Introduction
Recent genome-wide association studies (GWAS) have identified many common variants in the nuclear genome that are associated with type 2 diabetes [
1‐
3]. However, the identified variants explain only a small proportion of the heritability of type 2 diabetes. The unexplained heritability could be due partly to interactions with environmental and lifestyle factors, rare variants, epigenetic factors or possibly mitochondrial DNA (mtDNA) variants. Mutations in mtDNA such as the 3243A\G mutation in the mtDNA-encoded
tRNA
Leu(UUR)
gene (also known as
MTTL1) are associated with maternally inherited diabetes. Even though this mutation has a prevalence of 1 in 400, it causes fewer than 2% of type 2 diabetes cases [
4,
5]. As with many other monogenic forms of diabetes, it is possible that extreme functional mutations in mitochondrial DNA cause syndromic forms of diabetes while less severe common variants are more weakly associated with typical type 2 diabetes.
A common mtDNA variant that results in a T to C substitution at the nucleotide position 16189 (also called the 16189 variant) lies in the hypervariable D-loop region of mtDNA for replication and transcription [
6]. The mtDNA 16189 T>C transition frequently produces an uninterrupted polycytosine (polyC) tract between nucleotide positions 16184 and 16193 that is susceptible to replication errors in the important regulatory D-loop region. Because this variant maps precisely to the OriB origin of replication, we re-named it the ‘OriB variant’. In common with other homopolymeric C tracts in mtDNA [
7], when the unbroken tract exceeds 11 bp it generates heteroplasmic length variation. As well as this alteration, the mtDNA becomes less susceptible to DNase I, though this may be because of changes in DNA secondary structure or protein binding [
8].
The frequency of the OriB variant differs in ethnic groups, with 10% in Europids, 30% in Asians, 50% in Pima Indians and >95% in Polynesians; this distribution has intriguing similarities with the prevalence of diabetes [
9]. The association of this variant with type 2 diabetes has been studied previously, with apparently conflicting results [
8‐
27]. A previous review of four studies with a total of 1,455 type 2 diabetes cases and 3,132 controls reported that the OriB variant was not associated with type 2 diabetes in European populations [
18]. However, most of those data were from relatively small studies and although the meta-analysis was able to exclude a very strong association in European populations, it could not exclude the possibility that the variant was modestly associated. A modest association can only be detected by large, well-powered studies.
Our reassessment of the association of the OriB variant with type 2 diabetes used the following approach to maximise power and minimise bias: (1) we investigated the association between the OriB variant and type 2 diabetes in a large European population (the Norfolk Diabetes Case–Control Study), which included 5,574 type 2 diabetes cases and 6,950 healthy controls; and (2) we conducted a meta-analysis of studies of the OriB variant with type 2 diabetes in Europid populations (e.g. European continental ancestry), involving a total of 11,794 type 2 diabetes cases and 14,465 controls, over five time as many participants as in the previous review [
18].
Discussion
Previous genetic studies and earlier meta-analyses of the OriB variant have been dominated by small studies that are susceptible to various forms of bias. A previous meta-analysis concluded that the OriB variant was not associated with type 2 diabetes in the European population, but it did not include sufficient numbers of cases or controls to exclude a modest association of this variant with diabetes [
18]. Methods used by other studies were not specific for the homopolymeric tract and hence underestimated the magnitude of the association [
24]. Given the absence of large individual studies in Europid populations, we have conducted an updated systematic review and meta-analysis of new and published data, involving a total of 11,794 type 2 diabetes cases and 14,465 controls, which provides the most comprehensive assessment to date of the association of the OriB variant with type 2 diabetes in Europid populations. Our new data include the single largest study so far, the Norfolk Diabetes Case–Control Study, with a total of 5,574 type 2 diabetes cases and 6,950 controls. Our meta-analysis suggests that there is a statistically significant association of modest effect size between the OriB variant and the risk of type 2 diabetes in Europid populations. Our findings also suggest that the magnitude of the association may vary by ethnic background. The magnitude of the measure of association is comparable with that seen for variants identified by GWAS and associated with type 2 diabetes. The level of significance in this study is not as low as that seen in GWAS, but this is a function of the much smaller sample size, which, in itself, is a reflection of the much greater technical difficulty of typing the OriB variant. The accepted GWAS statistical thresholds for limiting false-positive associations do not apply to this study as only the OriB variant has been typed and this is not a genome-wide analysis. On the basis of the magnitude of the association and its overall statistical significance in Europid populations, the association of the OriB variant with type 2 diabetes deserves further investigation in additional case–control studies, including in other ethnic groups where the higher frequency of the OriB variant should increase the power to detect a true association.
