Physical mapping of probes proximal to the fragile X locus (FRAX) confirms the order F9-DXS105 (cX55.7)-DXS98 (4D8)-FRAXA

A woman with an abnormal karyotype, (46,X,der(X) (pter----q27::q27----q21), was analyzed using DNA probes in the region Xq27----qter. The results indicate that she is trisomic for the Factor IX locus, disomic for the locus DXS105 (cX55.7) and monosomic for the loci DXS98 (4D8), DXS52 (St14) and Factor VIII. This confirms the absence of the region Xq28 in the abnormal chromosome. Furthermore, the presence of only one copy of 4D8 and two copies of cX55.7 places the DXS98 locus distal to Factor IX and closer to the fragile X locus than DXS105.     

New polymorphisms at the DXS98 locus and confirmation of its location proximal to FRAXA by in situ hybridization.

The locus DXS98, detected with the 1.5-kb anonymous probe p4D-8, was recently shown to be closely linked and proximal to the locus for the fragile X syndrome, with theta = .05 at lod = 3.406, by utilizing a limited number of meioses informative for a two-allele MspI RFLP. Because DXS98 may be the closest available marker to the fragile X locus (FRAXA), we sought to increase its utility for linkage studies by extending its PIC and confirming its localization to Xq27, proximal to FRAXA. We have isolated 15 kb of genomic DNA (lambda 4D8-3) from the DXS98 locus by using p4D-8 to screen a genomic phage library containing partial Sau3A-digested human DNA. Three additional RFLPs for the enzymes BglII and XmnI were found by using the entire lambda 4D8-3 as probe. Combined heterozygosity for the four RFLPs in 25 unrelated females was 48%, as compared with only 28% when the MspI RFLP alone was used. In situ hybridization of unique sequences from lambda 4D8-3 was performed on metaphase chromosomes of lymphocytes and lymphoblasts from patients with the fragile X syndrome. Grains on the X chromosome were significantly clustered at band Xq27. Following fragile site induction, all nine grains in the q27-28 region were proximal to the fragile site. Confirmation of the location of DXS98 proximal to FRAXA and the new RFLPs at this locus make DXS98 more useful for linkage analysis and physical mapping in the region of the fragile X mutation.     

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.     

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.     

Myotubular Myopathy in a Girl with a Deletion at Xq27-q28 and Unbalanced X Inactivation Assigns the MTMI Gene to a 600-kb Region

A young girl with a clinically moderate form of myotubular myopathy was found to carry a cytogenetically detectable deletion in Xq27-q28. The deletion had occurred de novo on the paternal X chromosome. It encompasses the fragile X (FRAXA) and Hunter syndrome (IDS) loci, and the DXS304 and DXS455 markers, in Xq27.3 and proximal Xq28. Other loci from the proximal half of Xq28 (DXS49, DXS256, DXS258, DXS305, and DXS497) were found intact. As the X-linked myotubular myopathy locus (MTM1) was previously mapped to Xq28 by linkage analysis, the present observation suggested that MTM1 is included in the deletion. However, a significant clinical phenotype is unexpected in a female MTM1 carrier. Analysis of inactive X-specific methylation at the androgen receptor gene showed that the deleted X chromosome was active in ~80% of leukocytes. Such unbalanced inactivation may account for the moderate MTM1 phenotype and for the mental retardation that later developed in the patient. This observation is discussed in relation to the hypothesis that a locus modulating X inactivation may lie in the region. Comparison of this deletion with that carried by a male patient with a severe Hunter syndrome phenotype but no myotubular myopathy, in light of recent linkage data on recombinant MTM1 families, led to a considerable refinement of the position of the MTM1 locus, to a region of ~600 kb, between DXS304 and DXS497.     

