Molecular characterization of a new alpha-1-antitrypsin M variant allele, Mwhitstable: implications for DNA-based diagnosis.

The mother and second child from a family, already with one PI ZZ child, were typed PI MZ by isoelectric focusing and unexpectedly as PI ZZ using a commercial alpha-1-antitrypsin genotyping kit. Both methods typed the father and first child as PI MZ and PI ZZ, respectively. DNA sequence analysis identified a 26-base pair (bp) deletion and 2-bp insertion in intron IV of the normal PI*M allele from both the mother and second child. The majority of the binding site for an amplification primer of the genotyping kit was absent in the variant deletion-insertion allele. The apparent PI*Z/PI*Z genotype of the mother and second child therefore arose from amplification of the PI*Z allele alone. Two hundred random DNA samples were subsequently examined and 5 of these were found to be heterozygous for the same deletion-insertion allele. The authors have designated the previously undescribed PI*M allele that harbors this benign polymorphism PI*Mwhitstable. The genotyping kit has been redesigned and revalidated, and its performance is not affected by the presence of the PI*Mwhitstable allele. The Gen Bank accession number for the nucleotide sequence described is AF159454.

Genetic and physical mapping of the ataxia-telangiectasia locus on chromosome 11q22-q23.

The identification of A-T gene(s) using both positional and functional cloning techniques has been a major objective in A-T research over the past 10 years. Functional cloning, using complementation of the radiosensitivity phenotype, has met with some success, although technical problems remain to be overcome. Recent progress, however, in both genetic and physical mapping of the A-T locus on chromosome 11q22-q23, described in this review, suggests that the positional cloning of candidate genes should be achieved in the very near future. The region of the chromosome containing the gene(s) has been identified, and is no more than 1.6 Mb in size. The detailed physical characterization of this region, as a preliminary to candidate gene isolation, is now underway. There are, however, still some unresolved issues, most notably the existence of four A-T complementation groups, with the resulting supposition that these equate to a number of different genes. Although genetic linkage evidence does not support the hypothesis of genetic heterogeneity, the possibility of a cluster of genes at the 11q22-23 locus cannot be ruled out. It is likely that the explanations for this and other problems such as discrepancies in expected levels of consanguinity, and difficulties in the classification of atypical phenotypes will become much more obvious once a gene or genes have been cloned.

Genomic organization of the mouse dystrobrevin gene: comparative analysis with the dystrophin gene.

Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical.

A novel gene, DXS8237E, lies within 20 kb upstream of UBE1 in Xp11.23 and has a different X inactivation status.

A novel X-linked gene, DXS8237E, was isolated from human fetal brain cDNA, and its 3' end was mapped to within 20 kb upstream of UBE1 in Xp11.23. A 1.3-kb cDNA for DXS8237E detects homologous sequences in other mammals and a 3-kb mRNA that is widely expressed in human cell lines and mouse tissues. Sequence analysis indicated that the 1.3-kb cDNA contains the 3' end of the DXS8237E gene, but the sequence shows no significant homology to known genes. DXS8237E was shown to be subject to X inactivation in five somatic cell hybrids that contain an inactive human X chromosome but no active homologue. Since UBE1 escapes X inactivation, DXS8237E and UBE1 are the closest mapped genes with discordant X inactivation profiles. Sequences in the vicinity of these two genes may be important determinants of X inactivation status.

Tissue-selective expression of alpha-dystrobrevin is determined by multiple promoters.

alpha-Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a dystrophin-associated and dystrophin-related protein. Knockout of the gene in the mouse results in muscular dystrophy. The control of the alpha-dystrobrevin gene in the various tissues is therefore of interest. Multiple dystrobrevin isoforms differing in their domain content are generated by alternative splicing of a single gene. The data presented here demonstrate that expression of alpha-dystrobrevin from three promoters, that are active in a tissue-selective manner, also plays a role in the function of the protein in different tissues. The most proximal promoter A is active in brain and to a lesser extent in lung, whereas the most distal promoter B, which possesses several Sp1 binding sites, is restricted to brain. Promoter C, which contains multiple consensus myogenic binding sites, is up-regulated during in vitro myoblast differentiation. Interestingly, the organization and the activity of the alpha-dystrobrevin promoters is reminiscent of those in the dystrophin gene. Taken together we suggest that the multipromoter system, distributed over a region of 270 kilobases at the 5'-end of the alpha-dystrobrevin gene, has been developed to allow the regulation of this gene in different cell types and/or different developmental stages.

