Identification and characterization of an Xq26-q27 duplication in a family with spina bifida and panhypopituitarism suggests the involvement of two distinct genes

We investigated a family with a duplication, dup(X)q26-q27, that was present in two brothers, their mother, and their maternal grandmother. The brothers carrying the duplication displayed spina bifida and panhypopituitarism, whereas a third healthy brother inherited the normal X chromosome. Preferential inactivation of the X chromosome containing the duplication was evident in healthy carrier females. We determined the boundaries of the Xq26-q27 duplication. Via interphase FISH analysis we narrowed down each of the two breakpoint regions to approximately 300-kb intervals. The proximal breakpoint is located in Xq26.1 between DXS1114 and HPRT and is contained in YAC yWXD599, while the distal breakpoint is located in Xq27.3 between DXS369 and DXS1200 and contained in YAC yWXD758. The duplication comprises about 13 Mb. Evidence from the literature points to a predisposing gene for spina bifida in Xq27. We hypothesize that the spina bifida in the two brothers may be due to interruption of a critical gene in the Xq27 breakpoint region. Several candidate genes were mapped to the Xq27 critical region but none was shown to be disrupted by the duplication event. Recently, M. Lagerstr?m-Fermér et al. (1997, Am. J. Hum. Genet. 60, 910-916) reported on a family with X-linked recessive panhypopituitarism associated with a duplication in Xq26; however, no details were reported on the extent of the duplication. Our study corroborates their hypothesis that X-linked recessive panhypopituitarism is likely to be caused by a gene encoding a dosage-sensitive protein involved in pituitary development. We place the putative gene between DXS1114 and DXS1200, corresponding to the interval defined by the duplication in the present family.     

Further localization of X-linked hydrocephalus in the chromosomal region Xq28

X-linked hydrocephalus (HSAS) is the most frequent genetic form of hydrocephalus. Clinical symptoms of HSAS include hydrocephalus, mental retardation, clasped thumbs, and spastic paraparesis. Recently we have assigned the HSAS gene to Xq28 by linkage analysis. In the present study we used a panel of 18 Xq27-q28 marker loci to further localize the HSAS gene in 13 HSAS families of different ethnic origins. Among the Xq27-q28 marker loci used, DXS52, DXS15, and F8C gave the highest combined lod scores, of 14.64, 6.53 and 6.33, respectively, at recombination fractions of .04, 0, and .05, respectively. Multipoint linkage analysis localizes the HSAS gene in the telomeric part of the Xq28 region, with a maximal lod score of 20.91 at 0.5 cM distal to DXS52. Several recombinations between the HSAS gene and the Xq28 markers DXS455, DXS304, DXS305, and DXS52 confirm that the HSAS locus is distal to DXS52. One crossover between HSAS and F8C suggests that HSAS gene to be proximal to F8C. Therefore, data from multipoint linkage analysis and the localization of key crossovers indicate that the HSAS gene is most likely located between DXS52 and F8C. This high-resolution genetic mapping places the HSAS locus within a region of less than 2 Mb in length, which is now amenable to positional cloning.     

Multipoint linkage analysis of loci in the proximal long arm of the human X chromosome: application to mapping the choroideremia locus.

Choroideremia (McK30310), an X-linked retinal dystrophy, causes progressive night blindness, visual field constriction, and eventual central blindness in affected males by the third to fourth decade of life. The biochemical basis of the disease is unknown, and prenatal diagnosis is not available. Subregional localization of the choroideremia locus to Xq13-22 was accomplished initially by linkage to two restriction-fragment-length polymorphisms (RFLPs), DXYS1 (Xq13-q21.1) and DXS3 (Xq21.3-22). We have now extended our linkage analysis to 12 families using nine RFLP markers between Xp11.3 and Xq26. Recombination frequencies of 0%-4% were found between choroideremia and five markers (PGK, DXS3, DXYS12, DXS72, and DXYS1) located in Xq13-22. The families were also used to measure recombination frequencies between RFLP loci to provide parameters for the program LINKMAP. Multipoint analysis with LINKMAP provided overwhelming evidence for placing the choroideremia locus within the region bounded by DXS1 (Xq11-13) and DXS17 (Xq21.3-q22). At a finer level of resolution, multipoint analysis suggested that the choroideremia locus was proximal to DXS3 (384:1 odds) rather than distal to it. Data were insufficient, however, to distinguish between a gene order that puts choroideremia between DXS3 and DXYS1 and one that places choroideremia proximal to both RFLP loci. These results provide linkage mapping of choroideremia and RFLP loci in this region that will be of use for further genetic studies as well as for clinical applications in this and other human diseases.     

