Carrier determination for X-linked agammaglobulinemia using X inactivation analysis of purified B cells.

We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia (XLA). This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction (PCR) based assay for the determination of methylation status at the androgen receptor (AR) gene locus to assess whether X inactivation is random or non-random at this locus. We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders. In addition, we investigated four females from different families affected by XLA, two of whom were of unknown carrier status, and we discuss the results obtained with this and other X-inactivation assays. A similar assay has recently been described by Allen et al. (1992) and applied to members of one family affected by XLA.

Isolation and mapping of discrete DXS101 loci in Xq22 near the X-linked agammaglobulinaemia gene locus.

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22 in genetic linkage studies. The DXS101 locus has shown no recombinations with XLA in the ten informative meioses investigated so far. The DXS101 sequence, recognised by the cX52.5 plasmid, is moderately repeated in Xq22. We have isolated cosmids which contain this sequence; two copies of which have been found to lie near DXS178 and XLA, and a third copy which lies near the PLP gene, distal to these loci. We have used the cosmids to generate probes which should be of use for RFLP analysis, and thus in both prenatal diagnosis and carrier testing for XLA, and in constructing a genetic map of this region. These probes will also be used to complement the genetic map in the construction of a complete physical map of Xq22.

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22.

Genetic linkage analysis has been instrumental in mapping the gene for X-linked agammaglobulinemia (XLA) to the proximal long arm of the human X chromosome, to Xq22. Due to the relative rarity of this disease the localization of the gene within Xq22 has remained imprecise. We have investigated twenty-nine families affected by XLA and have found no recombinants with the DXS178 locus in over 30 informative meioses. DXS178 is now the most reliable and informative locus for use in pre-natal diagnosis and carrier detection of XLA. In addition, we have identified new closely linked proximal and distal flanking markers for XLA, DXS442 and DXS101, respectively. These loci are separated by 2cM, considerably reducing the extent of DNA within which the XLA locus can be contained. This will open up the way for more directed positional cloning efforts for the isolation of the XLA gene.

Pulsed-field gel electrophoresis and radiation hybrid mapping analyses enable the ordering of eleven DNA loci in Xq22.

The Xq22 region of the human X chromosome encompasses the loci of several genes and random DNA markers whose relative positions have not been determined. By a combination of PFGE mapping and the analysis of a selected panel of X chromosome radiation hybrid cell lines, we have constructed physical maps of Xq22 that order a total of 11 polymorphic and nonpolymorphic DNA markers. Ten of these probes have been linked physically into three separate clusters, spanning nearly 6 Mb of DNA in total. The DXS94, DXS147, DXS211, DXS17, and DXS87 loci are all present on a 2.7-Mb MluI fragment; PLP, DXS54, DXS24, and DXS83 are present on MluI fragments spanning over 1.6 Mb; and DXS178 is present on a 1.5-Mb MluI fragment. Mapping with additional enzymes has allowed the further ordering of these loci with respect to each other. Together with these data, analysis of a small set of radiation hybrids has suggested the following over-all order of loci within Xq22: centromere-DXS178-DXS94-DXS147-DXS211-DXS17++ +-DXS87- PLP-DXS54-DXS24-DXS83-COL4A5-telomere. The ordering of these random DNA markers, genes, and disease loci, including the genes responsible for Pelizaeus-Merzbacher disease and Alport syndrome, indicates DNA markers that could be of further use clinically for these diseases. Furthermore, this map should form a basis for the refinement of additional disease-associated loci in this region.

Identification of CpG islands around the DXS178 locus in the region of the X-linked agammaglobulinaemia gene locus in Xq22.

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22. Genetic linkage analysis has shown tight linkage between the disease and the DXS178 locus and that DXS3 and DXS94 are the closet proximal and distal flanking markers, respectively, separated by a genetic distance of 10-12 cM. We attempted to construct a physical map of Xq22 using pulsed field gel electrophoresis (PFGE) and rare-cutting restriction enzymes in order to obtain a finite physical value for the distance between DXS3 and DXS94. However, these attempts were hampered by the large number of rare-cutting restriction enzyme sites around the DXS178 locus, indicative of the presence of CpG rich regions of DNA. We were able to construct a physical map of the sites close to DXS178 that suggests the presence of at least three, and perhaps as many as five, CpG islands. These are arranged on either side of DXS178, extending over about 550kb of genomic DNA. Each of these regions must be considered as being associated with a potential "candidate" gene sequence for the XLA gene and we have initiated a chromosome walk from DXS178 to the nearest of these islands.

The murine heat-stable antigen: a differentiation antigen expressed in both the hematolymphoid and neural cell lineages.

The murine heat-stable antigen (HSA) and the p31 antigen are cell surface glycoconjugates which are transiently expressed during the development and differentiation of the hematolymphoid and neural cell lineages, respectively. We show here that monoclonal antibodies which react with these two species recognize a common antigenic determinant which is expressed on both HSA and p31, and the HSA and p31 share a common protein core. Differences in the molecular weights of the antigens most likely reflect variations in the extent of post-translational modifications. From these studies we conclude that these antigens are members of the same family of heat-stable antigens. Our results lead us to speculate on how these molecules are related, their function, and what role they play in cellular differentiation in hematolymphoid and neural cell development.

Characterization of the murine heat-stable antigen: an hematolymphoid differentiation antigen defined by the J11d, M1/69 and B2A2 antibodies.

The heat-stable antigen (HSA) is a marker of hematopoietic differentiation in both the B and T cell lineages. The antigen is recognized by a series of monoclonal antibodies which includes J11d, M1/69 and B2A2, and in addition YBM5.10.4. We show here that all these antibodies recognize the same antigenic determinant which is expressed on a variably glycosylated membrane protein. Tunicamycin experiments show that the antigen is not carbohydrate in nature as it is expressed on two unglycosylated protein core molecules of molecular mass ca. 20 kDa and 17 kDa. Furthermore, the antigenic determinant appears to be lost following phosphatidylinositol-specific phospholipase C cleavage. Although the molecular mass of HSA appears to be heterogenous on cells of different lineages, these variations in size appear to be due primarily to differences in the extent of N-linked glycosylation, since both protein core molecules were found in all cell types investigated which express the antigen. These findings have important implications for the structure and function of this antigen and its role in hematopoietic development.