Haplotype and linkage disequilibrium analysis to characterise a region in the calcium channel gene CACNA1A associated with idiopathic generalised epilepsy

Idiopathic generalised epilepsy (IGE) is a common form of epilepsy, including several defined and overlapping syndromes, and likely to be due to the combined actions of mutations in several genes. In a recent study we investigated the calcium channel gene CACNA1A for involvement in IGE, unselected for syndrome, by means of association studies using several polymorphisms within the gene. We reported a highly significant case/control association with a silent single nucleotide polymorphism (SNP) in exon 8 that we confirmed by within-family analyses. In this present study we screened the gene for novel SNPs within 25 kb of exon 8, which have enabled us to define the critical region of CACNA1A in predisposing to IGE. Several intronic SNPs were identified and three, within 1.5 kb of exon 8 and in strong linkage disequilibrium with each other and with the original SNP, were significantly associated with IGE (P=0.00029, P=0.0015 and P=0.010). The associations were not limited to an IGE syndrome or other subgroup. Another SNP, 25 kb away, in intron 6 was also significantly associated with IGE (P=0.0057) but is not in linkage disequilibrium with the SNPs around exon 8. Haplotype predictions revealed even more significant associations (3-marker haplotype: P<10(-6)). Logistic regression showed that all the data can be explained by two of the SNPs, which is consistent with two functionally significant variants being responsible for all five associations, although a single variant cannot be excluded. The functionally significant variant(s) are unlikely to be exonic and suggests an effect on expression or alternative splicing.     

Lupus susceptibility genes on human chromosome 1

During the past five years, there has been an intense interest in studying candidate susceptibility genes for systemic lupus erythematosus (SLE). Many such studies have been focused on candidates located on chromosome 1, demonstrating association of certain genetic variants with SLE. Some of the tested candidate genes were chosen because they encode molecules with relevant immunological functions that may play a role in the pathogenesis of SLE. More recently, the identification of genomic segments linked to SLE has suggested novel positional candidate genes. Thus far, there is considerable evidence supporting that multiple genes on this chromosome contribute to the development and expression of SLE. This review highlights the genetic loci located on chromosome 1 that have recently been associated with SLE. These include loci encoding the tumor necrosis factor receptor 2 (TNFR2), complement component C1q, Fcgamma receptors, T cell receptor zeta chain, interleukin-10 (IL-10), poly (ADP-ribose) polymerase (PARP), and HRES-1.     

Investigation of the HIN200 locus in UK SLE families identifies novel copy number variants

We undertook a candidate locus study of the HIN200 gene cluster on 1q21-23 in UK systemic lupus erythematosus (SLE) families. To date, despite mounting evidence demonstrating the importance of these proteins in autoimmune disease, cancer, apoptosis, inflammation, and cell cycle arrest, there has been a dearth of data with respect to the genetic characterisation of the HIN200 locus in SLE or any other disease. We typed 83 single nucleotide polymorphisms (SNPs) across 317 kb of the HIN200 cluster in 428 UK SLE families and sought replication from a European-American lupus cohort. We do not find strong evidence of SNP association in either cohort. Interestingly, we do observe a trend for association with certain HIN200 SNPs and serologic subphenotypes in UK SLE that parallels the association of lupus antibodies with the orthologous murine locus. Furthermore, we find the HIN200 locus to be unexpectedly complex in terms of genetic structural organisation. We have identified a number of copy number variants (CNVs) in this region in healthy French males, HapMap samples, and UK SLE families. In summary, candidate interferon signalling genes show evidence of common CNV in human SLE and healthy subjects. The impact of these CNVs in health and disease remains to be determined.     