Replication and transcription of mtDNA is complex, being mediated by nuclear-encoded proteins. The mechanisms of human mtDNA replication are far from resolved, and several origins of replication have been proposed. One of them, OriB, maps near one end of the major non-coding region (NCR) of mammalian mtDNA [
37]. Many molecules of mtDNA contain a triple strand or D-loop region, and OriB is located close to the 3′ end of a D-loop. The D-loop may act as a platform for recruiting essential components of the replisome to mtDNA, such as the β subunit of the mitochondrial γ polymerase [
38]. Therefore, the D-loop might regulate recruitment of replication initiation factors to OriB. Nevertheless, current understanding of the physiological roles of the various mtDNA origins is limited and nothing is known about the role of OriB in mtDNA replication in the beta cell [
37,
39]. OriB appears to be more important in mtDNA replication during recovery from transient mtDNA depletion than in mtDNA replication required for maintaining appropriate mtDNA during cell doubling in culture. The mtDNA content of blood cells is slightly lower in healthy individuals with the OriB variant than in those with wild type, suggesting that sequence variants might influence this origin’s activity [
40]. Furthermore, the sequence appears to influence the access of proteins to this region. For instance, the variant both restricts the DNase I footprint in the region [
9] and reduces the binding of the mitochondrial single-strand DNA binding protein (mtSSB) [
37]. These results suggest that the OriB variant may alter protein binding in the NCR and thereby affect mtDNA replication. Thus, our findings in the present meta-analysis should stimulate further investigation of the functional abnormalities underlying our observation, in particular the role of OriB and the homopolymeric C tract in the aetiology of type 2 diabetes.
Given that the prevalence of the OriB variant is about 10% in Europid populations, the small increase (10%) in diabetes risk associated with it would explain only a small proportion of people with type 2 diabetes. However, given that the prevalence of the variant is higher in Asian populations and that the magnitude of association with diabetes is also greater, it is possible that the population-attributable fraction related to this variant could differ markedly by ethnic group. Hence, it might explain an important component of variance in the worldwide distribution of diabetes [
9]. It has also been proposed that the association of the OriB variant with diabetes might be stronger in certain subgroups, such as those with obesity [
17]. Our analysis of the Norfolk Diabetes Case–Control Study does not provide support for the existence of such interactions with BMI or other characteristics. As our meta-analysis was based on published results and not individual participant data, we were unable to explore interactions by participant-level characteristics in the meta-analysis.
The strengths and limitations of the current study merit consideration. Our meta-analysis involved five times more data than the earlier review. These data provided greater power than previously available to quantify the magnitude of association. Although we have also identified a large effect of the OriB variant and type 2 diabetes in Asian populations, the sample sizes in the majority of studies in Asian populations were very small. Meta-analysis of these smaller studies is more likely to overestimate such associations and lead to biased results. There was evidence of heterogeneity identified between Europids and Asians (
I
2 = 45%; 95% CI 7%, 71%;
p = 0.02). Therefore, in this report, we focused on the association of the OriB variant and type 2 diabetes in Europid populations, which could give us more reliable results. In contrast to earlier studies that used PCR/RFLP and sequencing methods, we made use of a combination of pyrosequencing and Sanger sequencing methods to accurately genotype over 12,000 individuals for the OriB variant in the Norfolk Diabetes Case–Control Study, which should be less liable to misclassification bias in the OriB variant. Although the Norfolk study is the single largest study so far, it is still insufficient alone to enable reliable assessment of the moderate association between the OriB variant and the risk of type 2 diabetes in Europid populations. However, in a meta-analysis of this and other published studies, we were able to demonstrate a modest association. It is possible that studies that did not accurately identify the variations around the homopolymeric tract could have underestimated its effect [
24]. Because we did not have access to individual data in the published studies, we could not control for population stratification, adjust for possible confounding factors, explore heterogeneity by individual-level characteristics or perform haplogroup analyses.
In summary, this meta-analysis of new and previously published data suggests that the mitochondrial DNA OriB variant is modestly associated with an increased risk of type 2 diabetes in Europid populations. Further studies to investigate the functional abnormalities underlying this association are required, as are additional epidemiological investigations of ethnic group differences.
Acknowledgements
We thank all the study participants. We are grateful to the study team, Mike Sampson (Norfolk and Norwich University Hospital, Norwich, UK) and Elizabeth Young (Department of Public Health and Primary Care, University of Cambridge, UK) for help with the Norfolk Diabetes Case–Control Study. We thank staff from the MRC Epidemiology Functional Group Team for study coordination, data collection in the field by research nurses, data management and laboratory work and Di Hoult for carrying out the genotyping. We thank the general practitioners and the EPIC-Norfolk study team for their helpful input.