Genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci

Objective: To perform a 1-stage meta-analysis of genome-wide association studies (GWAS) of multiple sclerosis (MS) susceptibility and to explore functional consequences of new susceptibility loci. Methods: We synthesized 7 MS GWAS. Each data set was imputed using HapMap phase II, and a per single nucleotide polymorphism (SNP) meta-analysis was performed across the 7 data sets. We explored RNA expression data using a quantitative trait analysis in peripheral blood mononuclear cells (PBMCs) of 228 subjects with demyelinating disease. Results: We meta-analyzed 2,529,394 unique SNPs in 5,545 cases and 12,153 controls. We identified 3 novel susceptibility alleles: rs170934T at 3p24.1 (odds ratio [OR], 1.17; p ¼ 1.6 � 10�8) near EOMES, rs2150702G in the second intron of MLANA on chromosome 9p24.1 (OR, 1.16; p ¼ 3.3 � 10�8), and rs6718520A in an intergenic region on chromosome 2p21, with THADA as the nearest flanking gene (OR, 1.17; p ¼ 3.4 � 10�8). The 3 new loci do not have a strong cis effect on RNA expression in PBMCs. Ten other susceptibility loci had a suggestive p < 1 � 10�6, some of these loci have evidence of association in other inflammatory diseases (ie, IL12B, TAGAP, PLEK, and ZMIZ1). Interpretation: We have performed a meta-analysis of GWAS in MS that more than doubles the size of previous gene discovery efforts and highlights 3 novel MS susceptibility loci. These and additional loci with suggestive evidence of association are excellent candidates for further investigations to refine and validate their role in the genetic architecture of MS.

Multiple sclerosis susceptibility-associated SNPs do not influence disease severity measures in a cohort of Australian MS patients

Recent association studies in multiple sclerosis (MS) have identified and replicated several single nucleotide polymorphism (SNP) susceptibility loci including CLEC16A, IL2RA, IL7R, RPL5, CD58, CD40 and chromosome 12q13–14 in addition to the well established allele HLA-DR15. There is potential that these genetic susceptibility factors could also modulate MS disease severity, as demonstrated previously for the MS risk allele HLA-DR15. We investigated this hypothesis in a cohort of 1006 well characterised MS patients from South-Eastern Australia. We tested the MS-associated SNPs for association with five measures of disease severity incorporating disability, age of onset, cognition and brain atrophy. We observed trends towards association between the RPL5 risk SNP and time between first demyelinating event and relapse, and between the CD40 risk SNP and symbol digit test score. No associations were significant after correction for multiple testing. We found no evidence for the hypothesis that these new MS disease risk-associated SNPs influence disease severity.

An investigation of the C77G and C772T variations within the human protein tyrosine phosphatase receptor type C gene for association with multiple sclerosis in an Australian population

Multiple sclerosis (MS) is a common cause of neurological disability in young adults. The disease generally manifests in early to middle adulthood and causes various neurological deficits. Autoreactive T lymphocytes and their associated antigens have long been presumed important features of MS pathogenesis. The Protein tyrosine phosphatase receptor type C gene (PTPRC) encodes the T-cell receptor CD45. Variations within PTPRC have been previously associated with diseases of autoimmune origin such as type 1 diabetes mellitus and Graves' disease. We set out to investigate two variants within the PTPRC gene, C77G and C772T in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We employed high resolution melt analysis (HRM) and restriction length polymorphism (RFLP) techniques to determine genotypic and allelic frequencies. Our study found no significant difference between frequencies for PTPRC C77G by either genotype (Χ2 = 0.65, P = 0.72) or allele (Χ2 = 0.48, P = 0.49). Similarly, we did not find evidence to suggest an association between PTPRC C772T by genotype (Χ2 = 1.06, P = 0.59) or allele (Χ2 = 0.20, P = 0.66). Linkage disequilibrium (LD) analysis showed strong linkage disequilibrium between the two tested markers (D' = 0.9970, SD = 0.0385). This study reveals no evidence to suggest that these markers are associated with MS in the tested Australian Caucasian population. Although the PTPRC gene has a significant role in regulating CD4+ and CD8+ autoreactive T-cells, interferon-beta responsiveness, and potentially other important processes, our study does not support a role for the two tested variants of this gene in MS susceptibility in the Australian population.

