Conceived and designed the experiments: GZMM JS TJK MDB JF. Performed the experiments: GZMM JF. Analyzed the data: GZMM JS JF. Contributed reagents/materials/analysis tools: GZMM JS TJK MDB JF. Wrote the paper: GZMM JS TJK MDB JF.
¶ Membership of the ANZgene Consortium is provided in the Acknowledgments.
I have read the journal's policy and have the following conflicts: This work was funded by Perpetual Trustees Australia Ltd. This does not alter the authors' adherence to all the PloS ONE policies on sharing data and materials.
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 (
Multiple sclerosis (MS) is a chronic demyelinating and inflammatory disease of the central nervous system (CNS), affecting mainly individuals of European ancestry
The TAM receptors (
Previous work in our laboratory examined the course of cuprizone-induced demyelination in mice lacking the TAM receptor ligand GAS6. Cuprizone is a neurotoxin that when incorporated into the feed of mice, induces specific and focal T cell-independent demyelination within the CNS, particularly in the corpus callosum
The studies by Binder et al.
Given the aforementioned results, we hypothesized that polymorphisms in the TAM receptor or ligand genes would be associated with MS and thus also be involved in the aetiology of the disease. In a recent genome-wide association study (GWAS) conducted by the Australian and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene)
The Melbourne Health Human Research Ethics Committee and the Australian Bone Marrow Donor Registry Ethics Committee granted approval for this research. Written consent was given by the subjects for their information to be stored in the study database and used for research.
Genotype data from a GWAS conducted by the ANZgene Consortium
A sample of 1140 MS cases and 1140 healthy controls from Australia and New Zealand were used as a replication cohort in this study (
MS patients ( |
Controls ( |
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33.85±10.14 | N/A |
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Female | 816 | 669 |
Male | 247 | 450 |
Unknown | 77 | 21 |
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N/A | |
RRMS | 632 | |
SPMS | 369 | |
PPMS | 58 | |
Single | 1 | |
Unknown | 80 |
MS: multiple sclerosis; N/A: not applicable; RRMS: relapsing-remitting multiple sclerosis; SPMS: secondary-progressive multiple sclerosis; PPMS: primary-progressive multiple sclerosis; Single: first demyelinating event.
Age information only available for 1069 MS patients and the mean is calculated as such.
Of the top 7 directly genotyped SNPs in TAM receptor and ligand genes in the ANZgene GWAS, 6 were in the
Subjects in the replication cohort were genotyped using the Sequenom MassARRAY system and iPLEX Gold chemistry under conditions recommended by the manufacturer (Sequenom, California, USA). Genomic DNA from all subjects was extracted as described in the ANZgene GWAS
Imputation of tagged, un-genotyped SNPs in the GWAS cohort was performed with Beagle v3.2.0 (
For SNPs that were genotyped in both the GWAS and replication cohorts, association analysis was performed using the –assoc command in PLINK v1.07 (
For SNPs that were imputed in either the GWAS or replication subjects, posterior allele dosages were used to perform the association analysis. The posterior allele dosage is twice the posterior genotype probability of the AA genotype plus the posterior genotype probability of the AB genotype (where A represents one of the alleles for the marker and B the other allele). Linear regression of allele dosages on case-control status was performed to test for association, with adjustment for sample group (discovery/replication) in the combined dataset.