Genomic organization and refined mapping of the mouse beta-dystrobrevin gene.

beta-Dystrobrevin, a dystrophin-related protein that is expressed in non-muscle tissues, is highly homologous to alpha-dystrobrevin, a member of the dystrophin-associated protein complex (DPC). beta-Dystrobrevin associates with Dp71 and syntrophin and is believed to have a role in non-muscle DPCs. Here we report the characterization and mapping of the mouse beta-dystrobrevin gene. The mouse beta-dystrobrevin gene is organized into 21 exons spanning over 130 kb of DNA. We provide evidence that this gene is transcribed from at least two promoter regions but appears to utilize a common translation initiation site. We show that the similarity between beta-dystrobrevin and alpha-dystrobrevin is reflected in the conservation of their exon-intron junctions. beta-Dystrobrevin has been localized to proximal mouse Chromosome (Chr) 12 by backcross mapping. A database search revealed that two mouse genetic diseases involving tissues expressing beta-dystrobrevin have been mapped to this region, namely, congenital polycystic kidneys (cpk) and fatty liver dystrophy (fld). However, refined mapping analysis has excluded beta-dystrobrevin as a candidate gene for either disease.

Paired STSs amplified from radiation hybrids, and from associated YACs, identify highly polymorphic loci flanking the ataxia telangiectasia locus on chromosome 11q22-23.

The high resolution mapping of the ataxia telangiectasia (A-T) locus on chromosome 11q22-23 requires the generation of new polymorphic markers specifically within the segment of 11q22-23 to which the locus has been assigned. We have made use of a library of Alu-PCR clones, amplified from a radiation reduced somatic cell hybrid containing the relevant chromosome 11 segment, to generate sequence tagged sites (STS) within the 11q22-23 region and have used YAC clones to extend the loci identified by these STSs. The identification of paired polymorphisms (from Alu-PCR and the associated YAC derived clone), which are physically linked, but which show minimal linkage disequilibrium, provides a highly informative haplotype for use in genetic linkage analysis in A-T families. We describe the characterisation of 2 such polymorphic loci, D11S535 and D11S611, which map between existing flanking markers, and which provide additional information on the location of the major A-T locus.

The order and orientation of a cluster of metalloproteinase genes, stromelysin 2, collagenase, and stromelysin, together with D11S385, on chromosome 11q22-q23.

A cluster of metalloproteinase genes, stromelysin, fibroblast collagenase, and stromelysin 2 together with the anonymous DNA marker D11S385, was mapped using pulsed-field gel electrophoresis to a 135-kb region of chromosome 11q22-q23. The physical proximity of these markers was subsequently confirmed using two YAC clones, and their relative order was established as stromelysin 2-collagenase-stromelysin-D11S385. The pattern of marker representation in a panel of radiation-reduced chromosome 11 hybrids suggests that the metalloproteinase gene/D11S385 cluster is orientated with STMY2 closest to the centromere.

A member of the MAP kinase phosphatase gene family in mouse containing a complex trinucleotide repeat in the coding region.

We have identified a novel mouse gene encoding a protein that shows high homology to the dual-specificity tyrosine/threonine phosphatase family of proteins. The gene encodes a 5 kb transcript which is expressed predominantly in brain and lung and contains a translated complex trinucleotide repeat within the coding region. Using interspecific mouse backcross analysis, the gene has been localised to distal mouse chromosome 7. In human, homologous sequences are located in the syntenic region on distal chromosome 11p as well as to chromosome 10q11.2 and 10q22. The presence of a CG-rich trinucleotide repeat in the coding region provides a target for mutation which might result in loss of function or altered properties of this phosphatase.

A physical map across chromosome 11q22-q23 containing the major locus for ataxia telangiectasia.

We have constructed a long-range physical map for 12 markers, including genes for GRIA4, IL1BC, and ACAT, across 9 Mb of chromosome 11q22-q23 in the region of the major locus for ataxia-telangiectasia (A-T). The markers fall into proximal and distal groups with respect to the centromere. We have linked the proximal and distal groups by hybridization to a 2.7-Mb NotI fragment and a 4.6-Mb MluI fragment. The following locus order was obtained: centromere-CJ52.75-J12.1C2-Y11B11R-IL1BC-+ ++hbcDNA-GRIA4-CJ52.3-Y11B29L-ACAT- CJ52.193-J12.8-Y11B06R-telomere. We show that hbcDNA/GRIA4 and CJ52.3 are very closely linked to each end, respectively, of the 2.7-Mb NotI fragment, thereby fixing the position of the complete contig. Our results indicate that the gene for A-T is flanked by the markers GRIA4 and J12.8, which are no more than 3 Mb apart, on a 4.6-Mb MluI fragment. The physical map allows rapid positioning of markers, and this will facilitate the construction of a YAC contig across the region.