Partial deletions of a sequence family ("DXS278") and its physical linkage to steroid sulfatase as detected by pulsed-field gel electrophoresis

pCRI-S232 (DXS278) is a 7-kb genomic sequence that hybridizes to multiple polymorphic X-linked restriction fragments on standard Southern analysis. Physical mapping of pCRI-S232 by pulsed-field gel electrophoresis (PFGE) suggests that a sequence in S232 is repeated in multiple X-chromosomal regions in normal individuals. Steroid sulfatase (STS) and DXS237 each hybridize to two of six X-linked SfiI fragments detected by S232. Two independent familial STS deletions, one of which is associated with a phenotype of ichthyosis plus ocular albinism (XI/OA1) and the other with nystagmus plus Rud syndrome, lack some but not all of the normal S232 PFGE fragments. We isolated a DNA fragment, E25B1.8, from a cosmid that contains S232. E25B1.8 detects a subset of the S232 polymorphic fragments on standard Southern blots plus new constant fragments; some, but not all, of the E25B1.8-hybridizing fragments are deleted in the XI/OA1 and Rud syndrome/nystagmus males. The simpler, but highly informative, polymorphism detected by E25B1.8 (DXS452) also eliminates an "intralocus" recombination seen with S232. We conclude that (1) males with STS deletions and complex phenotypes are partially deleted for DXS278, (2) DXS237 and part of DXS278 lie within 800 kb of STS, and (3) a repeat sequence within or around pCRI-S232 is probably located in multiple X-chromosomal locations spanning at least 2-3 Mb.     

Regional localization of polymorphic DNA loci on the proximal long arm of the X chromosome using deletions associated with choroideremia

Summary In two unrelated families, males have been identified who suffer from choroideremia and at the same time have an interstitial deletion on the proximal long arm of the X chromosome. By high-resolution banding we have characterized the deletion chromosomes as del(X)(q21.1-q21.33) and del(X)(q21.2-q21.31) respectively. By Southern blot analysis we have mapped ten different polymorphic DNA loci relative to the position of the deletion and the choroideremia locus TCD. One probe, p31, was shown to cover one of the breakpoints of the smallest deletion. The following order of the loci was suggested by deletion mapping: cen-DXS106-DXS72-TCD-(DXYS1/DXYS23/DXYS5)-DXYS2-(DXYS12/DXS3)-(DXS17/DXS101)-Xqter.     

Linkage analysis in X-linked ocular albinism.

We studied the linkage of X-linked Nettleship-Falls ocular albinism (OA1) to Xp22.1-Xp22.3 RFLPs at 12 loci in five families, including one in which OA1 cosegregates with a deletion of steroid sulfatase (STS). We found evidence for tight linkage of OA1 to the Xp22.3 loci DXS143, STS, and DXS452. DXS452, a newly described polymorphism detected by the probe E25B1.8, is part of the sequence family "DXS278" (pCRI-S232), but represents a single genetic locus. Every female in this study was heterozygous for the DXS452 RFLP. Thus, this marker will be extremely useful for family studies and genetic counseling. Analysis of individual recombinations suggests that OA1 maps between DXS143 and DXS85. Multipoint linkage analysis was consistent with this localization but was not statistically significant. These data suggest that OA1 lies proximal to the deletion in a previously described family with OA1 and STS deletion, but maps within the Xp22.3-Xp22.2 region.     

Long-range physical mapping around the human steroid sulfatase locus

The region of the human X chromosome containing the steroid sulfatase locus was analyzed by pulsed-field gel electrophoresis. Restriction site maps were generated for the X chromosome in the blood of a normal male individual and that in the mouse-human hybrid cell line ThyB-X; these maps extend over approximately 4.3 Mb of DNA of the former, and 3.2 Mb of the latter. Physical linkage was defined between the STS locus and sequences detected by the probes GMGX9 (DXS237), GMGXY19 (DYS74), CRI-S232 (DXS278), and dic56 (DXS143), and the order telomere--(STS, DYS74)--DXS237--DXS278--DXS143--centromere was deduced. The pulsed-field maps were used to demonstrate a deletion of 180 kb of DNA from the X chromosome of an individual with X-linked ichthyosis. Also, possible locations for the Kallmann syndrome gene were revealed, and the distance between the steroid sulfatase locus and the pseudoautosomal region was estimated to be at least 4 Mb.     