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.     

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.     

Choroideremia is linked to the restriction fragment length polymorphism DXYS1 at XQ13-21.

Choroideremia (McK30310), an X-linked hereditary retinal dystrophy, causes night-blindness, progressive peripheral visual field loss, and, ultimately, central blindness in affected males. The location of choroideremia on the X chromosome is unknown. We have used restriction fragment length polymorphisms from the X chromosome to determine the regional localization of choroideremia by linkage analysis in families with this disease. One such polymorphic locus, DXYS1, located on the long arm (Xq) within bands q13-q21, shows no recombination with choroideremia at lod = 5.78. Therefore, with 90% probability, choroideremia maps within 9 centiMorgans (cM) of DXYS1. Another polymorphic locus, DXS11, located within Xq24-q26, also shows no recombination with choroideremia, although at a smaller lod score of 1.54 (90% probability limit theta less than 30 cM). This linkage with DXS11, a marker that is distal to DXYS1, suggests that the locus for choroideremia is also distal to DXYS1 and lies between these two markers in the region Xq13-q24. These results provide regional mapping for the disease that may be useful for prenatal diagnosis and, perhaps ultimately, for isolating the gene locus for choroideremia.     

Oto-palato-digital syndrome type I: further evidence for assignment of the locus to Xq28

Summary The oto-palado-digital syndrome (OPD) is a rare X-linked disease with diagnostic skeletal features, conduction deafness, cleft palate and mild mental retardation. Differences in clinical presentation between families have led investigators to classify OPD into two subtypes: type I and type II. A linkage study performed in one family segregating for OPD I has recently suggested linkage to three marker loci: DXS15, DXS52 at Xq28, and DXS86 at Xq26. We have investigated an additional OPD I family for linkage by using distal chromosome Xq DNA probes. The linkage data and the analysis of recombination events that have occurred in this family excluded, definitively, the Xq26 region for OPD I, and provide further support for mapping the mutant gene close to the cluster of tightly linked markers DXS15, DXS52 and DXS305 at Xq28.     

Assignment of Emery-Dreifuss muscular dystrophy to the distal region of Xq28: the results of a collaborative study.

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked humeroperoneal dystrophy associated with cardiomyopathy that is distinct from the Duchenne and Becker forms of X-linked muscular dystrophy. Linkage analysis has assigned EDMD to the terminal region of the human X chromosome long arm. We report here further linkage analysis in two multigenerational EDMD families using seven Xq28 marker loci. Cumulative lod scores suggest that EDMD is approximately 2 cM from DXS52 (lod = 15.67) and very close to the factor VIII (F8C) and the red/green color pigment (R/GCP) loci, with respective lod scores of 9.62 and 10.77, without a single recombinant. Several recombinations between EDMD and three proximal Xq28 markers suggest that the EDMD gene is located in distal Xq28. Multipoint linkage analysis indicates that the odds are 2,000:1 that EDMD lies distal to DXS305. These data substantially refine the ability to perform accurate carrier detection, prenatal diagnosis, and the presymptomatic diagnosis of at-risk males for EDMD by linkage analysis. The positioning of the EDMD locus close to the loci for F8C and R/GCP will assist in future efforts to identify and isolate the disease gene.     

Genetic mapping of X-linked albinism-deafness syndrome (ADFN) to Xq26.3-q27.1

X-linked albinism-deafness syndrome (ADFN) was described in one Israeli Jewish family and is characterized by congenital nerve deafness and piebaldness. The ADFN mutation probably affects the migration of neural crest-derived precursors of the melanocytes. As a first step toward identifying the ADFN gene, a linkage study was performed to localize the disease locus on the X chromosome. The family was found to be informative for 11 of 107 RFLPs along the X, and two-point analysis showed four of them--factor 9 (F9), DXS91, DXS37, and DNF1--to have definite or suggestive linkage with ADFN. Multipoint linkage analysis indicated two possible orders within this cluster of loci, neither of which was preferable. In both orders F9 was the most distal, and the best estimate for the location of ADFN was between F9 and the next proximal marker (8.6 cM from F9 [Z = 8.1] or 8.3 cM from F9 [Z = 7.9]). These results suggest that the ADFN is at Xq26.3-q27.1. Disagreement between our data and previous localization of DXS91 at Xq11-q13 was resolved by hybridization of the probe pXG-17, which detects the DXS91 locus, to a panel of somatic cell hybrids containing different portions of the X chromosome. This experiment showed that this locus is definitely at Xq24-q26. Together with the linkage data, our results place DXS91 at Xq26 and underscore the importance of using more than one mapping method for the localization of molecular probes.     