Toward a physical map of the Xq28 region in man: Linking color vision, G6PD, and coagulation factor VIII genes to an X-Y homology region

We are using pulsed-field gel electrophoresis (PFGE) to establish a physical map of the human Xq28 region. We have identified a new probe 35.239 (DXYS64), localized in Xq28 by somatic hybrid mapping and belonging to a region of greater than 99% homology between the X and the Y chromosomes. PFGE data show that probes 35.239 and the polymorphic locus DXS115 (probe 767) map within a common 300-kb BssHII fragment. Both probes, in addition, hybridize to 575-kb BssHII and 590-kb ClaI fragments that contain the gene coding for coagulation factor VIII (F8C). The order F8C-DXS115-DXYS64 could be determined. Our results also provide evidence for linkage between the red/green color vision locus (RCP,GCP) and probes MD13 and T1.7 (GdX, DXS254) within a 750-kb ClaI fragment. Although the latter two probes are located within 50 kb of the 3' end of the G6PD gene, a G6PD cDNA probe did not hybridize to this fragment. G6PD, on the other hand, could be linked to F8C on a 290-kb BssHII fragment. All these data allow us to propose the order (RCP,GCP)-MD13-GdX-G6PD-F8C-DXS115-DXYS 64. We also linked probes St14 (DXS52), MN12 (DXS33), and DX13 (DXS15) to a member of a small family of X-linked dispersed sequences (DNF22S3) within a 575-kb BssHII fragment. The preliminary physical map presented here should be useful for further fine mapping of disease genes in the Xq28 region and should be helpful in orientating efforts toward the cloning of sequences close to the fragile X syndrome.     

Genetic mapping of DNA segments relative to the locus for the fragile-X syndrome at Xq27.3.

We have tested linkage between the locus for the fragile-X [fra(X)] syndrome at Xq27.3 and five polymorphic restriction sites identified by four DNA probes mapping distal to Xq26.1. A maximum distance of approximately 15 centimorgans (cM) between Xq27.3 and the marker loci mapping to this region was predicted based on the physical chromosome length. Close linkage between the disease and marker loci was excluded for probes DXS19 and DXS37 (theta = .05, Z = -2.94 and Z = -4.17, respectively). These marker loci were estimated to be less than five cM apart but approximately 40 cM proximal to the fragile site, indicating that there is a significantly greater frequency of recombination in this region of the X chromosome than expected from the physical length. Linkage results for the other marker loci and the fra(X) syndrome were inconclusive. However, the pX45d probe locus appears very closely linked to the factor IX locus (Z = 1.94 at theta = 0) and is approximately 20 cM proximal to Xq27.3. A relative map of the polymorphic restriction sites, fra(X) syndrome locus, and factor IX locus was constructed by maximizing lod scores over the Xq26.1----q27.3 region.     

Localization by in situ hybridization of the coagulation factor IX gene and of two polymorphic DNA probes with respect to the fragile X site

Summary The coagulation factor IX gene and two other polymorphic loci corresponding to DNA probes 52 A and St 14 have been previously localized in the q27 to qter region of the human X chromosome. In order to study their localization with respect to the fragile site at Xq27-28, we have hybridized the three DNA probes to metaphase chromosomes of a boy with fragile X mental retardation. We show that probe 52A is located in the proximal part of the Xq27 band, while the coagulation factor IX gene is on the distal part of this band, but proximal to the fragile site. The very polymorphic St 14 probe is located in the distal part of the Xq28 band, on the other side of the fragile site.     

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.     

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.     

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.     

Linkage homogeneity near the fragile X locus in normal and fragile X families

The fragile X syndrome locus, FRAXA, is located at Xq27. Until recently, few polymorphic loci had been genetically mapped close to FRAXA. This has been attributed to an increased frequency of recombination at Xq27, possibly associated with the fragile X mutation. In addition, the frequency of recombination around FRAXA has been reported to vary among fragile X families. These observations suggested that the genetic map at Xq27 in normal populations was different from that in fragile X populations and that the genetic map also varied within the fragile X population. Such variability would reduce the reliability of carrier risk estimates based on DNA studies in fragile X families. Five polymorphic loci have now been mapped to within 4 cM of FRAXA--DXS369, DXS297, DXS296, IDS, and DXS304. The frequency of recombination at Xq26-q28 was evaluated using data at these loci and at more distant loci from 112 families with the fragile X syndrome. Two-point and multipoint linkage analyses failed to detect any difference in the recombination fractions in fragile X versus normal families. Two-point and multipoint tests of linkage homogeneity failed to detect any evidence of linkage heterogeneity in the fragile X families. On the basis of this analysis, genetic maps derived from large samples of normal families and those derived from fragile X families are equally valid as the basis for calculating carrier risk estimates in a particular family.     