Polymorphisms of the VDR gene are associated with presence of solar keratoses on the skin

Background Vitamin D has a range of biological effects including antiproliferative functions that are mediated through its receptors, encoded by the VDR gene. Objectives We investigated polymorphisms within the VDR gene for association with solar keratosis (SK), a biomarker for skin cancer, and examined interactions with skin phenotype. Methods Among participants of the community-based Nambour Skin Cancer Study, we genotyped 190 people with SKs and 190 without for ApaI, TaqI and FokI polymorphisms. Results We found a significant difference in genotype frequencies of the TaqI polymorphism between affected and unaffected populations (P = 0Æ008). The TT ⁄tt genotype group was associated with a twofold increase in odds of being affected by one or more SK. Individuals with fair skin and the TT ⁄tt genotype had about a sevenfold increase, whereas fair-skinned people with the Tt genotype had a fourfold increase in odds of being affected by SK. Individuals with the TT ⁄tt genotype who were prone to burn and not tan on acute sun exposure had about a sixfold increase in odds of SK. Fair-skinned people with VDR-Apa AA ⁄aa genotypes had about an eightfold increase in odds of being affected by SK compared with a fivefold increase in individuals with the Aa genotype and fair skin. Conclusions The trend for homozygote genotypes to increase the odds of SK suggests that intermediate VDR activity is important in protection or that the heterodimer formed by a heterozygous genotype may have an altered binding potential. Overall, these analyses indicate that VDR may be important in SK development.

Allelic variation investigation of the estrogen receptor within an Australian multiple sclerosis population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

No association between MTHFR A1298C and MTRR A66G polymorphisms, and MS in an Australian cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ2 = 0.005, P = 0.95, MTHFR χ2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS

No role for estrogen receptor 1 gene intron 1 Pvu II and exon 4 C325G polymorphisms in migraine susceptibility

Background We have previously reported an association between the estrogen receptor 1 (ESR1) gene exon 8 G594A polymorphism and migraine susceptibility in two independent Australian cohorts. In this paper we report results of analysis of two further single nucleotide polymorphisms (SNPs) in the ESR1 gene in the same study group, the T/C Pvu II SNP in intron 1 and the C325G SNP in exon 4, as well as results of linkage disequilibrium (LD) analysis on these markers. Methods We investigated these variants by case-control association analysis in a cohort of 240 migraineurs and 240 matched controls. The SNPs were genotyped using specific restriction enzyme assays. Results were analysed using contingency table methods incorporating the chi-squared statistic. LD results are presented as D' statistics with associated P values. Results We found no evidence for association of the Pvu II T/C polymorphism and the C325G polymorphism and migraine susceptibility and no evidence for LD between these two SNPs and the previously implicated exon 8 G594A marker. Conclusion We have found no role for the polymorphisms in intron 1 and exon 4 with migraine susceptibility. To further investigate our previously implicated exon 8 marker, we suggest the need for studies with a high density of polymorphisms be undertaken, with particular focus on markers in LD with the exon 8 marker.

A genetic analysis of serotonergic biosynthetic and metabolic enzymes in migraine using a DNA pooling approach

Migraine is a common debilitating primary headache disorder with significant mental, physical and social health implications. The brain neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) is involved in nociceptive pathways and has been implicated in the pathophysiology of migraine. With few genetic studies investigating biosynthetic and metabolic enzymes governing the rate of 5-HT activity and their relationship to migraine, it was the objective of this study to assess genetic variants within the human tryptophan hydroxylase (TPH), amino acid decarboxylase (AADC) and monoamine oxidase A (MAOA) genes in migraine susceptibility. This objective was undertaken using a high-throughput DNA pooling experimental design, which proved to be a very accurate, sensitive and specific method of estimating allele frequencies for single nucleotide polymorphism, insertion deletion and variable number tandem repeat loci. Application of DNA pooling to a wide array of genetic loci provides greater scope in the assessment of population-based genetic association study designs. Despite the application of this high-throughput genotyping method, negative results from the two-stage DNA pooling design used to screen loci within the TPH, AADC and MAOA genes did not support their role in migraine susceptibility.