Increased gene dosage at Xq26-q27 is associated with X-linked hypopituitarism

We have identified a novel interstitial duplication at Xq26.1-q27.3 in a previously reported family with X-linked recessive hypopituitarism [1]. Mapping of the duplication was carried out using interphase FISH analysis of over 60 bacterial genomic clones from Xq25-q28. The proximal and distal breakpoints of the duplication are contained within the 432N13 and 91O18 clones, respectively, and are separated by approximately 9 Mb. Comparison with a recently published 13-Mb duplication in another XH family [2] indicated that the duplication break-points in these families were different. Therefore, we conclude that X-linked hypopituitarism is caused by increased dosage of a gene that is critical for pituitary development and that the causative gene is located within the 9-Mb duplicated region that we have defined.     

Characterization of a deletion at Xq27-q28 associated with unbalanced inactivation of the nonmutant X chromosome.

We report the results of studies on the characterization of the mutation associated with marked unbalanced expression of the mutant X chromosome in a karyotypically normal girl with Hunter disease (mucopolysaccharidosis type II). Southern analysis of DNA extracted from somatic cell hybrids containing only the mutant X chromosome showed deletion of the Xq27.3-q28 loci: DXS297 (VK23AC), DXS293 (VK16), FRAXA (pfxa3), DXS296 (VK21A), and the 3' end of the iduronatesulfatase (IDS) gene. The flanking loci--DXS52 (St14-1), DXS304 (U6.2), and DXS369 (RN1)--were intact. On the basis of these results, we concluded that the mutation was a simple deletion extending a maximum of 3-5 cM to the centromeric side of the IDS gene. Both Southern analysis of DNA from somatic cell hybrids, using short segments of IDS cDNA, and PCR of reverse-transcribed RNA from cultured skin fibroblasts indicated that the telomeric terminus of the deletion was localized to a region near the middle of the coding sequences of the gene.     

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.     

Linkage studies in a new X-linked myopathy, suggesting exclusion of DMD locus and tentative assignment to distal Xq.

We here report linkage studies in a family suffering from a recently described hereditary muscle disease named X-linked myopathy with excessive autophagy (XMEA). Significant lod scores excluding linkage to the Duchenne-Becker muscular dystrophy locus were found. Several other loci on the short and long arms of the X chromosome produced negative lod scores, whereas probe DX13-7 defining locus DXS15 showed no recombinants and a lod score of z = 0.903 at theta = .0. Further studies should be done to determine whether the gene for XMEA is (1) located at Xq and (2) caused by a mutation of the Emery-Dreifuss muscular dystrophy gene, which has been assigned to the same region.     

Genetic and physical mapping of Xq24-q26 markers flanking the Lowe oculocerebrorenal syndrome

The Lowe oculocerebrorenal syndrome (OCRL) is characterized by congenital cataract, mental retardation, and renal tubular dysfunction. We are using the approaches of linkage analysis, mapping with somatic cell hybrids, and long-range restriction mapping to determine the order of Xq24-q26 markers with respect to each other and to the OCRL locus. DXS42 and DXS100 are proximal to the translocation breakpoint in a female patient with OCRL and a de novo translocation t(X;3)(q25;q27). DXS10, DXS86, HPRT, and DXS177 are distal to the breakpoint. These flanking markers show tight linkage to the disease locus in 11 families segregating for OCRL. Results from field inversion gel analysis show that DXS86 and DXS10 share a 460-kb BssHII fragment. Multipoint analysis to determine the position of HPRT with respect to (DXS10,DXS86) suggests that HPRT is proximal to (DXS10,DXS86). We propose the following order for markers in Xq24-q26: Xcen-(DXS42,DXS37,DXS100)-OCRL-DXS53 -HPRT-[(DXS10,DXS86),DXS177]-Xqter. The identification of additional tightly linked flanking markers extends the number of markers available for use in genetic counseling and begins to define the physical map of the region containing the gene for OCRL.     

PPM-X: a new X-linked mental retardation syndrome with psychosis, pyramidal signs, and macroorchidism maps to Xq28.

We report a three-generation family manifesting a previously undescribed X-linked mental retardation syndrome. Four of the six moderately retarded males have had episodes of manic-depressive psychosis. The phenotype also includes pyramidal signs, Parkinsonian features, and macroorchidism, but there are no characteristic dysmorphic facial features. Affected males do not show fragile sites at distal Xq on cytogenetic analysis, nor do they have expansions of the CGG repeats at the FRAXA, FRAXE, or FRAXF loci. Linkage analyses were undertaken, and a maximal LOD score of 3.311 at theta = .0 was observed with the microsatellite marker DXS1123 in Xq28. A recombination was detected in one of the affected males with DXS1691 (Xq28), which gives the proximal boundary of the localization. No distal recombination has been detected at any of the loci tested.     

