Increased frequency of the mannose-binding lectin LX haplotype in Chinese systemic lupus erythematosus patients.

Mannose-binding lectin (MBL) is an important complement-activating protein of the human immune system. As a result of one of three structural gene mutations in exon 1 (variants B, C and D) and/or the presence of a low-efficiency promoter polymorphism, MBL deficiency may be associated with increased susceptibility to infectious diseases and to autoimmune disorders, including systemic lupus erythematosus (SLE). Using a combined approach of heteroduplex generator and polymerase chain reaction, a systematic search for mutations in exon 1 and the promoter region of the MBL gene was performed in a Chinese study population comprising 41 SLE patients and 111 healthy controls. Two alleles, a wild-type allele A and a variant allele B (a previously reported mutation of GGC to GAC at codon 54), were identified in MBL exon 1. The frequency of the B allele (0.15) was higher in the SLE patients than in the healthy controls (0.09), but the difference did not attain statistical significance (P > 0.05). However, for two polymorphisms at positions -550 and -221 in the promoter region, the frequency of the low-MBL-producing haplotype (LX) in the patients (0.2073) was significantly higher than that in the controls (0.0855) (P = 0.003, relative risk = 2.79). Our results suggest that the LX haplotype represents a strong risk factor among Chinese SLE patients. Although of lesser importance, the MBL B allele also may be a risk component in the developing process of SLE in Chinese patients.

An interaction between the IL-4Ralpha gene and infection is associated with atopic eczema in young children.

BACKGROUND: A gain of function mutation (Q551- > R) in the IL-4 receptor alpha-chain (IL-4Ralpha) has been found to be associated with atopy in some studies but not others. The different results may be explained by interactions between the IL-4Ralpha polymorphism and environmental factors. OBJECTIVES: To identify interactions between the R551 mutation and environmental factors that are associated with atopy. METHODS: DNA from the Children in Focus (CiF) cohort of the Avon Longitudinal Study of Parents and Children (ALSPAC) was genotyped by heteroduplex formation for the presence of the R551 polymorphism. The data were then analysed for associations with flexural eczema as an indicator of atopic eczema, skin prick tests to allergens and serum IgE levels, and for interactions with environmental factors. RESULTS: A significant (P = 0.02) positive association was seen between the R551 polymorphism and flexural eczema in children up to 6 months of age who had not been given antibiotics, but not in children who had been given antibiotics. This association was maintained as a trend until 30 to 42 months of age but was no longer statistically significant. There was no significant association between the R551 polymorphism and positive skin prick tests or levels of serum IgE at 61 months of age, consistent with the effect of the R551 polymorphism being restricted to early life. CONCLUSION: There is an association between the R551 polymorphism and flexural eczema in children at 6 months of age who have not had infection requiring treatment with antibiotics. Restriction of the R551 association with eczema to children who have not had antibiotics lends support to the 'hygiene hypothesis', which states that exposure to infection in childhood can protect against allergic disease.

A new human genetic resource: a DNA bank established as part of the Avon longitudinal study of pregnancy and childhood (ALSPAC).

We describe a unique human DNA resource forming part of the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC), a longitudinal cohort study involving 14 000 children and their families living in a geographically defined area of England. The DNA bank will underpin the search for associations between genetic polymorphisms and common health outcomes. The opportunities to collect blood samples suitable for DNA extraction are necessarily limited, and the samples themselves have often been treated in different ways and have varied storage histories. With the need to maximise yields, the choice of DNA extraction method is critical to the success of the bank and we have investigated the suitability of various commercial and in-house methods of DNA extraction. Various steps have been taken to minimise errors in sample address and identification, including the use of a pipetting robot for dilution and transfer of samples between 96-well arrays to provide aliquots suitable for PCR. The robot has been programmed to cope with concentrated viscous DNA solutions.

Mannose-binding lectin: structure, function, genetics and disease associations.

Mannose-binding lectin (MBL), a serum protein characterised by both collagenous regions and lectin domains, plays an important role in innate immune defence. It binds to the repeating sugar arrays on many microbial surfaces through multiple lectin domains and, following binding, is able to activate the complement system via an associated serum protease, MASP-2. Serum levels of MBL are influenced by three mutations clustered in exon 1 of the gene and are further modulated by various promoter region polymorphisms. The exon 1 mutations lead to secondary structural abnormalities of the collagenous triple helix and a failure to form biologically functional higher order oligomers. There is an increased incidence of infections in individuals with such mutations and an association with the autoimmune disorders SLE and rheumatoid arthritis. Nevertheless, MBL genotyping of various populations has led to the suggestion that there may be some biological advantage associated with absence of the protein. These and other findings suggest that the concept of MBL as a protein involved solely in first line defence is an oversimplification and the protein should rather be viewed as having a range of activities including disease modulation.

Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates.

