor VIII gene (F8C). The best estimate of the recombination fraction for the DXS52-DXS305 interval is 0.014, with a lod score of 50.1. Multipoint analysis places DXS305 on the same side of F8C as DXS52, but complete ordering of the three loci was not possible with our present data. This highly informative marker should be useful in the precise mapping of the many disease genes that have been assigned to the Xq28 band.     

Assignment of the gene for complete X-linked congenital stationary night blindness (CSNB1) to Xp11.3

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by a presumptive defect of neurotransmission between the photoreceptor and bipolar cells. Carriers are not clinically detectable. A new classification for CSNB includes a complete type, which lacks rod function by electroretinography and dark adaptometry, and an incomplete type, which shows some rod function on scotopic testing. The refraction in the complete CSNB patients ranges from mild to severe myopia; the incomplete ranges from moderate hyperopia to moderate myopia. To map the gene responsible for this disease, we studied eight multigeneration families, seven with complete CSNB (CSNB1) and one with incomplete CSNB, by linkage analysis using 17 polymorphic X-chromosome markers. We found tight genetic linkage between CSNB1 and an Xp11.3 DNA polymorphic site, DXS7, in seven families with CSNB1 (LOD 7.35 at theta = 0). No recombinations to CSNB1 were found with marker loci DXS7 and DXS14. The result with DXS14 may be due to the small number of scored meioses (10). No linkage could be shown with Xq loci PGK, DXYS1, DXS52, and DXS15. Pairwise linkage analysis maps the gene for CSNB1 at Xp11.3 and suggests that the CSNB1 locus is distal to another Xp11 marker, TIMP, and proximal to the OTC locus. Five-point analysis on the eight families supported the order DXS7-CSNB1-TIMP-DXS225-DXS14. The odds in favor of this order were 9863:1. Removal of the family with incomplete CSNB (F21) revealed two most favored orders, DXS7-CSNB1-TIMP-DXS255-DXS14 and CSNB1-DXS7-TIMP-DXS255-DXS14. Heterogeneity testing using the CSNB1-M27 beta and CSNB1-TIMP linkage data (DXS7 was not informative in F21) was not significant to support evidence of genetic heterogeneity (P = 0.155 and 0.160, respectively).     

Fragile X syndrome: diagnosis using highly polymorphic microsatellite markers.

We describe two highly polymorphic microsatellite AC repeat sequences, VK23AC and VK14AC, which are closely linked to the fragile X at Xq27.3. Both VK23AC (DXS297) and VK14AC (DXS292) are proximal to the fragile site. Two-point linkage analysis in 31 fragile X families gave (a) a recombination frequency of 1% (range 0.00%-4%) with a maximum lod score of 32.04 for DXS297 and (b) a recombination frequency of 7% (range of 3%-15%) with a maximum lod score of 12.87 for DXS292. Both of these polymorphisms are applicable to diagnosis by linkage in families with fragile X syndrome. A multipoint linkage map of genetic markers at Xq27.3 was constructed from genotyping these polymorphisms in the CEPH pedigrees. The DXS292 marker is in the DXS98-DXS297 interval and in 3 cM proximal to DXS297.     

Mapping of the Emery-Dreifuss gene through reconstruction of crossover points in two Italian pedigrees

Summary Two unrelated pedigrees, which show recurrence of Emery-Dreifuss muscular dystrophy (EDMD) in three generations, have been studied using 13 X-linked DNA polymorphisms and somatic cell hybrids to establish the phase of the corresponding alleles in some obligate carriers. The reconstruction of cross-over points on the X chromosomes carrying the EDMD gene excludes from mapping most regions of the X chromosome except for the terminal portion of Xq. Pooled linkage data from the two pedigrees confirm the linkage previously reported with locus DXS15. A cross-over in a carrier female suggests that the EDMD gene is probably located distally to DXS15. In addition the recombinant meioses from one of the two pedigrees suggest the following order for some Xq polymorphic loci: DXS1 (DXYS1-DXS178) DXS42 (F9-DXS15).     