Association analysis of a highly polymorphic CAG Repeat in the human potassium channel gene KCNN3 and migraine susceptibility

Background Migraine is a polygenic multifactorial disease, possessing environmental and genetic causative factors with multiple involved genes. Mutations in various ion channel genes are responsible for a number of neurological disorders. KCNN3 is a neuronal small conductance calcium-activated potassium channel gene that contains two polyglutamine tracts, encoded by polymorphic CAG repeats in the gene. This gene plays a critical role in determining the firing pattern of neurons and acts to regulate intracellular calcium channels. Methods The present association study tested whether length variations in the second (more 3') polymorphic CAG repeat in exon 1 of the KCNN3 gene, are involved in susceptibility to migraine with and without aura (MA and MO). In total 423 DNA samples from unrelated individuals, of which 202 consisted of migraine patients and 221 non-migraine controls, were genotyped and analysed using a fluorescence labelled primer set on an ABI310 Genetic Analyzer. Allele frequencies were calculated from observed genotype counts for the KCNN3 polymorphism. Analysis was performed using standard contingency table analysis, incorporating the chi-squared test of independence and CLUMP analysis. Results Overall, there was no convincing evidence that KCNN3 CAG lengths differ between Caucasian migraineurs and controls, with no significant difference in the allelic length distribution of CAG repeats between the population groups (P = 0.090). Also the MA and MO subtypes did not differ significantly between control allelic distributions (P > 0.05). The prevalence of the long CAG repeat (>19 repeats) did not reach statistical significance in migraineurs (P = 0.15), nor was there a significant difference between the MA and MO subgroups observed compared to controls (P = 0.46 and P = 0.09, respectively), or between MA vs MO (P = 0.40). Conclusion This association study provides no evidence that length variations of the second polyglutamine array in the N-terminus of the KCNN3 channel exert an effect in the pathogenesis of migraine.

Investigation of the low-density lipoprotein receptor gene and cholesterol as a risk factor for migraine

The Low-Density Lipoprotein Receptor (LDLR) gene is a cell surface receptor that plays an important role in cholesterol homeostasis. We investigated the (TA)n polymorphism in exon 18 of the LDLR gene on chromosome 19p13.2 performing an association analysis in 244 typical migraine-affected patients, 151 suffering from migraine with aura (MA), 96 with migraine without aura (MO) and 244 unaffected controls. The populations consisted of Caucasians only, and controls were age- and sex-matched. The results showed no significant difference between groups for allele frequency distributions of the (TA)n polymorphism even after separation of the migraine-affected individuals into subgroups of MA and MO affected patients. This is in contradiction to Mochi et al. who found a positive association of this variant with MO. Our study discusses possible differences between the two studies and extends this research by investigating circulating cholesterol levels in a migraine-affected population.

Large-scale association study identifies ICAM gene region as breast and prostate cancer susceptibility locus

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

Association analysis of somatostatin receptor (SSTR1 and SSTR2) polymorphisms in breast cancer and solar keratosis

The presence of somatostatin receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms. We have performed association studies on breast cancer and solar keratosis populations to determine whether these genes play a role in the development of these conditions. Results showed that there was no significant difference between SSTR1 and SSTR2 polymorphism frequencies in the tested breast cancer population (P = 0.59 and P = 0.54, respectively) nor the solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to unaffected populations. Hence, these studies do not support a role for these receptor genes in either breast cancer or solar keratosis lesions.

