Study samples
The first part of our study aimed to identify common alleles associated with increased type 2 diabetes susceptibility using DIAGRAM consortium data and a tagging SNP approach. For this we genotyped several European samples.
The first sample was from the Hoorn Study (NL1) [
7], a Dutch population-based study from the city of Hoorn, in the north-west of the Netherlands, from which we selected 519 participants with normal glucose tolerance (NGT) and 480 type 2 diabetes patients. Glucose tolerance was assessed using a fasting OGTT, according to 1999 WHO criteria [
8]. This sample was used to analyse common variation in
LARS2 gene with a tagging SNP approach. Variants in
LARS2 identified from the DIAGRAM meta-analysis and the tagging SNP approach were then taken forward for replication in three Dutch samples.
The second Dutch sample (NL2) included 1,517 controls and 821 type 2 diabetic patients [
9,
10]. The 1,517 controls were randomly selected from the New Hoorn Study (NHS), which is an ongoing, population-based study from the city of Hoorn which does not overlap with the original NL1 sample. Of the type 2 diabetes patients, 147 were from the NHS and the remainder (
n = 674) were recruited from the diabetes clinics of the Leiden University Medical Centre and the Vrije Universiteit medical centre, Amsterdam. All participants in this replication sample were Dutch whites. All NGT participants underwent an OGTT and were classified according to WHO criteria [
8].
The third replication sample was ascertained from the Breda study (NL3) [
11,
12]. This is a case–control study from the city of Breda, in the south of the Netherlands. The 920 controls were from the Dutch blood bank and self-reported a non-diabetic state. The 501 cases had type 2 diabetes diagnosed on the basis of WHO criteria [
8].
For the fourth replication sample we selected 5,183 NGT participants and 1,222 type 2 diabetes patients from the population-based ERGO study (NL4) from Rotterdam in the south-west region of the Netherlands [
13].
In total 8,139 controls and 3,024 type 2 diabetes patients were included in our replication study in the Netherlands.
The second part of this study was focused on the follow up of two low-frequency variants in LARS2, for which we carried out replication in samples from the Netherlands (NL1–NL4) as well as samples from the UK (UK sample 1 [UK1], UK sample 2 [UK2]), Denmark (Denmark sample 1 [DK1]), Finland (Finland sample 1 [FL1], Finland sample 2 [FL2]) and Sweden (Sweden [SE1]).
Our DK1 sample [
14] consisted of 514 NGT controls randomly selected from public registers at the Steno Diabetes Center and the Research Centre for Prevention and Health, Copenhagen, Denmark. The 706 type 2 diabetes patients were recruited from the Steno Diabetes Center. NGT participants underwent an OGTT according to WHO criteria [
8].
Of the two UK samples, the first (UK1) was the United Kingdom Type 2 Diabetes Genetics Consortium case–control sample, comprising 4,124 type 2 diabetes patients and 5,126 controls ascertained in Tayside, Scotland. Details of the ascertainment scheme and recruitment criteria for this sample have been described elsewhere [
15,
16]. The enlarged sample used here represents continuing recruitment to this resource under precisely the same criteria. The second sample, UK2, consisted of 1,853 type 2 diabetes patients ascertained as part of the BDA Warren 2 collection (Exeter, London, Oxford, Norwich and Newcastle) and 10,220 control samples. The latter represent the full British 1,958 Birth Cohort (
n = 7,133) and the United Kingdom Blood Services Collection of Common Controls (
n = 3,087), a subset of which featured in the Wellcome Trust Case Control Consortium (WTCCC) genome-wide association scan (both samples were collected throughout the UK) [
15,
16].
Finally, we included samples from Finland and Sweden. The FL1 sample was a case–control sample from the Botnia region of Finland, consisting of 353 controls and 402 type 2 diabetes patients. The sample from Sweden, SE1, was from a case–control study from Skara and Malmö, and consisted of 468 controls and 480 type 2 diabetes patients. We also included a set of trios originating from the Botnia region of Finland. This sample, FL2, consisted of 211 probands (multiple diabetic sibs) and 370 parents [
17,
18]. All study samples are summarised in Table
1.
Table 1
Description of study samples
NL1 | 519 (55) | 480 (52) | 65 (8) | 67 (8) | 26.4 (4.5) | 28.8 (4.6) |
NL2 | 1,517 (44) | 821 (50) | 53 (7) | 61 (11) | 25.5 (3.6) | 29.0 (4.6) |
NL3 | 920 (61) | 501 (46) | 48 (13) | 71 (10) | n.a. | 27.8 (4.1) |
NL4 | 5,183 (41) | 1,222 (39) | 69 (9) | 73 (9) | 26.0 (3.9) | 27.4 (4.0) |
DK1 | 514 (46) | 706 (48) | 57 (10) | 59 (10) | 25.9 (3.8) | 29.3 (5.1) |
UK1 | 5,126 (51) | 4,124 (55) | 60 (13) | 66 (6) | 26.9 (11.4) | 31.2 (13.8) |
UK2 | 10,220 (50) | 1,853 (61) | 42 (7) | 57 (9) | 27.2 (6.4)a
| 31.8 (6.7) |
FL1 | 353 (53) | 402 (55) | 60 (10) | 61 (10) | 26.1 (3.6) | 28.7 (4.5) |
FL2 | 370 (50)b
| 211 (47)c
| n.a. | 40 (9) | 28.5 (5.5) | n.a. |
SE1 | 468 (52) | 480 (53) | 66 (12) | 67 (11) | 27.5 (4.1) | 27.9 (4.1) |
In total 25,191 controls and 10,800 type 2 diabetes patients were included for follow up of the low-frequency variants.
All studies were approved by the appropriate medical ethical committees and were in accordance with the principles of the Declaration of Helsinki. All participants provided written, informed consent for this study.