A chromosomal translocation causing multiple abnormalities including open eyelids at birth and glomerulonephritis

We have characterized the phenotype of a mouse with a t(2;13) reciprocal translocation induced by chlorambucil. It results in abnormal eyelid formation as well as a series of neurological, physiological, and immunological abnormalities. This mutant has been termed T(2;13)1Fla/+. T(2;13)1Fla/+ mice exhibit open eyelids at birth, a dilute coat color, hyperactivity, and occasional circling and stargazing activity. At 1-6 months, T(2;13)1Fla/+ mice show signs of immune complex-mediated glomerulonephritis and die prematurely. Additionally, double-stranded DNA autoantibodies have been found in sera of T(2;13)1Fla/+ mice. Cytogenetic analysis situated the translocation breakpoint at the proximal end of Chromosome (chr) 2 at band A2, and on Chr 13 at band A4. The mutant phenotype completely correlated with the presence of the translocation. Additional genetic studies have mapped the mutation and translocation breakpoint to Chr 13 between D13Mit16 and D13Mit64, and to Chr 2 proximal to D2Mit5. By fluorescent in situ hybridization (FISH), the position of this mutation/translocation on Chr 13 has been mapped to a region less than 1cM from D13Mit61.     

Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

我国人红细胞膜MiⅢ血型糖蛋白的基因结构分析

在１１４名青年人中筛查出的两例血型糖蛋白（ＧＰ）变种,携有ＭｉⅢ基因特殊的变异片段．先证者均为海南省黎族男性,属杂合子．借助聚合酶链式反应（ＰＣＲ）获得跨越ＭｉⅢ基因外显子２～４的基因组序列,再进行被扩增ＤＮＡ的直接测序,确证其基因结构属（δ－α－δ）杂化体．通过基因的微转换机理,δ基因的假外显子在其３＇端同显示５＇剪接信号的α基因外显子３及内含子３融合,前两者形成一组合外显子３．ＭｉⅢ基因的近端（δ－α）断点（Ｂｒｅａｋｐｏｉｎｔ）在组合外显子３内（第８２５～８５０位核苷酸）;而远端（α－δ）断点则在内含子３内（第９０４～９６８位核苷酸）．     

Characterization of the recombination hot spot involved in the genomic rearrangement leading to the hybrid D-CE-D gene in the D(VI) phenotype.

In the Caucasian population, the RH locus of RhD-positive individuals is composed of two homologous genes, RHD and RHCE, arranged in tandem but of a single gene, RHCE, in RhD-negative individuals. Many variants recently characterized carry rearranged RH genes, most often by an unidirectional segmental DNA-exchange (gene-conversion) event. In D(VI) variants of type II, RHD is a D-CE-D hybrid gene in which the DNA fragment carrying exons 4-6 has been replaced by the corresponding sequences from the RHCE gene. To identify precisely and characterize the two transition sites, we have studied, by both PCR and sequence analysis, a genomic region between the 3' end of intron 3 and exon 7 in normal RHCE and RHD genes as well as in D(VI) DNA. We show that the D-CE breakpoint is located in intron 3, within a 250-bp fragment comprising an Alu S sequence, and that the CE-D breakpoint lies within a 39-bp fragment in intron 6. This Alu S sequence (and the 100-bp region immediately downstream) most likely defines a recombination hot spot, since there lies also the 5' breakpoint of different rearrangement events leading to D-CE and CE-D transitions in hybrid D(VI),DFR and Dc-,R(N) gene complexes, respectively.     

The human hyaluronan synthase genes: genomic structures,proximal promoters and polymorphic microsatellite markers

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.     

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.     

Localization of the gene for the erythroid anion exchange protein, band 3 (EMPB3), to human chromosome 17

We have isolated genomic DNA clones which code for the human erythroid membrane protein band 3 (EMPB3). The identification of the gene has been confirmed by comparison of the amino acid sequence derived from the nucleotide sequence for two restriction fragments from the 5' end of the gene. Two exons have been identified. One exon encodes 20 amino acids which are identical to residues 36 to 56 of the band 3 protein, and the other encodes 44 amino acids homologous to residues 118 to 162. Southern analysis of genomic DNA derived from a panel of rodent-human somatic cell hybrids, which retain different complements of human chromosomes, with band 3 probes has allowed us to localize EMPB3 to human chromosome 17. The gene has been further localized between 17q21 and qter by analysis of DNA from somatic cell hybrids which carry derivative chromosomes from translocations involving chromosome 17 and either chromosome 15 or 21.     