Efficient isolation of X chromosome-specific single-copy probes from a cosmid library of a human X/hamster hybrid-cell line: mapping of new probes close to the locus for X-linked mental retardation.

We isolated X-chromosomal DNA probes from a cosmid library constructed from a single human X/hamster hybrid-cell line (C12D). One hundred human clones were isolated and used to construct a pool of X-chromosomal DNA. This DNA was digested into 0.15-2-kb fragments and subcloned into plasmids allowing the rapid characterization of new single-copy probes. These were regionally mapped and used for the detection of restriction-site polymorphisms. Together with a series of subcloned probes from individually isolated cosmids, we found seven polymorphic probes among 53 tested. Thirty-one of the probes were physically localized to different regions of the X chromosome. Four polymorphic probes map to Xq27-Xq28: DXS102 (cX38.1), DXS105(cX55.7), DXS107(cpX234), and DXS134(cpX67). These were genetically mapped by multipoint analysis relative to previously characterized loci, a mapping that resulted in the following order: DXYS1, DXS107, DXS51/DXS102, F9, DXS105, Fra-X, F8/DXS52, DXS15, DXS134. The mapping of DXS105 between F9 and Fra-X makes this probe useful for Fra-X analysis. For the linkage between FraX and DXS105, a maximum lod score of 5.01 at 4 cMorgans has been obtained in one large Dutch pedigree.     

Two sisters with a distal deletion at the Xq26/Xq27 interface: DNA studies indicate that the gene locus for factor IX is present

Summary Two sisters with premature menopause and a small deletion of the long arm of one of their X chromosomes [del (X)(pterq26.3:)] were investigated with polymorphic DNA probes near the breakpoint. The deleted chromosome retained the factor IX (F9) locus and the loci DXS51 (52A) and DXS100 (pX45h), which are proximal to F9. However, the factor VIII (F8) locus was not present, nor were two loci tightly linked to this locus, DXS52 (St14) and DXS15 (DX13) This deletion refines the location of the F9 locus to Xq26 or to the interface Xq26/Xq27, thus placing it more proximally than has been previously reported. The DNA obtained from these patients should be valuable in the mapping of future probes derived from this region of the X chromosome.     

Linkage analysis of families with fragile-X mental retardation, using a novel RFLP marker (DXS 304).

A new polymorphic DNA marker U6.2, defining the locus DXS304, was recently isolated and mapped to the Xq27 region of the X chromosome. In the previous communication we describe a linkage study encompassing 16 fragile-X families and using U6.2 and five previously described polymorphic markers at Xq26-q28. One recombination event was observed between DXS304 and the fragile-X locus in 36 informative meioses. Combined with information from other reports, our results suggest the following order of the examined loci on Xq: cen-F9-DXS105-DXS98-FRAXA-DXS304-(DXS52-F8 -DXS15). The locus DXS304 is closely linked to FRAXA, giving a peak lod score of 5.86 at a corresponding recombination fraction of .00. On the basis of the present results, it is apparent that U6.2 is a useful probe for carrier and prenatal diagnosis in fragile-X families.     

Genetic mapping of 12 marker loci in the Xp22.3-p21.2 region

Summary To provide a more precise genetic map of the p22.3–p21.2 region on the short arm of the human X chromosome, we performed multilocus linkage studies in an expanded database including 31 retinoschisis families and 40 normal families. Twelve loci from this region were examined. Although significant lod scores were observed between various pairs of markers by two-point linkage analysis, the confidence limits were found to be broad. The most likely gene order on the basis of multilocus analysis was Xpter-DXS89-DXS85-DXS16-(DXS207, DXS43)-DXS274-(DXS41, DXS92)-ZFX-DXS164-Xcen. All other alternative orders were excluded by odds of at least 401.     

Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.