cDNA sequence, map, and expression of the murine homolog of GTBP, a DNA mismatch repair gene.

DNA mismatch repair genes (MMR) have been actively studied in humans for their role in cancer susceptibility. Recently, GTBP, a new member of the MMR gene family, has been cloned and found to encode a 160-kDa protein that forms a heterodimer with hMSH2. Our group has isolated and characterized the mouse homolog of GTBP. The transcript, termed Gtmbp (G/T mismatch-binding protein), encodes a protein highly similar to its human counterpart. The gene is markedly conserved across species and maps to the distal portion of mouse chromosome 17, in a region homologous to human 2p. One Gtmbp-related sequence maps to proximal mouse chromosome 4. Studies of the expression of Gtmbp suggest that its regulated transcription is tightly linked to the level of DNA replication, consistent with the protein's DNA proofreading function.

Cloning and characterization of a new human Xq13 gene, encoding a putative helicase.

We describe the cloning and characterization of a new human Xq13 gene (XH2), extending over a 220 kb genomic stretch between MNK and DXS56. The gene, which undergoes X-inactivation, contains a 4 kb open reading frame and encodes a putative NTP-binding nuclear protein homologous to several members of the helicase II superfamily. The murine homologue maps to the syntenic genetic interval, between Pgk1 and Xist. In situ hybridization studies in mouse reveal precocious, widespread expression of the murine homologue of XH2 at early stages of embryogenesis, and more restricted expression during late developmental stages and at birth. XH2 is a new member of an expanding family of proven and putative helicases, sharing six conserved, collinear domains. In particular, the XH2 protein shows homology with yeast RAD54. Type II helicases have been implicated in nucleotide excision repair and the initiation of transcription. This new gene, represents a potential candidate for several genetic disorders mapped to human Xq13.

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.

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.

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.

Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region.