Mapping DNA sequences in a human X-chromosome deletion which extends across the region of the Duchenne muscular dystrophy mutation.

A somatic cell hybrid has been constructed and characterized using fibroblasts from a phenotypically normal woman who possesses an X chromosome with an interstitial deletion of the short arm. High-resolution banding indicates that the deleted segment is either Xp22.13-p11.4 or Xp22.11-p11.23. Southern blot hybridization to previously mapped DNA sequences confirms that the missing segment of the X chromosome is a deletion and not an interstitial translocation and supports the cytogenetic interpretation that the deletion extends proximal of Xp11.3 and therefore probably comprises Xp22.11-p11.23. Three further DNA sequences have been localized to the region of the deleted segment. The following order has been assigned to the seven probes used: Xpter-RC8-pXUT22-(OA1,C7,M2C)-L1.28-RD6 -Xcen.     

Genetic and physical mapping around the properdin P gene.

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

Molecular cloning of Xp11 breakpoints in two unrelated mentally retarded females with X;autosome translocations

Mental retardation is a very common and extremely heterogeneous disorder that affects about 3% of the human population. Its molecular basis is largely unknown, but many loci have been mapped to the X chromosome. We report on two mentally retarded females with X;autosome translocations and breakpoints in Xp11, viz., t(X;17)(p11;p13) and t(X;20)(p11;q13). (Fiber-) FISH analysis assigned the breakpoints to different subbands, Xp11.4 and Xp11.23, separated by approximately 8 Mb. High-resolution mapping of the X- chromosome breakpoints using Southern blot hybridization resulted in the isolation of breakpoint-spanning genomic subclones of 3 kb and 0. 5 kb. The Xp11.4 breakpoint is contained within a single copy sequence, whereas the Xp11.23 breakpoint sequence resembles an L1 repetitive element. Several expressed sequences map close to the breakpoints, but none was found to be inactivated. Therefore, mechanisms other than disruption of X-chromosome genes likely cause the phenotypes.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Localization of the properdin structural locus to Xp11.23-Xp21.1

Properdin is a serum protein belonging to the alternative pathway of complement activation whose absence is often associated with fatal bacterial infections. Properdin deficiency segregates with an X-linked recessive pattern and its position has been recently refined by genetic linkage analysis to the proximal part of the X-chromosome short arm near the OTC and DXS7 loci. We have hybridized an 0.8-kb genomic clone encoding part of the human properdin gene to a panel of somatic cell hybrids retaining different portions of the human X chromosome and thereby localized the probe to Xcen-Xp21.1. Furthermore, in situ hybridization of the same probe to replication banded metaphase chromosomes refined this localization to the region Xp11.23-Xp21.1 (with a peak grain distribution in the region equivalent to Xp11.4). As OTC and DXS7 map to Xp21.1 and Xp11.3, respectively, the data presented here strongly suggest that the X-linked deficiency syndrome is due to a defect in the locus encoding the structural properdin gene or in a physically close regulatory locus.     

Genetic Analysis of a Kindred With X-linked Mental Handicap and Retinitis Pigmentosa

A kindred is described in which X-linked nonspecific mental handicap segregates together with retinitis pigmentosa. Carrier females are mentally normal but may show signs of the X-linked retinitis pigmentosa carrier state and become symptomatic in their later years. Analysis of polymorphic DNA markers at nine loci on the short arm of the X chromosome shows that no crossing-over occurs between the disease and Xp11 markers DXS255, TIMP, DXS426, MAOA, and DXS228. The 90% confidence limits show that the locus is in the Xp21-q21 region. Haplotype analysis is consistent with the causal gene being located proximal to the Xp21 loci DXS538 and 5'-dystrophin on the short arm of the X chromosome. The posterior probability of linkage to the RP2 region of the X chromosome short arm (Xp11.4-p11.23) is .727, suggesting the possibility of a contiguous-gene-deletion syndrome. No cytogenetic abnormality has been identified.     