Isolation and characterization of a highly polymorphic human locus (DXS455) in proximal Xq28

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.     

In situ hybridization studies using a molecular probe that maps to Xq27–Xq28

Summary The locus DXS98, which is recognized by the sequence p4D-8, is closely linked to the FRAXA locus. In this study we present data that confirm the existing mapping data, sublocalizing this sequence to the Xq27 region immediately proximal to the fragile site at Xq27.3.     

Linear order of new and established DNA markers around the fragile site at Xq27.3

We have used recombinant clones derived from microdissection of the fragile X region to characterize breakpoints around the fragile site at Xq27.3. So far, no microdissection markers derived from Xq28 material have been found, thus allowing a rapid screening for clones surrounding the fragile site by their presence in a somatic cell hybrid containing Xq27.2-Xqter. A total of 43 new DNA markers from Xq27 have been sublocalized within this chromosome band. Of these new DNA markers, 5 lie in an interval defined as containing the fragile X region. The saturation of Xq27 with DNA markers by microdissection demonstrates the power of this technique and provides the resources for generating a complete physical map of the region.     

Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability.     

Repeated DNA sequences in the distal long arm of the human X chromosome

Summary Two DNA probes from within a single large insert from a recombinant phage-DNA library that was constructed from flow-sorted chromosomes enriched for the human X chromosome were shown to hybridize with repeated X-specific and autosomal DNA sequences. The X-chromosomal repeated sequences were assigned to the distal long arm of the X chromosome by both hybrid mapping and in situ hybridization. Fine mapping places these repeats in a region of Xq28 between DX13 (DXS15, in distal Xq28) and factor VIII (F8C, in proximal Xq28). The location of the X-specific repeats makes them potentially useful for future investigations of discases mapping to the distal long arm of the X chromosome, such as the fragile X syndrome.     

Isolation and mapping of discrete DXS101 loci in Xq22 near the X-linked agammaglobulinaemia gene locus

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22 in genetic linkage studies. The DXS101 locus has shown no recombinations with XLA in the ten informative meioses investigated so far. The DXS101 sequence, recognised by the cX52.5 plasmid, is moderately repeated in Xq22. We have isolated cosmids which contain this sequence; two copies of which have been found to lie near DXS178 and XLA, and a third copy which lies near the PLP gene, distal to these loci. We have used the cosmids to generate probes which should be of use for RFLP analysis, and thus in both prenatal diagnosis and carrier testing for XLA, and in constructing a genetic map of this region. These probes will also be used to complement the genetic map in the construction of a complete physical map of Xq22.     

Deletion (X)(q26.1-->q28) in a proband and her mother: molecular characterization and phenotypic-karyotypic deductions.

During a routine prenatal diagnosis we detected a female fetus with an apparent terminal deletion of an X chromosome with a karyotype 46,X,del(X)(q25); the mother, who later underwent premature ovarian failure, had the same Xq deletion. To further delineate this familial X deletion and to determine whether the deletion was truly terminal or, rather, interstitial (retaining a portion of the terminal Xq28), we used a combination of fluorescence in situ hybridization (FISH) and Southern analyses. RFLP analyses and dosage estimation by densitometry were performed with a panel of nine probes (DXS3, DXS17, DXS11, DXS42, DXS86, DXS144E, DXS105, DXS304, and DXS52) that span the region Xq21 to subtelomeric Xq28. We detected a deletion involving the five probes spanning Xq26-Xq28. FISH with a cosmid probe (CLH 128) that defined Xq28 provided further evidence of a deletion in that region. Analysis with the X chromosome-specific cocktail probes spanning Xpter-qter showed hybridization signal all along the abnormal X, excluding the possibility of a cryptic translocation. However, sequential FISH with the X alpha-satellite probe DXZ1 and a probe for total human telomeres showed the presence of telomeres on both the normal and deleted X chromosomes. From the molecular and FISH analyses we interpret the deletion in this family as 46,X,del(X) (pter-->q26::qter). In light of previous phenotypic-karyotypic correlations, it can be deduced that this region contains a locus responsible for ovarian maintenance.     

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.