Evidence for unique association signals in SLE at the CD28-CTLA4-ICOS locus in a family-based study

CD28, CTLA4 (cytotoxic T lymphocyte-associated protein 4) and ICOS (inducible T cell co-stimulator) are good candidate genes for systemic lupus erythematosus (SLE) because of their role in regulating T cell activation. CTLA4 inhibits CD28-mediated T cell activation. CTLA4 is expressed on CD4+ and CD8+ activated T cells, and also B cells, but CD28 and ICOS are largely restricted to T cells. An interval encompassing the CD28-CTLA4-ICOS locus on chromosome 2q33 was linked to lupus in two genome-wide linkage scans. This large family-based association study in 532 UK SLE families represents the first high-density genetic screen of 80 SNPs at this locus. There are seven haplotype blocks across the locus. In CTLA4, the strongest signal comes from two variants, located 2.1 kb downstream from the 3'-UTR. These polymorphisms, rs231726 (SNP 43) and rs231726 (SNP 44), are in complete linkage disequilibrium (LD) (r(2)=1) and are associated with SLE P=0.0008 (GH) and P=0.01 (family-based association test). There is also a signal in the distal 3' flanking region of CTLA4/ICOS promoter (P=0.003). There was no confirmation of published associations for SLE in the promoter or coding region of CTLA4. These SLE risk alleles are more distal than those identified in Graves' disease and are in LD with Graves' disease protective alleles identified in both of these regions of CTLA4 (Ueda et al. 2003). These factors suggest an SLE-specific pattern of association. The functional consequences of the associated polymorphisms are likely to influence CTLA4 expression, although it is possible that genetically modulated ICOS expression is involved in SLE susceptibility.     

Evidence for two independent associations with type 1 diabetes at the 12q13 locus

Genome-wide association studies have identified associations between type 1 diabetes and single-nucleotide polymorphisms (SNPs) at chromosome 12q13, surrounding the gene ERBB3. Our objective was to fine map this region to further localize causative variants. Re-sequencing identified more than 100 putative SNPs in an 80-kb region at 12q13. By genotyping 42 SNPs, spanning ～214?kb, in 382 affected sibling pair type 1 diabetes families, we were able to genotype or tag 67 common SNPs (MAF≥0.05) identified from HapMap CEU data and CEU data from the 1000 Genomes Project, plus additional rare coding variants identified from our re-sequencing efforts. In all, 15 SNPs provided nominal evidence for association (P≤0.05), with type 1 diabetes. The most significant associations were observed with rs2271189 (P=4.22 × 10(-5)), located in exon 27 of the ERBB3 gene, and an intergenic SNP rs11171747 (P=1.70 × 10(-4)). Follow-up genotyping of these SNPs in 2740 multiplex type 1 diabetes families validated these findings. After analyzing variants spanning more than 200?kb, we have replicated associations from previous GWAS and provide evidence for novel associations with type 1 diabetes. The associations across this region could be entirely accounted for by two common SNPs, rs2271189 and rs11171747.     

Monocyte-dependent cell death of T lymphocyte subsets in chronic hepatitis C

Much evidence indicates that atherosclerotic lesions are largely of an inflammatory nature. Activated macrophages and macrophage-derived foam cells laden with cholesterol esters are a major constituent of these lesions and can influence lesion formation via several potential mechanisms. One such mechanism is Fcgamma receptor activation and/or Fcgamma receptor-mediated clearance of immune complexes containing cholesterol, such as lipoprotein immune complexes. That this mechanism contributes to lesion formation would be further supported if Fcgamma receptor expression in arterial lesions were demonstrated. We therefore used monoclonal antibodies and immunocytochemical methods to analyze frozen sections of human arterial lesions for expression of each of the three primary classes of mononuclear phagocyte Fcgamma receptors. Approximately 800 sections of aorta, carotid, and coronary arteries obtained from five elderly donors were analyzed. The presence of macrophages was determined by assaying reactivity of a monoclonal antibody specific to CD163, which is expressed only on cells of the human mononuclear phagocyte lineage. Results indicate that highly cellular preatheromatous lesions contained numerous macrophages in the zone of proliferation that expressed each class of Fcgamma receptor (FcgammaRIA, FcgammaRIIA, and FcgammaRIIIA). Fcgamma receptor-positive cells were also present in medial and adventitial areas. Fcgamma receptor staining was both punctate and diffuse, the latter suggesting that soluble receptors were present in the extracellular matrix. These data further support that Fcgamma receptor-mediated clearance of immune complexes can occur in arterial lesions during atherogenesis. Expression of both the high affinity (FcgammaRIA) and lower affinity (FcgammaRIIA/FcgammaRIIIA) receptors indicates that mono- and multivalent IgG-containing immune complexes could engage Fcgamma receptors and influence lesion formation through several different inflammatory mechanisms triggered by receptor activation.     