An 18-locus linkage map of the pericentromeric region of the human X chromosome: Genetic framework for mapping X-linked disorders

We report a high-resolution genetic linkage map of the region Xp11.4 to Xq13.3, spanning the centromere of the X chromosome and encompassing approximately 30 cM. This 18-locus map is composed of 11 intervals that are spaced on average about 3 cM apart. Markers incorporated into the map together detect 19 distinct polymorphisms and include five genes (TIMP, SYP, AR, CCG1, PGK1), the OATL1 cluster, the hypervariable locus DXS255, the centromeric locus DXZ1, and 10 other anonymous DNA segments. Given that this map spans roughly one-fifth of the length of the X chromosome and includes many loci currently used in both diagnosis and mapping of X-linked disorders, it should be useful for genetic counseling and for guiding efforts to clone disease genes in this region.     

A deletion map of the human Yq11 region: implications for the evolution of the Y chromosome and tentative mapping of a locus involved in spermatogenesis.

A deletion map of Yq11 has been constructed by analyzing 23 individuals bearing structural abnormalities (isochromosomes, terminal deletions and X;Y, Y;X, or A;Y translocations) in the long arm of the Y chromosome. Twenty-two Yq-specific loci were detected using 14 DNA probes, ordered in 11 deletion intervals, and correlated with the cytogenetic map of the chromosome. The breakpoints of seven translocations involving Xp22 and Yq11 were mapped. The results obtained from at least five translocations suggest that these abnormal chromosomes may result from aberrant interchanges between X-Y homologous regions. The use of probes detecting Yq11 and Xp22.3 homologous sequences allowed us to compare the order of loci within these two chromosomal regions. The data suggest that at least three physically and temporary distinct rearrangements (pericentric inversion of pseudoautosomal sequences and/or X-Y transpositions and duplications) have occurred during evolution and account for the present organization of this region of the human Y chromosome. The correlation between the patient' phenotypes and the extent of their Yq11 deletions permits the tentative assignment of a locus involved in human spermatogenesis to a specific interval within Yq11.23.     

A gene for X-linked idiopathic congenital nystagmus (NYS1) maps to chromosome Xp11.4-p11.3

Congenital nystagmus (CN) is a common oculomotor disorder (frequency of 1/1,500 live births) characterized by bilateral uncontrollable ocular oscillations, with onset typically at birth or within the first few months of life. This condition is regarded as idiopathic, after exclusion of nervous and ocular diseases. X-linked, autosomal dominant, and autosomal recessive modes of inheritance have been reported, but X-linked inheritance is probably the most common. In this article, we report the mapping of a gene for X-linked dominant CN (NYS1) to the short arm of chromosome X, by showing close linkage of NYS1 to polymorphic markers on chromosome Xp11.4-p11.3 (maximum LOD score of 3.20, over locus DXS993). Because no candidate gene, by virtue of its function, has been found in this region of chromosome Xp, further studies are required, to reduce the genetic interval encompassing the NYS1 gene. It is hoped that the complete gene characterization will address the complex pathophysiology of CN.     

Gene structure of the human DDX3 and chromosome mapping of its related sequences.

The human DDX3 gene (GenBank accession No. U50553) is the human homologue of the mouse Ddx3 gene and is a member of the gene family that contains DEAD motifs. Previously, we mapped the gene to the Xp11.3-11.23. In this report, we describe the structural organization of the human DDX3 gene. It consisted of 17 exons that span approximately 16 kb. An Alu element was present in the intron 13. Its organization was the same as that of the human DBY gene, a closely related sequence present on the Y chromosome. We also identified two processed pseudogenes (DDX3) with a sequence that is highly homologous to those of DDX3 cDNAs, but contain a translation termination codon within its open-reading frame. Pseudogenes are mapped on human chromosomes 4 and X, respectively. In this paper, we discuss the relationships between DDX3 and its related sequences that have been isolated.     