Characterisation of a Xp21 microdeletion syndrome in a 2-year-old boy with muscular dystrophy,glycerol kinase deficiency and adrenal hypoplasia congenita

Summary We report a 2-year-old boy with Duchenne muscular dystrophy (DMD), glycerol kinase deficiency (GK) and adrenal hypoplasia congenita (AHC). At three weeks of age, the patient was hospitalized for the first time with symptoms of hypotone dehydration because of AHC, At present, he shows severe muscular hypotonia and developmental delay. The patient and his family were referred to us for prenatal diagnosis and carrier testing in the mother of the patient and the mother's sister, respectively. The patient's DNA was examined by Southern blot and polymerase chain reaction analyses, using cDNA and genomic probes within and around the dystrophin (DYS) locus. A deletion was revealed, spanning DXS28, the whole dystrophin locus, DXS84 and DXS148, whereas DXS67, DXS68 (pter) and OTC (cen) were found to be retained. The cytogenetically visible microdeletion was also seen in the patient's mother, but not in the mother's sister or the patient's maternal grandmother. Our findings support the locus order pter-DXS67-DXS68-DXS28-AHC-GK-DMD-cen.     

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.     

Phenotypic variability in X-linked ocular albinism: relationship to linkage genotypes.

One hundred nineteen individuals from 11 families with X-linked ocular albinism (OA1) were studied with respect to both their clinical phenotypes and their linkage genotypes. In a four-generation Australian family, two affected males and an obligatory carrier lacked cutaneous melanin macroglobules (MMGs); ocular features were identical to those of Nettleship-Falls OA1. Four other families had more unusual phenotypic features in addition to OA1. All OA1 families were genotyped at DXS16, DXS85, DXS143, STS, and DXS452 and for a CA-repeat polymorphism at the Kallmann syndrome locus (KAL). Separate two-point linkage analyses were performed for the following: group A, six families with biopsy-proved MMGs in at least one affected male; group B, four families whose biopsy status was not known; and group C, OA-9 only (16 samples), the family without MMGs. At the set of loci closest to OA1, there is no clear evidence in our data set for locus heterogeneity between groups A and C or among the four other families with complex phenotypes. Combined multipoint analysis (LINKMAP) in the 11 families and analysis of individual recombination events confirms that the major locus for OA1 resides within the DXS85-DXS143 interval. We suggest that more detailed clinical evaluations of OA1 individuals and families should be performed for future correlation with specific mutations in candidate OA1 genes.     

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.     

Mapping of the gene for X-linked amelogenesis imperfecta by linkage analysis.

X-linked Amelogenesis imperfecta (AI) is a genetic disorder affecting the formation of enamel. In the present study two families, one with X-linked dominant and one with X-linked recessive AI, were studied by linkage analysis. Eleven cloned RFLP markers of known regional location were used. Evidence was obtained for linkage between the AI locus and the marker p782, defining the locus DXS85 at Xp22, by using two-point analysis. No recombination was scored between these two loci in 15 informative meioses, and a peak lod score (Zmax) of 4.45 was calculated at zero recombination fraction. Recombination was observed between the more distal locus DXS89 and AI, giving a peak lod score of 3.41 at a recombination fraction of .09. Recombination was also observed between the AI locus and the more proximal loci DXS43 and DXS41 (Zmax = 0.09 at theta max = 0.31 and Zmax = 0.61 at theta max = 0.28, respectively). Absence of linkage was observed between the AI locus and seven other loci, located proximal to DXS41 or on the long arm of the X chromosome. On the basis of two-point linkage analysis and analysis of crossover events, we propose the following order of loci at Xp22: DXS89-(AI, DXS85)-DXS43-DXS41-Xcen.     

DXS26 (HU16) is located in Xq21.1

Summary We have localized a single-copy DNA probe, HU16 (locus DXS26), to Xq21.1. The probe was isolated from a human-mouse hybrid X;13 library and mapped with human-mouse hybrids containing different portions of the human X chromosome and DNA from male patients with different X-chromosomal deletions. The following order of loci is proposed: Xcen-(DXS72, DXS169)-(DXS232,DXS26)-DXS121-DXS233-DXS165 TCD-DXS95-DXYSl-Xqter. HU16 will be useful in the study of the putative genes that reside in Xq21 and whose defects lead to deafness and mental retardation.     

Refinement of linkage of human severe combined immunodeficiency (SCIDX1) to polymorphic markers in Xq13.

The most common form of human severe combined immunodeficiency (SCID) is inherited as an X-linked recessive genetic defect, MIM 300400. The disease locus, SCIDX1, has previously been placed in Xq13.1-q21.1 by demonstration of linkage to polymorphic markers between DXS159 and DXS3 and by exclusion from interstitial deletions of Xq21.1-q21.3. We report an extension of previous linkage studies, with new markers and a total of 25 SCIDX1 families including female carriers identified by nonrandom X chromosome inactivation in their T lymphocytes. SCIDX1 was nonrecombinant with DXS441, with a lod score of 17.96. Linkage relationships of new markers in the SCIDX1 families were consistent with the linkage map generated in the families of the Centre d'Etude du Polymorphisme Humain (CEPH) and with available physical map data. The most likely locus order was DXS1-(DXS159,DXS153)-DXS106-DXS132-DXS4 53-(SCIDX1,PGK1, DXS325,DXS347,DXS441)-DXS447-DXS72-DXYS 1X-DXS3. The SCIDX1 region now spans approximately 10 Mb of DNA in Xq13; this narrowed genetic localization will assist efforts to identify gene candidates and will improve genetic management for families with SCID.     