Association of estrogen receptor and glucocorticoid receptor gene polymorphisms with sporadic breast cancer

We have utilized a cross-sectional association approach to investigate sporadic breast cancer. Polymorphisms in 2 candidate genes, ESRalpha and GRL, were examined in an unrelated breast cancer-affected and age-matched control population. Several polymorphic regions within the ESRalpha gene have been identified, and some alleles of these polymorphisms have been found to occur at increased levels in breast-cancer patients. Additionally, variations in GRL have the potential to disrupt cell transcription and may be associated with cancer formation. We analyzed 3 polymorphisms, from codons 10 (TCT to TCC), 325 (CCC to CCG) and 594 (ACA to ACG) of ESRalpha, and a highly polymorphic dinucleotide repeat, D5S207, located within 200 kb of the GRL. When allelic frequencies of the codon 594 (exon 8) ESR polymorphism were compared between affected and unaffected populations, a significant difference was observed (p = 0.005). Results from the D5S207 dinucleotide repeat located near GRL also indicated a significant difference between the tested case and control populations (p = 0.001). Allelic frequencies of the codon 10 and codon 325 ESR polymorphisms were not significantly different between populations (p = 0.152 and 0.181, respectively). Our results indicate that specific alleles of the ESR gene (alpha subtype) and a marker for the GRL gene locus are associated with sporadic breast-cancer development in the tested Caucasian population and justify further investigation of the role of these and other nuclear steroid receptors in the etiology of breast cancer.

Chromosome 17 and the inducible nitric oxide synthase gene in human essential hypertension

Essential hypertension is a common multifactorial trait that results in a significantly increased risk for heart attack and stroke. The condition has a genetic basis, although at present the number of genes is unknown. In order to identify such genes, we are utilising a linkage scanning approach using microsatellite markers and affected sibships. Here we provide evidence for the location of at least one hypertension susceptibility locus on chromosome 17. Analysis of 177 affected sibpairs gave evidence for significant excess allele sharing to D17S949 (SPLINK: P=0.0029; MAPMAKER SIBS: P=0.0033; ASPEX: P=0.0061; GENEHUNTER: P=0.0096; ANALYZE (SIBPAIR): P=0.0025) on 17q22–24, with significant allele sharing also indicated for an additional marker, D17S799 (SPLINK: P=0.025; MAPMAKER SIBS: P= 0.025) located close to the centromere. Since these two genomic regions are well separated, our results indicate that there may be more than one chromosome 17 locus affecting human blood pressure. Moreover, further investigation of this chromosome, utilizing a polymorphism within the promoter of the iNOS candidate gene, NOS2A, revealed both increased allele sharing among sibpairs (SPLINK: P=0.02; ASPEX: P=0.00004) and positive association (P= 0.034) of NOS2A to essential hypertension. Hence these results indicate that chromosome 17 and, more specifically, the NOS2A gene may play a role in human essential hypertension.

Investigation of glutathione S-transferase zeta and the development of sporadic breast cancer

Background Certain genes from the glutathione S-transferase superfamily have been associated with several cancer types. It was the objective of this study to determine whether alleles of the glutathione S-transferase zeta 1 (GSTZ1) gene are associated with the development of sporadic breast cancer. Methods DNA samples obtained from a Caucasian population affected by breast cancer and a control population, matched for age and ethnicity, were genotyped for a polymorphism of the GSTZ1 gene. After PCR, alleles were identified by restriction enzyme digestion and results analysed by chi-square and CLUMP analysis. Results Chi-squared analysis gave a χ2 value of 4.77 (three degrees of freedom) with P = 0.19, and CLUMP analysis gave a T1 value of 9.02 with P = 0.45 for genotype frequencies and a T1 value of 4.77 with P = 0.19 for allele frequencies. Conclusion Statistical analysis indicates that there is no association of the GSTZ1 variant and hence the gene does not appear to play a significant role in the development of sporadic breast cancer.