Biomedical subjects
Publications and source records attributed to I W Craig.
The human gene for aromatic L-amino acid decarboxylase (DDC) was previously assigned to chromosome 7 by analysis of a panel of somatic cell hybrids. We report here refinement of this localisation, by in situ hybridisation, to 7p13-->p11.
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X-chromosome inactivation results in the strictly cis-limited inactivation of many but not all genes on one of the two X chromosomes during early development in somatic cells of mammalian females. One feature of virtually all models of X inactivation is the existence of an X-inactivation centre (XIC) required in cis for inactivation to occur. This concept predicts that all structurally abnormal X chromosomes capable of being inactivated have in common a defineable region of the X chromosome. Here we report an analysis of several such rearranged human X chromosomes and define a minimal region of overlap. The results are consistent with models invoking a single XIC and provide a molecular foothold for cloning and analysing the XIC region. One of the markers that defines this region is the XIST gene, which is expressed specifically from inactive, but not active, X chromosomes. The localization of the XIST gene to the XIC region on the human X chromosome implicates XIST in some aspect of X inactivation.
We report on a thrombocytopenic female belonging to a pedigree with the Wiskott-Aldrich syndrome (WAS). Restriction fragment length polymorphism (RFLP) analysis with probe M27 beta, closely linked to the WAS gene, demonstrated that she is a carrier of WAS. Both small-sized and normal-sized platelets were present, suggesting that, unlike the vast majority of WAS carriers, she does not manifest nonrandom X-chromosome inactivation in the thrombopoietic cell lineage. Study of X-chromosome inactivation by means of RFLP and methylation analysis demonstrated that the pattern of X-chromosome inactivation was nonrandom in T lymphocytes, but random in granulocytes. While this is the first complete report on the occurrence of thrombocytopenia in a carrier female of WAS as the result of atypical lyonization, it also suggests that expression of the WAS gene occurs at (or extends up to) a later stage than the multipotent stem cell along the hematopoietic differentiation pathway.
Interspersed repetitive sequence polymerase chain reaction (IRS-PCR) has become a powerful tool for the rapid generation of DNA probes from human chromosomes present in rodent somatic cell hybrids. We have constructed a somatic cell hybrid containing a major portion of the mouse X chromosome in a human background (clone 8.0). IRS-PCR was developed for the specific amplification of mouse DNA using either of two primers from the rodent-specific portion of the murine B1 repeat. Amplification was subsequently performed with clone 8.0 and a subclone, 8.1/1, which retains a small murine X-chromosomal fragment including Hprt and the Gdx locus. A total of 15-20 discrete PCR products ranging in size from less than 500 to greater than 3000 bp were obtained from clone 8.0 with each primer. In clone 8.1/1, a subset of these bands plus some additional bands were observed. Nine bands amplified from clone 8.1/1 have been excised from gels and used as probes on Southern blots. All of the fragments behaved as single-copy probes and detected domesticus/spretus variation. They have been regionally mapped using an interspecific backcross. The probe locations are compatible with those of markers known to be present in clone 8.1/1. These results demonstrate the feasibility of this method as applied to the mouse genome and the high likelihood of generating useful DNA probes from a targeted region.
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.
The human gene for tryptophan hydroxylase has been previously assigned to chromosome 11 by analysis of a panel of somatic cell hybrids. We report here on the refinement of this localization by in situ hybridization.
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X-linked immunodeficiency with hyperimmunoglobulinemia M (XHM) reflects an impairment of the immunoglobulin (Ig) heavy (H) chain class switch of B lymphocytes from IgM to IgG and IgA. XHM is recessive; female carriers manifest normal IgG and IgA production. Due to random X chromosome inactivation in all somatic cells of females, about half of the lymphocytes of XHM carriers are not able to express an intact XHM gene. An intrinsic defect of the Ig H chain class switch mechanism in XHM B lymphocytes would thus lead to a skewed X chromosome inactivation pattern in the IgG- and IgA-expressing B lymphocytes of female carriers. IgM-, IgG- and IgA-expressing B lymphoblastoid cells (BLC) were established by Epstein-Barr virus transformation of peripheral blood mononuclear cells of two female XHM carriers. In an analysis of differential methylation of the polymorphic DXS255 loci, random X chromosome inactivation patterns were found in populations of T lymphocytes, in IgM-expressing B lymphocytes and in IgG- or IgA-expressing B lymphocytes. The heterogeneity of Ig H chain rearrangements and the Ig light chain usage in the IgA- or IgG-expressing BLC clones that had inactivated the X chromosome which carries the intact XHM gene and in BLC clones with the homologous X chromosome inactivated were similar. The results indicated that the intrinsic Ig H chain class switch mechanism in XHM B lymphocytes is fully intact. We conclude that the XHM gene encodes a class switch inducer that is transferred to B lymphocytes.
Linkage between X-linked congenital stationary night blindness (CSNB1) and seven markers on the X chromosome was investigated in a large four-generation Albertan kindred. We detected significant linkage between the CSNB1 locus and the locus DXS255 (maximum lod score = 6.73 at a recombination fraction of 6%; confidence interval of 1% to 18%), which anchors the CSNB1 locus to the proximal region near p11.22 on the short arm of the X chromosome.
A cosmid containing the entire coding region for human carcinoembryonic antigen has been isolated. Detailed analysis and sequencing have determined an organization comprising nine exons encoding amino acids and one for a 3' untranslated fragment. Comparison with other family members reveals a complex pattern of homology at the 3' end of the gene. The 5' noncoding region is rich in purine-rich motifs and possible enhancer elements and has a region with properties similar to those of HTF islands.
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.
X-linked retinitis pigmentosa is a hereditary retinal degenerative disorder which has been localised to the proximal short arm of the X chromosome. Recent evidence suggests that the disorder is heterogeneous with two possible loci for the disease mutation. DNA analysis on the family presented in this paper showed that the mutation mapped to the more telomeric locus (RP3), thus enabling two flanking polymorphic DNA probes (XJ1.1 and M27 beta) to be used for the detection of female carriers in the family. In none of the carriers was a tapetal reflex (metallic sheen) observed.
We have localised the human cDNA for neurone-specific enolase (ENO2) to chromosome region 12p13 by in situ hybridisation. Two additional smaller peaks of hybridisation to specific chromosomal subregions were observed. That on chromosome 1p36 probably represents cross-hybridisation to the locus for nonneuronal enolase (ENO1), which has been previously localised to this chromosome and with which ENO2 shares homology. A further gene for a member of the enolase family may be responsible for the hybridisation to chromosome 17.
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A hypervariable DNA marker is closely linked to one of the most severe forms of night blindness, X-linked retinitis pigmentosa (RP). Affected individuals with X-linked RP, obligate carriers, and ophthalmologically identifiable carriers of the disease were included in a linkage study. The diagnosis was established in five sibships by funduscopic and electrophysiological investigations. When the X-linked probe M27 beta was used, 2 recombinants out of 29 informative meioses were detected (theta = 0.07 at a maximum lod of 4.75). The hypervariable probe detected two different alleles in 38 of 39 females tested. M27 beta is therefore a potentially very useful probe for carrier detection and prenatal diagnosis, as well as for addressing the question of heterogeneity of X-linked RP.