To investigate linkage disequilibrium (LD) between SNPs, data from Hapmap phase II CEU individuals (release 24, build 36, forward strand) was analysed using Haploview v4.2 (
Hardy-Weinberg equilibrium of SNPs was determined by using the –hardy command in PLINK v1.07. SNPs in significant Hardy-Weinberg disequilibrium (
Our discovery dataset consisted of genotype data of 1618 MS cases and 3413 controls of European ancestry from a recent GWAS
A. Schematic of the
GWAS | Replication | Combined | |||||||||||||||||||
SNP ID | Chr | Position |
Gene | Major Allele | Minor Allele | Gen/Imp | MAF cases | MAF controls | HWE |
OR | Gen/Imp | MAF cases | MAF controls | HWE |
OR | MAF cases | MAF controls | OR | |||
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rs1516640 | 2 | 112,674,859 |
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G | A | Gen | 0.479 | 0.445 | 0.755 | 1.57×10−3 | 1.15 | Gen | 0.480 | 0.456 | 0.061 | 0.10 | 1.10 | 0.461 | 0.459 | 4.45×10−4 | 1.01 |
rs4848901 | 2 | 112,710,829 |
|
G | A | Gen | 0.476 | 0.443 | 0.627 | 1.88×10−3 | 1.14 | Gen | 0.467 | 0.447 | 0.069 | 0.17 | 1.09 | 0.458 | 0.453 | 7.91×10−4 | 1.02 |
rs13419523 | 2 | 112,781,918 |
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A | G | Gen | 0.086 | 0.069 | 0.894 | 3.32×10−3 | 1.26 | Gen | 0.077 | 0.066 | 0.224 | 0.15 | 1.18 | 0.078 | 0.070 | 1.27×10−3 | 1.13 |
rs6730521 | 2 | 112,783,958 |
|
A | G | Gen | 0.221 | 0.247 | 0.462 | 4.20×10−3 | 0.86 | Gen | 0.217 | 0.232 | 1.000 | 0.24 | 0.92 | 0.235 | 0.234 | 1.83×10−3 | 1.00 |
rs3811632 | 2 | 112,754,829 |
|
C | A | Gen | 0.272 | 0.293 | 0.934 | 0.03 | 0.90 | Gen | 0.264 | 0.273 | 0.082 | 0.50 | 0.96 | 0.283 | 0.279 | 0.03 | 1.02 |
SNPs in bold are the 12 SNPs that show association in both the discovery and replication cohorts. Un-bolded SNPs are SNPs genotypes in the discovery dataset that showed suggestive association, but failed to replicate. SNP, single nucleotide polymorphism; GWAS, genome-wide association study; Chr, Chromosome; Gen, genotyped; Imp, imputed; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium; OR, odds ratio.
SNP positions are from the Ensembl Genome Browser (
Analysis of the combined dataset was adjusted for sample group (discovery/replication).
We included the top 6 genotyped SNPs from the discovery dataset with
Although the SNPs interrogated in this study do not reach the level of genome-wide significance (
The TAM receptors have been shown to be major players in regulation of the immune response
Multiple sclerosis has often been classified as an autoimmune disease, and the obvious inflammatory response observed in the disease strongly supports this hypothesis. In light of previous studies implicating the TAM receptors in regulating both demyelination and autoimmune responses in both experimental animals and human MS lesions (for review, see ref.
In conclusion, this candidate gene study has identified an association of MS with 12 SNPs in the
SNP associations of all directly genotyped TAM receptor and ligand genes in the top 300,000 SNPs of the discovery (GWAS) dataset.
(DOC)
We thank individuals in Australia and New Zealand for supporting this research. We thank Laura Johnson for technical assistance. Replication genotyping was conducted at the Murdoch Children's Research Institute Sequenom Platform Facility.
¶
1 The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, AUS, 3050.
2 The Westmead Millenium Institute, Westmead, NSW, Australia 2145.
3 School of Medicine, Griffith University, QLD, Australia, 4222.
4 Department of Neurology, Gold Coast Hospital, QLD, Australia 4215.
5 The Diamantina Institute of Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, University of Queensland, Brisbane, QLD, Australia 4102.
6 Botnar Research Centre, Nuffield Department of Orthopaedic Surgery, University of Oxford, Oxford, OX3 7BN, UK.
7 Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
8 Department of Biostatistics, University of Washington, Seattle, WA 98195, USA.
9 Department of Medicine, University of Melbourne, Melbourne, VIC, Australia.
10 Florey Neuroscience Institutes, University of Melbourne, Melbourne, VIC, Australia.
11 Department of Neurology, Box Hill Hospital, VIC, Australia.
12 Sir Charles Gairdner Hospital, Nedlands, WA, AUS 6009.
13 Australian Neuromuscular Research Institute, Nedlands WA, AUS 6009.
14 Centre for Neuromuscular Research and Neurological Disorders, University of Western Australia, Nedlands, WA, Australia.
15 Centre for Neuroscience, University of Melbourne, VIC, Australia 3010.
16 Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia. 7001.
17 Genomics Research Centre, Griffith University, QLD, Australia 4222.
18 Royal Melbourne Hospital, Parkville, VIC, Australia 3050.
19 John Hunter Hospital, Hunter New England Health Service, Newcastle NSW, Australia 2310.
20 Hunter Medical Research Institute, Newcastle, NSW, Australia 2308.
21 Centre for Bioinformatics, Biomarker Discovery and Information-based Medicine, University of Newcastle, NSW, Australia 2308.
22 School of Medicine, Department of Neurology, Flinders University of South Australia, Bedford Park, Adelaide, SA, Australia 5042.