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Ritchie M.E.,Walter and Eliza Hall Institute of Medical Research | Ritchie M.E.,University of Melbourne | Liu R.,Walter and Eliza Hall Institute of Medical Research | Carvalho B.S.,University of Cambridge | And 66 more authors.
BMC Bioinformatics | Year: 2011

Background: Illumina's Infinium SNP BeadChips are extensively used in both small and large-scale genetic studies. A fundamental step in any analysis is the processing of raw allele A and allele B intensities from each SNP into genotype calls (AA, AB, BB). Various algorithms which make use of different statistical models are available for this task. We compare four methods (GenCall, Illuminus, GenoSNP and CRLMM) on data where the true genotypes are known in advance and data from a recently published genome-wide association study.Results: In general, differences in accuracy are relatively small between the methods evaluated, although CRLMM and GenoSNP were found to consistently outperform GenCall. The performance of Illuminus is heavily dependent on sample size, with lower no call rates and improved accuracy as the number of samples available increases. For X chromosome SNPs, methods with sex-dependent models (Illuminus, CRLMM) perform better than methods which ignore gender information (GenCall, GenoSNP). We observe that CRLMM and GenoSNP are more accurate at calling SNPs with low minor allele frequency than GenCall or Illuminus. The sample quality metrics from each of the four methods were found to have a high level of agreement at flagging samples with unusual signal characteristics.Conclusions: CRLMM, GenoSNP and GenCall can be applied with confidence in studies of any size, as their performance was shown to be invariant to the number of samples available. Illuminus on the other hand requires a larger number of samples to achieve comparable levels of accuracy and its use in smaller studies (50 or fewer individuals) is not recommended. © 2011 Ritchie et al; licensee BioMed Central Ltd.


Patsopoulos N.A.,Brigham and Women's Hospital | Patsopoulos N.A.,Harvard University | Patsopoulos N.A.,The Broad Institute of MIT and Harvard | Esposito F.,San Raffaele Scientific Institute | And 88 more authors.
Annals of Neurology | Year: 2011

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: rs170934 T at 3p24.1 (odds ratio [OR], 1.17; p = 1.6 × 10-8) near EOMES, rs2150702G in the second intron of MLANA on chromosome 9p24.1 (OR, 1.16; p = 3.3 × 10-8), and rs6718520A in an intergenic region on chromosome 2p21, with THADA as the nearest flanking gene (OR, 1.17; p = 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. Copyright © 2011 American Neurological Association.


Jensen C.J.,Howard Florey Institute | Jensen C.J.,Monash University | Stankovich J.,Menzies Research Institute | Van der Walt A.,Royal Melbourne Hospital | And 44 more authors.
PLoS ONE | Year: 2010

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. © 2010 Jensen et al.


Zin R.,University of Western Australia | Pham K.,Australian Neuromuscular Research Institute | Ashleigh M.,University of Western Australia | Ashleigh M.,King Edward Memorial Hospital | And 3 more authors.
Cancer Genetics | Year: 2012

Wilms' tumors have characteristic chromosomal abnormalities, such as the 11p13 deletion, in a subset of cases. This is one of the very few reports comparing single nucleotide polymorphism (SNP) array analysis with conventional karyotyping of Wilms' tumors. A total of 43 frozen tumor samples were analyzed using the Affymetrix Cytogenetics Whole-Genome 2.7. M array. The findings from the SNP array analysis were then compared with those from conventional karyotyping. A comparison between SNP array and conventional karyotype findings was possible in 38 of 43 specimens (88.4%). The SNP array and classic cytogenetic results were concordant in 33 of 38 specimens (87%). SNP array analysis was able to support the findings of classic cytogenetics. The SNP array detected regions of loss of heterozygosity (LOH) in 41 of 43 (95%) specimens. However, it did not detect balanced translocations and inversions that were observed by conventional cytogenetics. Our results show that the data generated from these platforms are complementary. The SNP array also detected additional gains and losses as well as regions of LOH with associated disomy, which are likely to represent segmental uniparental disomy. The observed discrepancies can be explained by the inherent limitations of each technique. © 2012 Elsevier Inc.