Single nucleotide polymorphism screening and association analysis – exclusion of integrin β7 and vitamin D receptor (chromosome 12q) as candidate genes for asthma

BACKGROUND: The human genes coding for integrin beta 7 (ITGB7) and vitamin D receptor (VDR) are two of the several candidate genes for asthma and related phenotypes found in a promising candidate region on chromosome 12q that has been identified in multiple genomewide screens and candidate gene approaches. METHODS: All exons, including parts of the neighbouring introns, and the predicted promoter region of the ITGB7 gene were screened for common polymorphisms in 32 independent asthmatic and healthy probands, resulting in the detection of two single nucleotide polymorphisms (SNPs) unknown so far. In addition to these SNPs, five already described SNPs of the ITGB7 and one in the human VDR gene were analysed in a Caucasian sib pair study of 176 families with at least two affected children, using matrix assisted laser desorption/ionization time of flight mass spectrometry. All confirmed SNPs were tested for linkage/association with asthma and related traits (total serum IgE level, eosinophil cell count and slope of the dose-response curve after bronchial challenge). RESULTS: Two new variations in the ITGB7 gene were identified. The coding SNP in exon 4 causes a substitution of the amino acid GLU by VAL, whereas the other variation is non-coding (intron 3). None of the eight analysed SNPs, of either the ITGB7 or the VDR genes, showed significant linkage/association with asthma or related phenotypes in the family study. CONCLUSIONS: These findings indicate that neither the human ITGB7 nor the VDR gene seem to be associated with the pathogenesis of asthma or the expression of related allergic phenotypes such as eosinophilia and changes in total IgE level.     

Haplotype analysis of tumour necrosis factor receptor genes in 1p36: no evidence for association with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with partially understood aetiology. The 1p36 region has been previously linked with SLE and harbours tumour necrosis factor receptor (TNFR) genes. Functional and genetic data implicate their gene products in SLE and other autoimmune diseases. In all, single-nucleotide polymorphisms (SNPs) across TNFRSF14 (HVEM), and 43 SNPs across the TNFRSF8 (CD30) and TNFRSF1B (CD120B) locus were investigated for linkage disequilibrium (LD) and haplotype analysis in European-Caucasians. Strong LD was observed across HVEM and CD120B, and little LD and recombination across CD30. We also examined the association of SNPs and haplotypes in HVEM, CD30 and CD120B with SLE in European-Caucasians. There was no evidence of association for these genes in 456 European-Caucasian families with SLE from UK. Haplotype tagging SNPs are made known across areas of strong LD, which will facilitate analysis for susceptibility in other diseases.     

High density SNP association study of a major autism linkage region on chromosome 17

A region on chromosome 17 has recently been highlighted as linked to autism (MIM[209850]) in multiple studies and evidence has accumulated suggesting that male-only families (those families that have produced only affected males) provide the major contribution to linkage at this locus. In an attempt to comprehensively test for association of common variants to autism within the region on chromosome 17 defined in Stone et al. (Stone, J.L., Merriman, B., Cantor, R.M., Yonan, A.L., Gilliam, T.C., Geschwind, D.H. and Nelson, S.F. (2004) Evidence for sex-specific risk alleles in autism spectrum disorder. Am. J. Hum. Genet., 75, 1117-1123), a dense panel of single nucleotide polymorphisms (SNPs) was selected across the linkage peak and analyzed in a trio-based study design. SNPs were genotyped in 219 independent trios at an average intermarker distance of 6.1 kb across the 13.7 Mb interval. This provided ~80% coverage of common HapMap variation present in Caucasians, testing exonic, intronic, promoter and intergenic regions, as knowledge of important functional regions within the genome is currently limited. In this comprehensive association study of a linkage region in autism, no single SNP or haplotype association was sufficient to account for the initial linkage signal. Nominally significant single SNP and/or haplotype-based association results were detected in 15 genes, of which, MYO1D, ACCN1 and LASP1 stand out as genes with autism risk alleles requiring further study, with potential GRRs in the range of 1.34-2.29.     