Localization of the gene for the Wiskott-Aldrich syndrome between two flanking markers, TIMP and DXS255, on Xp11.22-Xp11.3.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive genetic disease in which the basic molecular defect is unknown. We previously located the WAS gene between two DNA markers, DXS7 (Xp11.3) and DXS14 (Xp11), and mapped it to the proximal short arm of the human X chromosome (Kwan et al., 1988, Genomics 3:39-43). In this study, further mapping was performed on 17 WAS families with two additional RFLP markers, TIMP and DXS255. Our data suggest that DXS255 is closer to the WAS locus than any other markers that have been previously described, with a multipoint maximum lod score of Z = 8.59 at 1.2 cM distal to DXS255 and thus further refine the position of the WAS gene on the short arm of the X chromosome. Possible locations for the WAS gene are entirely confined between TIMP (Xp11.3) and DXS255 (Xp11.22). Use of these markers thus represents a major improvement in genetic prediction in WAS families.     

Complex Segmental Duplications Mediate a Recurrent dup(X)(p11.22-p11.23) Associated with Mental Retardation, Speech Delay, and EEG Anomalies in Males and Females

Submicroscopic copy-number variations make a considerable contribution to the genetic etiology of human disease. We have analyzed subjects with idiopathic mental retardation (MR) by using whole-genome oligonucleotide-based array comparative genomic hybridization (aCGH) and identified familial and de novo recurrent Xp11.22-p11.23 duplications in males and females with MR, speech delay, and a peculiar electroencephalographic (EEG) pattern in childhood. The size of the duplications ranges from 0.8–9.2 Mb. Most affected females show preferential activation of the duplicated X chromosome. Carriers of the smallest duplication show X-linked recessive inheritance. All other affected individuals present dominant expression and comparable clinical phenotypes irrespective of sex, duplication size, and X-inactivation pattern. The majority of the rearrangements are mediated by recombination between flanking complex segmental duplications. The identification of common clinical features, including the typical EEG pattern, predisposing genomic structure, and peculiar X-inactivation pattern, suggests that duplication of Xp11.22-p11.23 constitutes a previously undescribed syndrome.     

Mapping of the glycine receptor alpha 2-subunit gene and the GABAA alpha 3-subunit gene on the mouse X chromosome.

We have mapped the gene for the alpha 2-subunit of the inhibitory glycine receptor (Glra2) to the telomeric end of the mouse X chromosome by backcross analysis of a Mus musculus/Mus spretus interspecific cross. In addition, we have extended the mapping of the GABAA alpha 3-subunit receptor gene (Gabra3). A deduced gene order of cen-Cybb-Hprt-DXPas6-Gabra3-Rsvp-Gdx/Cf-8- Dmd-Pgk-1-DXPas2-Plp-DXPas1-Glra2-tel places Gabra3 proximal to the visual pigment gene Rsvp and Glra2 in the region of loci for hypophosphatemia (Hyp), steroid sulfatase (Sts), and the E1 alpha-subunit of pyruvate dehydrogenase (Pdha1). This establishes the XF region of the mouse X chromosome as homologous with the Xp22.1-p22.3 region of the human X chromosome and indicates the presence of an evolutionary breakpoint in the region of Xp21.3.     

A multipoint linkage map of the distal short arm of the human X chromosome.