Field J.,University of Melbourne | Browning S.R.,University of Auckland | Johnson L.J.,University of Melbourne | Danoy P.,University of Queensland | And 42 more authors.
PLoS ONE | Year: 2010

We conducted an association study across the human leukocyte antigen (HLA) complex to identify loci associated with multiple sclerosis (MS). Comparing 1927 SNPs in 1618 MS cases and 3413 controls of European ancestry, we identified seven SNPs that were independently associated with MS conditional on the others (each P≤4×10-6). All associations were significant in an independent replication cohort of 2212 cases and 2251 controls (P≤0:001) and were highly significant in the combined dataset (P≤6 × 10-8). The associated SNPs included proxies for HLA-DRB1*15:01 and HLA-DRB1*03:01, and SNPs in moderate linkage disequilibrium (LD) with HLA-A*02:01, HLA-DRB1*04:01 and HLA-DRB1*13:03. We also found a strong association with rs9277535 in the class II gene HLA-DPB1 (discovery set P = 9 × 10-9, replication set P = 7 × 10-4, combined P=2 × 10-10). HLA-DPB1 is located centromeric of the more commonly typed class II genes HLA-DRB1, -DQA1 and -DQB1. It is separated from these genes by a recombination hotspot, and the association is not affected by conditioning on genotypes at DRB1, DQA1 and DQB1. Hence rs9277535 represents an independent MS-susceptibility locus of genome-wide significance. It is correlated with the HLA-DPB1*03:01 allele, which has been implicated previously in MS in smaller studies. Further genotyping in large datasets is required to confirm and resolve this association. © 2010 Field et al.


Ma G.Z.M.,University of Melbourne | Stankovich J.,Menzies Research Institute | Kilpatrick T.J.,University of Melbourne | Binder M.D.,University of Melbourne | And 34 more authors.
PLoS ONE | Year: 2011

Multiple sclerosis (MS) is a debilitating, chronic demyelinating disease of the central nervous system affecting over 2 million people worldwide. The TAM family of receptor tyrosine kinases (TYRO3, AXL and MERTK) have been implicated as important players during demyelination in both animal models of MS and in the human disease. We therefore conducted an association study to identify single nucleotide polymorphisms (SNPs) within genes encoding the TAM receptors and their ligands associated with MS. Analysis of genotype data from a genome-wide association study which consisted of 1618 MS cases and 3413 healthy controls conducted by the Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene) revealed several SNPs within the MERTK gene (Chromosome 2q14.1, Accession Number NG_011607.1) that showed suggestive association with MS. We therefore interrogated 28 SNPs in MERTK in an independent replication cohort of 1140 MS cases and 1140 healthy controls. We found 12 SNPs that replicated, with 7 SNPs showing p-values of less than 10-5 when the discovery and replication cohorts were combined. All 12 replicated SNPs were in strong linkage disequilibrium with each other. In combination, these data suggest the MERTK gene is a novel risk gene for MS susceptibility. © 2011 Ma et al.


Tschochner M.,Murdoch University | Leary S.,Murdoch University | Cooper D.,Murdoch University | Strautins K.,Murdoch University | And 12 more authors.
PLoS ONE | Year: 2016

Background: Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1∗15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome. Methods: Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1∗1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins. Results EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cutoff). In silico epitope mapping revealed two known HLA-DRB1∗1501-restricted epitopes ('AEG': aa 481-496 and 'MVF': aa 562-577), and two putative epitopes between positions 502-543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1∗15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis. Conclusions: This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of immunogenic regions of EBNA-1 as well as known and novel targets for autoreactive HLA-DRB1∗15-restricted T cells within the central nervous system that could arise as a result of cross-reactivity with EBNA-1-specific immune responses. © Copyright 2016 Tschochner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


PubMed | Guiyang Medical University, Australian Neuromuscular Research Institute and University Pierre and Marie Curie
Type: Journal Article | Journal: Molecular neurobiology | Year: 2016