A high-density SNP map for the FRAX region of the X chromosome. Single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation within the human genome, occurring approximately once every kilobase. However, for association studies, SNPs are not as informative as microsatellite markers and a large number of SNPs and substantial population sizes are required for linkage and mapping studies. A SNP map was generated for the FRAX region of the X chromosome, approximately 0.8 Mb proximal and 1.8 Mb distal to the FRAXA repeat, at a density of at least 1 SNP every 100 kb. SNPs were identified in a population of 28 women with a FRAXA expan-sion (including three women with a FRAXE expansion) on a background of different DXS548, CA1 and CA2 haplotypes, and a normal X chromosome with a different microsatellite haplotype. Fifty-four polymorphisms were identified in a total of 52 257 bp distributed over 2.6 Mb. This represented about 1 SNP every 1024 bp, which was consistent with a nondesert region (1 : 1000 bp). Because the SNPs identified in this study have haplotype and frequency data from an affected population, they should provide a useful resource for researchers to investigate the genetic mechanisms behind instability and expansion of both FRAXA and FRAXE triplet repeats.     

A cSNP map and database for human chromosome 21

Single nucleotide polymorphisms (SNPs) are likely to contribute to the study of complex genetic diseases. The genomic sequence of human chromosome 21q was recently completed with 225 annotated genes, thus permitting efficient identification and precise mapping of potential cSNPs by bioinformatics approaches. Here we present a human chromosome 21 (HC21) cSNP database and the first chromosome-specific cSNP map. Potential cSNPs were generated using three approaches: (1) Alignment of the complete HC21 genomic sequence to cognate ESTs and mRNAs. Candidate cSNPs were automatically extracted using a novel program for context-dependent SNP identification that efficiently discriminates between true variation, poor quality sequencing, and paralogous gene alignments. (2) Multiple alignment of all known HC21 genes to all other human database entries. (3) Gene-targeted cSNP discovery. To date we have identified 377 cSNPs averaging ~1 SNP per 1.5 kb of transcribed sequence, covering 65% of known genes in the chromosome. Validation of our bioinformatics approach was demonstrated by a confirmation rate of 78% for the predicted cSNPs, and in total 32% of the cSNPs in our database have been confirmed. The database is publicly available at http://csnp.unige.ch or http://csnp.isb-sib.ch. These SNPs provide a tool to study the contribution of HC21 loci to complex diseases such as bipolar affective disorder and allele-specific contributions to Down syndrome phenotypes.     

Presence of four major haplotypes in human BCMA gene: lack of association with systemic lupus erythematosus and rheumatoid arthritis

BCMA (TNFRSF17), along with TACI, has recently been demonstrated to be a receptor for BLyS (TNFSF13B). Recent studies indicated substantial role of BLyS signaling pathway for systemic lupus erythematosus (SLE). In the present study, we made an attempt to screen for polymorphisms of human BCMA, and to test their possible association with SLE and rheumatoid arthritis (RA). Two single nucleotide polymorphisms (SNPs) were detected within the coding sequence, both of which were synonymous substitutions. In addition, two SNPs within the promoter, two SNPs in the 5'-untranslated region (UTR), one SNP and one single nucleotide deletion in the 3'UTR and four rare variations were detected. From the combination of the polymorphisms, it was elucidated that four major haplotypes account for most of the genotypes in the Japanese population. Association with SLE and RA was not detected, although a slight tendency for the increase of BCMA.03 in SLE was observed (P = 0.089). These results indicated that human BCMA is conserved with respect to the amino acid sequence, and evidence for association with SLE and RA was not observed.     