Physical mapping of two Xp markers DXS16 and DXS143

Summary Lymphocyte karyotyping of an infant girl with the clinical features of microphthalmia, iridoschisis, goiter, hip joint dysplasia, labium synechia and craniotabes revealed an Xp deletion. The lymphocyte karyotypes of the parents were normal. Bromodeoxyuridine incorporation studies showed that, in 42 out of 43 metaphases, the deleted X chromosome was late replicating. In one metaphase, the normal X chromosome was observed to be allocyclic. Using DNA markers from the Xp22 region, the breakpoint was assigned distal to DXS16 (pXUT23) and proximal to DXS143 (dic56). Dosage intensity measurements confirmed that the STS gene and the DNA marker DXS31 were involved in the deleted area. Restriction fragment length polymorphism analysis revealed that the paternally derived X-chromosome was deleted.     

Linkage relationships and gene order around the locus for X-linked retinoschisis.

X-linked recessive retinoschisis (RS) is a hereditary disorder with variable clinical features. The main symptoms are poor sight; radial, cystic macula degeneration; and peripheral superficial retinal detachment. The disease is quite common in Finland, where at least 300 hemizygous males have been diagnosed. We used nine polymorphic DNA markers to study the localization of RS on the short arm of the X chromosome in 31 families comprising 88 affected persons. Two-point linkage results confirmed close linkage of the RS gene to the marker loci DXS43, DXS16, DXS207, and DXS41 and also revealed close linkage to the marker loci DXS197 and DXS9. Only one recombination was observed between DXS43 and RS in 59 informative meioses, giving a maximum lod score of 13.87 at the recombination fraction .02. No recombinations were observed between the RS locus and DXS9 and DXS197 (lods between 3 and 4), but at neither locus was the number of informative meioses sufficient to provide reliable estimates of recombination fractions. The most likely gene order on the basis of multilocus analysis was Xpter-DXS85-(DXS207,DXS43)-RS-DXS41-DXS 164-Xcen. Because multilocus linkage analysis indicated that the most probable location of RS is proximal to DXS207 and DXS43 and distal to DXS41, these three flanking markers are the closest and most informative markers currently available for carrier detection.     

DNA studies in a family with Duchenne muscular dystrophy and a deletion at Xp21.

We have performed Southern blot analysis on a large, four-generation kindred with Duchenne muscular dystrophy (DMD). Probes 754 (DXS 84), pERT87-1, pERT87-8, pERT87-15 (DXS164), and pXJ-1.1 did not hybridize to digested genomic DNA of affected males. Obligate-carrier mothers and unaffected brothers showed signals of a single X-chromosome copy intensity, and suspected noncarrier sisters demonstrated either a single band of two-copy intensity or informative polymorphisms. Uniform hybridization was seen with probes C7 (DXS28) and D2 (DXS43), which map distal to the DMD locus, and with OTC, which maps proximally. This deletion was present in six affected individuals and has been transmitted through 3 generations to date. On high-resolution chromosome analysis, a deletion within band Xp21 was consistently observed in one affected male studied and in one of the two X chromosomes in obligate carriers. This large molecular and cytogenetically visible deletion in affected DMD individuals without glycerol kinase deficiency, chronic granulomatous disease, retinitis pigmentosa (RP), or ornithine transcarbamylase deficiency is a very rare finding and should prove useful in specifically cloning additional probes within and flanking the DMD locus.     

Localization of X chromosome short arm markers relative to synovial sarcoma- and renal adenocarcinoma-associated translocation breakpoints

A series of thirteen different DNA markers was mapped relative to papillary renal cell carcinoma- and synovial sarcoma-associated translocation breakpoints in Xp11.2 using a panel of tumor-derived somatic cell hybrids in conjunction with Southern blot analysis. Our results indicate that the two translocation breakpoints differ from each other and that the chromosomal break in t(X; 1)-positive papillary renal cell carcinoma is located between the markers PFC-TIMP-OATL1-SYP-TFE3 and DXS226-DXS146-DXS255-OATL2-DXS14. In addition, our current breakpoint analysis has resulted in a revision of the regional localization of the proximal Xp marker DXS226.     

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.     

Unusual X chromosome inactivation in a mentally retarded girl with an interstitial deletion Xq27: implications for the fragile X syndrome

Summary A de novo interstitial deletion (X)(q27.1q27.3), between the loci DXS 105 and F8, has been found in a mentally retarded female. The deleted X chromosome is preferentially early replicating in fibroblasts, B cells and T cells, suggesting that the missing region plays a role in inactivation of the X chromosome. None of the available DNA probes except DXS 98 maps to the deleted region of about 10000kb. The locus FRAXA is either included in the deletion, or located close to the distal break point.     

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).     

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.     

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.