The purpose of this study was to determine whether or not aquaporin-4 (AQP4) gene mutations are related to the pathogenesis of inflammatory demyelinating diseases in the central nervous system. Polymorphisms of AQP4 exons 1-5 were determined by sequencing DNA from 67 patients with central nervous system inflammatory demyelinating diseases, including neuromyelitis optica (NMO), multiple sclerosis, recurrent or simultaneous bilateral optic neuritis, and longitudinally extensive transverse myelitis. A plasmid with the identified new missense mutation was constructed, and human embryonic kidney cells (HEK293A) were transfected with either the pEGFP-N1-AQP4-M23 vector (bearing the identified mutated cDNA sequence) or with the plasmid bearing the wild-type AQP4 gene sequence. AQP4 protein expression was analyzed in both experimental groups using Western Blot analysis following protein extraction from transfected cells. A synonymous mutation (rs1839318) was detected on exon 3, and an additional synonymous mutation was detected on the exon 2-2 (rs72557968). Most importantly, a new missense mutation was detected on exon 2-1. According to Western blot analysis, the mutated cDNA sequence yielded increased AQP4 protein expression in comparison with the wild-type cDNA sequence (P<0.05). AQP4 gene mutations are uncommon, occurring in only 3 out of 67 patients. Although it is possible that the mutations contributed to an increased risk of inflammatory central nervous system disease in these individuals, it is unlikely that mutations are a significant contributor to most patients with NMO spectrum disorders in China.


Craig A.J.,University of Western Australia | Meloni B.P.,University of Western Australia | Meloni B.P.,Sir Charles Gairdner Hospital | Meloni B.P.,Australian Neuromuscular Research Institute | And 3 more authors.
International Journal of Peptide Research and Therapeutics | Year: 2011

Using acute and delayed in vitro ischaemia models we evaluated the neuroprotective efficacy of five peptides (PYC19D-TAT, PYC35D-TAT, PYC36D-TAT, PYC38D-TAT, PYC41D-TAT) previously demonstrated to down-regulate AP-1 activation (e.g. c-Jun/c-Fos activation), and inhibit neuronal death in vitro following glutamate and kainic acid excitotoxicity. The JNK inhibitor peptide (JNKI-1D-TAT) and the TAT cell-penetrating-carrier peptide (D-TAT) were used as controls. In the acute model, all five AP-1 inhibitory peptides, JNKI-1D-TAT, and D-TAT provided neuroprotection by increasing neuronal viability from ≈5 to 23-53%. In the delayed model, three of the five AP-1 inhibitory peptides (PYC35D-TAT, PYC36D-TAT, PYC38D-TAT) and JNKI-1D-TAT provided neuroprotection by increasing neuronal viability from ≈10 to 35-80%. This study not only highlights a group of peptides with therapeutic potential, but also the need to assess putative therapeutics in multiple in vitro models to achieve a comprehensive representation of their neuroprotective capacity. © 2010 Springer Science+Business Media, LLC.


Takechi R.,Curtin University Australia | Takechi R.,Curtin Health Innovation Research Institute | Galloway S.,Curtin University Australia | Galloway S.,Curtin Health Innovation Research Institute | And 9 more authors.
British Journal of Nutrition | Year: 2010

Some dietary fats are a risk factor for Alzheimer's disease (AD) but the mechanisms for this association are presently unknown. In the present study we showed in wild-type mice that chronic ingestion of SFA results in blood-brain barrier (BBB) dysfunction and significant delivery into the brain of plasma proteins, including apo B lipoproteins that are endogenously enriched in amyloid-(A). Conversely, the plasma concentration of S100B was used as a marker of brain-to-blood leakage and was found to be increased two-fold because of SFA feeding. Consistent with a deterioration in BBB integrity in SFA-fed mice was a diminished cerebrovascular expression of occludin, an endothelial tight junction protein. In contrast to SFA-fed mice, chronic ingestion of MUFA or PUFA had no detrimental effect on BBB integrity. Utilising highly sensitive three-dimensional immunomicroscopy, we also showed that the cerebral distribution and co-localisation of A with apo B lipoproteins in SFA-fed mice are similar to those found in amyloid precursor protein/presenilin-1 (APP/PS1) amyloid transgenic mice, an established murine model of AD. Moreover, there was a strong positive association of plasma-derived apo B lipoproteins with cerebral A deposits. Collectively, the findings of the present study provide a plausible explanation of how dietary fats may influence AD risk. Ingestion of SFA could enhance peripheral delivery to the brain of circulating lipoprotein-A and exacerbate the amyloidogenic cascade. © 2010 The Author.

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