Analysis on the association of human BLYS (BAFF,TNFSF13B) polymorphisms with systemic lupus erythematosus and rheumatoid arthritis

Recent studies indicated a substantial role of BLyS (BAFF, TNFSF13B) in the pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in humans and in animal models. This study was conducted to screen for polymorphisms of human BLYS, and to examine whether they are involved in the genetic susceptibility to human SLE and RA. A systematic polymorphism screening was performed in the coding region, 5' and 3' untranslated regions, and promoter region of human BLYS. Association of the detected polymorphisms with SLE and RA was analyzed in 221 Japanese patients with RA, 156 with SLE, and 227 healthy individuals, using the case-control approach. Four single nucleotide polymorphisms (SNPs) in the promoter, one SNP in intron 1, and one rare nonsynonymous substitution (Ala105Thr) in the coding region were detected. The BLYS SNPs were found to form three common haplotypes. Significant association with the susceptibility to SLE or RA was not observed. However, a tendency for the increase of -871T/T genotype was observed in SLE patients with anti-Sm antibody (P=0.082). BLYS mRNA level was significantly elevated in the monocytes from individuals carrying -871T (P=0.010). In addition, although statistically not significant, 105Thr allele was slightly increased in patients with RA compared with controls (P=0.058). Characterizing the functional and clinical significance of these new SNPs requires further study.     

Linkage disequilibrium of HLA-A11 and A1 with one of the polymorphisms of the gamma-aminobutyric acid receptor type B

The gamma-aminobutyric acid receptor type B 1 (GABA(B) R1) is located approximately at 200 kb telomeric to HLA-A on chromosome 6. It has 11 single-nucleotide polymorphisms (SNPs). We studied the most common of its SNPs (T1974C) in a panel of 118 normal Caucasians from New England and 161 epileptic patients of Caucasian ancestry residing in USA. The frequency of the polymorphism did not differ between patients and controls. Here, we report that the allele C of this SNP in the GABA(B) R1 gene is in linkage disequilibrium with HLA-A11 (P<0.00001) and to a lesser extent with HLA-A1 (P<0.01).     

Prolactin and prolactin receptor gene polymorphisms in multiple sclerosis and systemic lupus erythematosus

Genes encoding for prolactin (PRL) and its receptor (PRLR) are possible candidates for multiple sclerosis (MS) and systemic lupus erythematosus (SLE) susceptibility. In fact: (1) a prolactin secretion dysfunction has been described in several autoimmune diseases including SLE and MS and their animal models; (2) both PRL and PRLR are structurally related to members of the cytokine/hematopoietin family and have a role in the regulation of the immune response; and (3) both PRL and PRLR genes map in genomic regions that showed linkage with autoimmunity. Prolactin maps on chromosome 6p, about 11-kb telomeric to HLA-DRB1 and PRLR in 5p12-13, which revealed evidence of linkage with MS in different populations. To evaluate a possible role of these two genes in SLE and MS we performed an association study of 19 PRL and PRLR single nucleotide polymorphisms (SNPs). These were directly searched by DHPLC in a panel of SLE and MS patients and selected from databases and the literature. The SNP allele frequencies were determined on patient and control DNA pools by primer-extension genotyping and HPLC analysis. Moreover a panel of HLA typed SLE and control individuals were individually genotyped for the PRL G-1149T polymorphism previously described to be associated with SLE. No statistically significant difference in the allele distribution was observed for any of the tested variations.     

Comparative study of the haplotype structure and linkage disequilibrium of chromosome 1p36.2 region in the Korean and Japanese populations

The patterns of linkage disequilibrium (LD) in the human genome provide important information for disease gene mapping. LDs may vary depending on chromosomal regions and populations. We have compared LD and haplotypes defined by SNPs in the chromosome 1p36.2 region of the Korean and Japanese populations. Fifty-eight SNPs in about 418 kb ranging from tumor necrosis factor receptor 2 (TNFR2:TNFRSF1B) to procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) gene were examined in 96 healthy Koreans and Japanese each by direct sequencing and fluorescence correlation spectroscopy combined with the PCR-sequence specific primer method (PCR-SSP-FCS), respectively. Upon pair-wise LD analysis, a total of 25 and 16 out of 58 SNPs greater than MAF 10% were included in LD blocks, encompassing almost 81 kb and 55 kb in total, in Koreans and Japanese, respectively. Both similarities and differences were observed in LD strength and haplotype frequencies between the populations. Considerable similarities were observed in the telomeric region where a long-range block of approximately 80 kb including three genes was found to have strong LDs in both Koreans and Japanese. Significant difference in LD strength was present near the TNFR2 region between the Japanese and Korean populations.     