The TATA-binding protein (TBP) is a factor required for the transcription of all classes of eukaryotic genes. Here, we demonstrate that in the mouse the TBP-encoding gene (Tbp) resides next to the proteasomal subunit C5-encoding gene (Psmb1). The genes are located on mouse chromosome 17 in the t complex within the Hybrid sterility 1 (Hst1) region. We demonstrate that the homologous human genes (TBP AND PSMB1) are tightly linked on the long arm of chromosome 6, in a region syntenic with the proximal part of mouse chromosome 17. The mouse Tbp and Psmb1 and the human TBP and PSMB1 genes are transcribed in the opposite orientation. The TATA-binding protein and proteasomal subunit C5 genes are also linked on chromosome III of Caenorhabditis elegans, and together they are linked to other genes whose homologs map to human chromosome 6 and mouse chromosome 17. In the Drosophila genome, the housekeeping TATA-binding protein gene maps close to two other genes with homologs in the mammalian major histocompatibility complex. There thus exists conserved synteny of unrelated genes between mammals and invertebrates.

Isolation of candidate hybrid sterility 1 genes by cDNA selection in a 1.1 megabase pair region on mouse chromosome 17.

The Hybrid sterility 1 (Hst1) gene causes male infertility in crosses between certain inbred strains of the laboratory and wild mouse, Mus musculus. To identify the causative gene, we have searched YAC clones encompassing the Hst1 region for testis-expressed sequences, using the cDNA selection method. We isolated 12 non-overlapping cDNA clones, sequenced them, and placed them on a physical map based on the analysis of YAC clones and total genomic DNA. The cDNA clones map to ten loci. Three cDNA sequences correspond to the proteasome subunit C5 (locus Psmb1), ornithine decarboxylase (Odc-rs15), and penta-zinc finger (Zfp91-rs1) transcripts. Three of the ten testis-expressed loci described in this report (D17Ph4e, Psmb1, and Zfp91-rs1) co-segregate with all Hst1 recombinants and, together with the Tbp gene, are therefore potential candidates for the Hst1 gene. The presented physical and genetic mapping data indicate there are no gross rearrangements distinguishing the Hst1(f) and Hst1(s) alleles.

Xist expression from an Xist YAC transgene carried on the mouse Y chromosome.

We have constructed mouse transgenic lines carrying a YAC clone encompassing the Xist gene in order to investigate the factors influencing Xist expression and the initiation of X-inactivation. Two transgenic lines were derived, one carrying four copies integrated at an autosomal site and a second line carrying four copies integrated at a single site on the Y chromosome. Xist expression was not observed in mice carrying the autosomal insertion. However, Xist expression from the Y-inserted transgenes was observed and at levels commensurate with that found in normal female mice. Methylation sites in the autosomal transgene both 5' and 3' of the Xist gene are hypermethylated and appear to reflect methylation patterns observed on the active X chromosome. For the Y-linked transgene, methylation sites 5' and 3' of the Xist gene are hypomethylated reflecting patterns found on the inactive X chromosome. However, the 5' and 3' methylation levels have been decoupled at the active transgenic locus. The data suggest that sequences in the vicinity of Xist can initiate some of the features that are associated with the initiation process of X-inactivation.

Sub-milliMorgan map of the proximal part of mouse Chromosome 17 including the hybrid sterility 1 gene.

We have generated a high-resolution genetic map, 0.071 cM per backcross animal, of the 13 cM T-H2 region of the mouse Chromosome (Chr) 17. The map contains two phenotypic loci, T and Hst1, 12 RFLP markers, and 24 microsatellite loci. The Hst1 gene was mapped to a chromosomal interval contained within a single 580-kb YAC clone. The FFEH11 YAC is 0.44 cM long and carries, besides the Hst1 gene, five polymorphic DNA markers and recombination breakpoints of six backcross animals. Two candidate genes for Hst1 were identified based on their location and testicular expression. These are Tbp and D17Ph4e. The submilliMorgan map of the T-H2 region revealed significant clustering of (CA)n loci. The clustering, if shown to be a common feature in the mouse genome, may cause gaps in the physical map of the mouse genome.

Repetitive sequence fingerprinting in the long range mapping of mammalian genomes.

This review presents some properties of interspersed repeats, particularly human and mouse repeats, and shows how these have been utilized in long-range genome mapping. The link between the distribution of such repeats and their relationship with genome organization is discussed.

Physical map of mouse chromosome 17 in the region relevant for positional cloning of the Hybrid sterility 1 gene.

Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mapped and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene.

A YAC contig spanning the hypophosphatemic rickets disease gene (HYP) candidate region.