The distal portion of the short arm of the human X chromosome (Xp) exhibits many unique and interesting features. Distal Xp contains the pseudoautosomal region, a number of disease loci, and several cell-surface markers. Several genes in this area have also been observed to escape X-chromosomal inactivation. The characterization of new polymorphic loci in this region has permitted the construction of a refined multipoint linkage map extending 15 cM from the Xp telomere. This interval is known to contain the loci for the diseases X-linked ichthyosis, chondrodysplasia punctata, and Kallmann syndrome, as well as the cell-surface markers Xg and 12E7. This region also contains the junction between the pseudoautosomal region and strictly X-linked sequences. The locus MIC2 has been demonstrated by linkage analysis to be indistinguishable from the pseudoautosomal junction. The steroid sulfatase locus has been mapped to an interval adjacent to the DXS278 locus and 6 cM from the pseudoautosomal junction. The polymorphic locus (STS) DXS278 was shown to be informative in all families studied, and linkage analysis reveals that the locus represents a low-copy repeat with at least one copy distal to the STS gene. The generation of a multipoint linkage map of distal Xp will be useful in the genetic dissection of many of the unique features of this region.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.     

An evolutionary rearrangement of the Xp11.3-11.23 region in 3p21.3, a region frequently deleted in a variety of cancers.

In searching for a tumor suppressor gene in the 3p21.3 region, we isolated two genes, RBM5 and RBM6. Sequence analysis indicated that these genes share similarity. RBM5 and-to a lesser extent-RBM6 also have similarity to DXS8237E at Xp11.3-11.23, which maps less than 20 kb upstream of UBE1. A homologue of UBE1, UBE1L, is located at 3p21. 3. FISH analysis showed that the distance between UBE1L and RBM5 in 3p21.3 is about 265 kb. DXS8237E and UBE1 on the X chromosome have the same orientation, whereas on chromosome 3 the orientation of RBM5 and that of RBM6 are opposite to the orientation of UBE1L. Presumably, part of the Xp11.3-11.23 region has duplicated to chromosome 3. Part of this region on chromosome 3 may subsequently have duplicated again within the same chromosomal region. Inversion at some stage of the evolution of the human genome would explain the change in orientation of the genes on chromosome 3 compared with that of the genes on the X chromosome.     

Physical mapping and exclusion of<Emphasis Type="Italic"> GPR34</Emphasis> as the causative gene for congenital stationary night blindness type 1

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by impaired night vision, variably involving high myopia, nystagmus, decreased visual acuity, and strabismus. Linkage studies have identified two distinct loci for X-linked CSNB1 and CSNB2 on the short arm of chromosome X. The gene mutated in families displaying the "incomplete phenotype" of CSNB (i.e., CSNB2) has recently been identified. To identify novel candidate genes for the "complete form" of CSNB (i.e., CSNB1) we screened the physically vast region Xp11.3-Xp11.4 for cDNA sequences. This led us to identify and map the G protein coupled receptor (GPCR) gene GPR34 to Xp11.4 within 650 kb of the marker DXS993. Deletion screening via Southern blotting and direct sequencing of GPR34 revealed no mutations in 19 unrelated men with CSNB1, excluding a causal role in the disease. However, because of its expression in retinal and neural tissue and the involvement of GPCRs in transmembrane signal transduction, GPR34 remains a putative candidate gene for a number of ocular diseases which also map to the Xp11.4 region.     

Construction of a 1.5-Mb Bacterial Artificial Chromosome Contig in Xp11.23, a Region of High Gene Content

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.     

The gene coding for the p68 calcium-binding protein is localised to bands q32–q34 of human chromosome 5, and to mouse chromosome 11

Summary The gene encoding a tissue inhibitor of metalloproteinases, TIMP, has previously been shown to be X-linked in both the human and mouse genomes. We have used a series of somatic cell hybrids segregating translocation and deletion X chromosomes to map the TIMP gene on the human X chromosome. In combination with previous data, the gene can be assigned to Xp11.23Xp11.4. Genetic linkage analyses demonstrate that TIMP is linked to the more distal ornithine transcarbamylase (OTC) locus at a distance of about 22 centimorgans. The data are consistent with the conclusion that TIMP maps to a conserved synteny and linkage group on the proximal short arm of the human X chromosome and on the pericentric region of the mouse X chromosome, including loci for synapsin-1, a member of the raf oncogene family, OTC, and TIMP.