Novel Olig1‐coding variants and susceptibility to multiple sclerosis

Olig1 is a basic helix–loop–helix (bHLH) transcription factor expressed in cells of the oligodendrocyte lineage in the nervous system. Its role during normal development has not yet been fully resolved, but it is known that in adult life the protein is crucial in the process of remyelination after injury.¹,²,³ Olig1 translocates from the cytoplasm to the nucleus in early remyelinating lesions in rodent models of demyelinating disease as well as in oligodendrocyte precursor cells at the edge of multiple sclerosis lesions.¹ Olig1 specifically regulates the expression of the major myelin‐specific genes during oligodendrocyte maturation in the brain,² and remyelination after injury is impaired in Olig1^‐/‐ mice.¹ In patients with multiple sclerosis, remyelinating capacity is limited even though oligodendrocyte precursor cells are often efficiently recruited.⁴ These findings raise the question whether genetic variants in the Olig1 gene (Olig1) influence remyelinating capacity and vulnerability to the consequences of a demyelinating event.⁵

To investigate this hypothesis, we first aimed at identifying any Olig1‐coding variants by resequencing the coding region in 20 patients with multiple sclerosis having at least one affected first‐degree relative. Three coding variants were identified: a (TCC)_n repeat between nucleotides 230 and 247, leading to a variable number (3–9) of serine residues starting from codon 43 in the protein sequence; a synonymous C/T single‐nucleotide polymorphism (SNP) at position 707 (codon 202); and a non‐synonymous C/A SNP at position 840, leading to a substitution of threonine by asparagine at codon 246. Inspection of the public databases showed a further five SNPs in the 3′ untranslated region (3′UTR), but no other variant in the coding region. A robust assay could not be designed for three of these UTR SNPs.

Subsequently, we elected to investigate the novel variants, remaining 3′UTR variants, and the first SNP upstream and downstream of the gene typed in the HapMap project for association with susceptibility to multiple sclerosis. We typed these seven variants in 937 UK trio families (an affected individual and both parents). Patients satisfied Poser criteria for the diagnosis of multiple sclerosis and had typical demographic features, with a mean age of 37.8 years, mean Expanded Disability Status Scale of 4.3, mean disease duration of 11.9 years and a male:female ratio of 1:3. All participants gave written informed consent and the study was approved by the local ethics committee. Four SNPs (707CT, 840AC, rs11554599 and rs11554600) were typed with Taqman Assays‐by‐Design and two (rs928736 and rs7278725) with Assays‐on‐Demand (Applied Biosystems). The serine repeat polymorphism was amplified in a polymerase chain reaction and fluorescently labelled fragments were separated on a 3700 capillary sequencer (Applied Biosystem, Foster City, California, USA).

No markers deviated significantly from Hardy–Weinberg equilibrium, and genotyping success rates were >98.5% for all SNPs and 91.8% for the serine repeat polymorphism. For each marker, 166 participants were typed in duplicate. No inconsistency was seen for any of the SNPs and not more than two inconsistent alleles were seen for the serine repeat microsatellite. Only two mendelian errors (one for rs11554600 and rs7278725 each) were observed. The minor allele frequency for the 840C/A coding variant was only 0.1%, thereby preventing a meaningful analysis. For the serine repeat polymorphism, alleles with a frequency of <5% were grouped together. Evidence for association was sought by transmission disequilibrium testing using the TRANSMIT program, with 10 000 bootstrap replicates used to provide an empirical estimate of statistical significance. Marker rs7278735 showed borderline nominally significant evidence for association (table 1).

Table 1 Individual marker analysis for association with multiple sclerosis susceptibility.

	Location	MAF	p*	pcorr†
rs928736	−2561	0.306	0.84	–
Ser repeat	230	0.048	0.80	–
707C/T	707	0.066	0.72	–
840C/A	840	0.001	–	–
rs11554599	1311	0.065	0.63	–
rs11554600	1688	0.128	0.15	–
rs7278735	2633	0.180	0.022	0.11

Location relative to transcription start; MAF, minor allele frequency in 3748 independent parental chromosomes; Ser, serine.

*p Value in a TRANSMIT test (http://www‐gene.cimr.cam.ac.uk/clayton/software/) with 10 000 bootstrap replicates; †p value after multiple testing correction according to the method suggested by Nyholt (http://genepi.qimr.edu.au/general/daleN/SNPSpD/), which indicates that the six analysed markers behave as five independent ones.

The data were also analysed by using the Haploview program (http://www.broad.mit.edu/mpg/haploview/). All markers except rs928736 were found to lie in the same haplotype block (accepting the program's default definitions), within which five haplotypes with a frequency of ⩾5% were observed. A haplotype transmission disequilibrium test on these showed overtransmission of the most frequent haplotype, but again this trend was not significant after appropriate correction for multiple testing (table 2).

Table 2 Haplotype analysis for markers serine repeat−707C/T−rs11554599−rs11554600−rs7278735.

Haplotype	Frequency	p*
6‐C–C–G–C	0.705	0.021
6‐C–C–A–T	0.129	0.037
6‐T–T–G–C	0.065	0.65
6‐C–C–G–T	0.051	0.19
X†‐C–C–G–C	0.049	0.73
Global		0.072

*Uncorrected p values in a TRANSMIT test with 10 000 bootstrap replicates; †X, any alternative allele (3, 4, 7, 8 or 9 serine repeats).

In conclusion, we have identified novel coding variants in the Olig1 gene, including a trinucleotide repeat, but have found no evidence to support the hypothesis that genetic variation in Olig1 influences susceptibility to multiple sclerosis.

Acknowledgements

This work was supported by the Wellcome Trust (grant 057097), the Multiple Sclerosis Society of the United States (grant RG3500‐A‐1) and the Multiple Sclerosis Society of Great Britain and Ireland (grant 730/02).