Novel exonic mu-opioid receptor gene (OPRM1) polymorphisms not associated with opioid dependence.

The mu-opioid receptor (MOR) mediates reward and dependence associated with opioids and other commonly abused substances. Variability in the MOR gene, OPRM1, may influence risk for opioid dependence. In this study, associations between two single nucleotide polymorphisms (SNPs), dbSNP rs540825 and dbSNP rs562859, and opioid dependence were investigated. The two SNPs are located in the protein coding region of the novel exon X of an alternative splice variant of OPRM1, and can be detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Genotyping at the two SNPs was performed for 170 severe opioid dependent individuals and 128 carefully screened controls. Although no differences were found between cases and controls, there were significant prevalence differences between African-American (AA) subjects and European-American (EA) subjects for SNP 540825 allele and genotype frequencies. The 540825 and 562859 polymorphisms were found to be in complete linkage disequilibrium (LD) for both ethnic groups, and LD existed between the 562859 SNP and the A(-1320)G SNP in the promoter region of OPRM1 in AAs, based on genotyping data previously carried out on the same subjects. LD between these two markers, separated by 55 kb, links the entire distance studied in this project. The results indicate that polymorphisms in the novel splice variant are not associated with opioid dependence, but are in LD with other polymorphisms in OPRM1.     

Characterization of the FcgammaRIIA promoter and 5'UTR sequences in patients with systemic lupus erythematosus

FcgammaRIIA is a candidate gene involved in the predisposition to systemic lupus erythematosus (SLE). The presence of low binding alleles in patients with SLE is not sufficient to explain the lower phagocytic capacity observed in SLE patients. We considered the possibility that nucleotide polymorphisms in the FcgammaRIIA promoter that cause alterations in receptor expression might be present in SLE patients. In the present study, a 2.0 kb region of the human FcgammaRIIA 5'UTR from 20 normal donors and 53 SLE patients was examined. The results demonstrate that the sequence of the human FcgammaRIIA 5' region differs from the published sequence. Two novel SNPs have been identified in the distal region of the FcgammaRIIA promoter. The polymorphisms are present in both disease-free and SLE donors and do not associate with quantitative changes in FcgammaRIIa phagocytic function.     

Toward the evaluation of function in genetic variability: characterizing human SNP frequencies and establishing BAC-transgenic mice carrying the human CYP1A1_CYP1A2 locus

Interindividual differences in human CYP1A1 and CYP1A2 expression appear to be associated with variability in risk toward various types of environmental toxicity and cancer. These two genes are oriented head-to-head on human chromosome 15; the 23.3-kb spacer region might contain distinct regulatory regions for CYP1A1 and distinct regulatory regions for CYP1A2, or the regulatory regions for the two genes might overlap one another. From 24 unrelated subjects of five major, geographically-isolated subgroups, we resequenced both genes (all exons and all introns) plus some 3' flanking sequences and the entire spacer region (39.6 kb total); 85 SNPs were found, 49 of which were not currently in the National Center for Biotechnology Information (NCBI) database. Of the 57 double-hit SNPs, we carried out SNP-typing in 94 Africans, 96 Asians, and 83 Caucasians and found striking ethnic differences in SNP frequencies and haplotype evolution; the two CYP1A1 SNPs and the one CYP1A2 SNP that are most commonly used in epidemiological studies were shown not to be representative haplotype tag SNPs across these three human subgroups. Four BAC-transgenic mouse lines, carrying the human CYP1A2 and 15,190 bp of 5' flank, expressed only negligible basal or inducible CYP1A2 mRNA. A fifth BAC-transgenic mouse line, carrying both the human CYP1A1 and CYP1A2 genes and ample amounts of 3' flanking sequences, plus all of the spacer region--in the absence of the mouse Cyp1a1 or Cyp1a2 genes--expressed the human CYP1A1 and CYP1A2 mRNA, protein and enzyme activities in liver and nonhepatic tissues very similar to that of the mouse. Comparison of this hCYP1A1_1A2 transgenic line with hCYP1A1_1A2 lines carrying other common human haplotypes will enable us to evaluate function in human CYP1A1_CYP1A2 locus variability, with regard to toxicity and cancer caused by combustion products.