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.     

An Alu-mediated deletion at Xp11.23 leading to Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema and immunodeficiency of varying severity. The WASP gene, mutations of which are responsible for the phenotype, maps to Xp11.23. We describe here a patient with a large deletion in the Xp11.23 region. The deletion, which totals 15.8 kb, begins downstream of DXS1696 and encompasses 13 kb upstream of WASP and includes the distal and proximal promoters and exons 1-6. Analysis of the 5'-boundary region identified sequences missing in the Human Genome database and, as a result, the normal DNA sequence was revised to include 743 bp of novel sequence (AF466616). The patient's upstream breakpoint was localized to an AluSg element within a highly repetitive DNA region containing other Alu elements. A 26-bp recombinogenic element is located downstream of the 5' breakpoint. A 16-bp sequence just upstream of the 5' breakpoint shares close homology with the sequence that spans the 3' breakpoint in intron 6. A heptanucleotide of unknown origin, CAGGGGG, links the 5' and 3' breakpoints. To our knowledge this is the largest deletion in a WAS patient.     

An interstitial deletion in Xp22.3 in a family with X-linked recessive chondrodysplasia punctata and short stature

Summary In a four-generation family, chondrodysplasia punctata was found in a boy and one of his maternal uncles. These two patients also have short stature, as do all female members of the family. DNA molecular analysis of the pseudoautosomal and Xp22.3-specific loci revealed the presence of an interstitial deletion that cosegregates with the phenotypic abnormalities. The proximal breakpoint of this deletion was located distal to the DXS31 locus and the distal breakpoint in the pseudoautosomal region between DXYS59 and DXYS17. This maps the recessive X-linked form of chondrodysplasia punctata between the proximal boundary of the pseudoautosomal region and DXS31, and an Xp gene controlling growth between DXYS59 and DXS31.     

The structure and population genetics of the breakpoints associated with the cosmopolitan chromosomal inversion In(3R)Payne in Drosophila melanogaster

We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

Subcongenic analyses reveal complex interactions between distal chromosome 4 genes controlling diabetogenic B cells and CD4 T cells in nonobese diabetic mice

Autoimmune type 1 diabetes (T1D) in humans and NOD mice results from interactions between multiple susceptibility genes (termed Idd) located within and outside the MHC. Despite sharing ～88% of their genome with NOD mice, including the H2(g7) MHC haplotype and other important Idd genes, the closely related nonobese resistant (NOR) strain fails to develop T1D because of resistance alleles in residual genomic regions derived from C57BLKS mice mapping to chromosomes (Chr.) 1, 2, and 4. We previously produced a NOD background strain with a greatly decreased incidence of T1D as the result of a NOR-derived 44.31-Mb congenic region on distal Chr. 4 containing disease-resistance alleles that decrease the pathogenic activity of autoreactive B and CD4 T cells. In this study, a series of subcongenic strains for the NOR-derived Chr. 4 region was used to significantly refine genetic loci regulating diabetogenic B and CD4 T cell activity. Analyses of these subcongenic strains revealed the presence of at least two NOR-origin T1D resistance genes within this region. A 6.22-Mb region between rs13477999 and D4Mit32, not previously known to contain a locus affecting T1D susceptibility and now designated Idd25, was found to contain the main NOR gene(s) dampening diabetogenic B cell activity, with Ephb2 and/or Padi2 being strong candidates as the causal variants. Penetrance of this Idd25 effect was influenced by genes in surrounding regions controlling B cell responsiveness and anergy induction. Conversely, the gene(s) controlling pathogenic CD4 T cell activity was mapped to a more proximal 24.26-Mb region between the rs3674285 and D4Mit203 markers.