Waisman syndrome, a human X-linked recessive basal ganglia disorder with mental retardation: localization to Xq27.3-qter.

Linkage of the gene responsible for an X-linked early onset parkinsonism disorder with mental retardation (McKusick 311510) to DNA probes that detect restriction fragment length polymorphisms is described. The disease gene is linked to the F8C gene, and to DNA probes detecting polymorphic loci DXS52, DXS15, DXS134, and DXS374 with maximum lod scores at theta = 0 of 5.08, 5.19, 5.00, 5.03, and 4.46, respectively. Multipoint linkage analysis gives a maximum multipoint lod score of 6.75 at the F8C gene. This places the disease gene in chromosomal region Xq27.3-qter.     

Dystonia-parkinsonism syndrome (XDP) locus: flanking markers in Xq12-q21.1.

The study of rare genetic forms of dystonia and parkinsonism permits positional cloning of genes potentially involved in more common, multifactorial forms of these diseases. One movement disorder amenable to molecular genetic analysis is the X-linked dystonia-parkinsonism syndrome (XDP). This disease is endemic to the Philippines where it originated by a genetic founder effect. Linkage analysis was performed with DNA from 14 XDP kindreds by using 12 polymorphic DNA sequences in Xp11-Xq22. Two-point analysis demonstrated maximum lod scores of 5.45, 4.95, 4.28, and 5.99 for DXS106, DXS159, PGK1, and DXS72, respectively, at recombination fractions of zero (DXS106 and DXS159), .01 (PGK1), and .04 (DXS72). Multipoint analysis resulted in a maximum-likelihood score (Zmax) of 8.41 with a (Zmax - 1) support interval of 9 cM between DXS159 and DXS72 (Xq12-q21.1). In 19 XDP kindreds significant linkage disequilibrium was found for loci DXS72 (delta = .47), PGK1 (delta = .36), DXS95 (delta = .30), DXS106 (delta = .28), and DXS159 (delta = .26). These data indicate that the gene mutated in XDP (locus DYT3) is located in Xq12-q21.1.     

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 mapping studies on the human X chromosome in the region Xq27-Xqter

We have characterized three terminal deletions of the long arm of the X chromosome. Southern analysis using Xq27/q28 probes suggests that two of the deletions have breakpoints near the fragile site at Xq27.3. Flow karyotype analysis provides an estimate of 12 X 10(6) bp for the size of the deleted region. We have not detected the deletion breakpoints by pulsed-field gel electrophoresis (PFGE) using the closet DNA probes, proximal to the fragile site. The physical distance between the breakpoints and the probes may therefore be several hundred kilobases. The use of the deletion patients has allowed a preliminary physical map of Xq27/28 to be constructed. Our data suggest that the closest probes to the fragile site on the proximal side are 4D-8 (DXS98), cX55.7 (DXS105), and cX33.2 (DXS152). PFGE studies provide evidence for the physical linkage of 4D-8, cX55.7, and cX33.2. We have also found evidence for the physical linkage of F8C, G6PD, and 767 (DXS115), distal to the fragile site.     

Genetic heterogeneity of FG syndrome: a fourth locus (FGS4) maps to Xp11.4-p11.3 in an Italian family

FG syndrome (FGS, MIM 305450) is a rare X-linked recessive disorder comprising mental retardation and multiple malformations. Various families have been described to date, increasing our knowledge of the phenotype variability and making the clinical diagnosis complex, especially in sporadic patients. The first locus for FG syndrome (FGS1) was linked to chromosome region Xq12-q21.31, but other families have been excluded from this locus. The genetic heterogeneity of FG syndrome has been confirmed by analysis of an X chromosome inversion [inv(X)(q11q28)] in an affected boy and in his mentally retarded maternal uncle, suggesting that an additional locus for FG syndrome (FGS2, MIM 300321) is located at either Xq11 or Xq28. Recently, a third locus (FGS3) has been mapped to Xp22.3. We have identified and clinically characterized an Italian FG family, including 31 members with three affected males in two generations and two obligate carriers. We have excluded linkage to known FGS loci, whereas an extensive study of the whole X chromosome has yielded a maximum LOD score (Z(max)) of 2.66 (recombination fraction=0) for markers between DXS8113 and sWXD805. This new locus for FG syndrome corresponds to a region of approximately 4.6 Mb on the X chromosome.