Dominant X-linked hypophosphatemic rickets (HYP) is the most common form of familial rickets. Linkage studies have localized the gene for this disorder to Xp22.1 between the markers DXS365 and DXS274, a region estimated to be approximately 3.5 cM. We have constructed a 1.5-Mb YAC contig encompassing this region by hybridization screening of high-density YAC clone filters. Rapid chromosome walking was achieved by direct hybridization of a pool of Alu-PCR products derived from a YAC containing DXS365 to the filter grids. Overlaps between YACs in the contig were estimated by hybridization of end probes to YAC digest blots and by analysis of cosmid fingerprints obtained by hybridization of YAC inserts to a flow-sorted chromosome X cosmid library. All YACs in the contig have been verified by fluorescence in situ hybridization. Several YACs spanning the HYP gene candidate region were selected for further analysis by rare-cutter enzyme digestion and pulsed-field gel electrophoresis. We estimate that the markers flanking the disease region, DXS365 and DXS274, are less than 1 Mb apart. This clone contig map provides an essential resource for the isolation of the HYP gene.

A 1.2-Mb YAC contig spans the quaking region.

We describe here a 1.2-Mb yeast artificial chromosome (YAC) contig within the region of mouse chromosome 17 between Brachyury (T) and D17Rp17e, and spanning the quaking (qk) region. We describe six new probes distributed across 1.2 Mb: D17Leh502, D17Leh503, D17Leh504, D17Leh505, D17Leh506, and D17Leh507. Probes D17Leh502 and D17Leh507 are at the extreme ends of the YAC contig. With the exception of D17Leh507, all of these probes are within a deletion associated with the quaking(viable) (qkv) allele of quaking. We have positioned these probes on a detailed YAC physical map together with two previously published probes, D17Leh508 and D17Aus119. We show here that D17Leh508 is also within the qkv deletion. Genetic mapping of D17Leh504 and D17Leh507 on two high-resolution genetic crosses carrying qkv and quaking(lethal-1) (qkl-1) alleles shows that these probes do not recombine with quaking and are therefore within 0.04 cM of qkv and 0.05 cM of qkl-1 mutations. The deletion breakpoint contained within the YAC contig has been positioned to within 90 kb by restriction mapping of wildtype and mutant DNA. This contig will form the basis for identification and mapping of expressed sequences and for an investigation of genome organization.

An integrated YAC-overlap and 'cosmid-pocket' map of the human chromosome 21.

We describe here the construction of an ordered clone map of human chromosome 21, based on the identification of ordered sets of YAC clones covering > 90% of the chromosome, and their use to identify groups of cosmid clones (cosmid pockets) localised to subregions defined by the YAC clone map. This is to our knowledge the highest resolution map of one human chromosome to date, localising 530 YAC clones covering both arms of the chromosome, spanning > 36 Mbp, and localising more than 6300 cosmids to 145 intervals on both arms of the chromosome. The YAC contigs have been formed by hybridising a 6.1 equivalents chromosome 21 enriched YAC collection displayed on arrayed nylon membranes to a series of 115 DNA markers and Alu-PCR products from YACs. Forty eight mega-YACs from the previously published CEPH-Genethon map of sequence tagged sites (STS) have also been included in the contig building experiments. A YAC tiling path was then size-measured and confirmed by gel-fingerprinting. A minimal tiling path of 70 YACs were then used as probes against the 7.5 genome equivalents flow sorted chromosome 21 cosmid library in order to identify the lists of cosmids mapping to alternating shared--non-shared intervals between overlapping YACs ('cosmid pockets'). For approximately 1/5 of the minimal tiling path of YACs, locations and non-chimaerism have been confirmed by fluorescence in situ hybridisation (FISH), and approximately 1/5 of all cosmid pocket assignments have independent, confirmatory marker hybridizations in the ICRF cosmid reference library system. We also demonstrate that 'pockets' contain overlapping sets of cosmids (cosmid contigs). In addition to being an important logical intermediate step between the YAC maps published so far and a future map of completely ordered cosmids, this map provides immediately available low-complexity cosmid material for high resolution FISH mapping of chromosomal aberrations on interphase nuclei, and for rapid positional isolation of transcripts in the highly resolved regions of genetic interest.

Identification of YAC and cosmid clones encompassing the ZFX-POLA region using irradiation hybrid cell lines.

The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions. To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used. Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH). Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chimerism by FISH. Cosmid content analysis of the YACs defined two contigs, one with 12 YACs covering about 1.5 Mb and one with 3 YACs. Five YACs from the 12-YAC cluster had been previously recognized by DNA polymerase alpha (POLA). ZFX identified a single YAC; hence, the physical linkage of ZFX and POLA was demonstrated within the contig. Four YACs had been isolated previously with ZFX and these extend the contig to 2 Mb. Restriction mapping of several YACs demonstrates that ZFX and POLA are about 700 kb apart, a distance similar to that reported in the mouse between Zfx and Pola. The order of these two loci and two additional loci identified by homologous mouse linking clones was found to be conserved between human and mouse: tel-ZFX-DXCrc57-DXCrc140-POLA-cen. We have shown that YAC contigs can be rapidly constructed from targeted regions without the need for time-consuming YAC end rescue and chromosomal walking.(ABSTRACT TRUNCATED AT 250 WORDS)