Download Warren 2 Sib Pairs (Wiltshire et al 2001)

Detailed description of study populations used in
“Association analysis of 6736 UK subjects provides
replication and confirms TCF7L2 as a type 2 diabetes
susceptibility gene with a substantial effect on
individual risk” (Groves et al).
T2D Case Resources
The type 2 diabetes (T2D) case samples used in this study came from three related
UK collections. These were:
(a) probands from the Warren 2 sibpair studies (denoted “W2SP”)
(b) cases from the Warren 2 case collection (denoted “W2C”)
(c) probands from the Warren 2 trios collection (denoted “W2TP”)
Though there were some differences in the ascertainment scheme for each of these
(see below), most of the inclusion and exclusion criteria were similar. All cases were
of British/Irish European descent [except for 38 probands from the W2TP collection –
see below], with all four grandparents having exclusively British and/or Irish origin,
both by self-reported ethnicity and by place of birth. Each of the collections involved
a network of multiple centres across the UK (coordinated from London, Oxford,
Exeter, Cambridge/Norwich and Newcastle), and therefore provides a regionallyrepresentative sample of T2D cases across the UK. Analysis of over 1M microsatellite
genotypes from the Warren 2 sibpair scan has previously revealed no evidence for
population substructure associated with this recruitment strategy (M.McCarthy,
S.Wiltshire, L.Cardon, unpublished data).
For all cases used, validation of the diagnosis of diabetes was based on either current
prescribed treatment with sulfonylureas, biguanides, and/or insulin or, in the case of
individuals treated with diet alone, historical or contemporary laboratory evidence
of hyperglycemia (as defined by World Health Organization [1985] the guidelines in
place at the time of recruitment). Other forms of diabetes (e.g., maturity-onset
diabetes of the young, mitochondrial diabetes, and type 1 diabetes) were excluded by
standard clinical criteria based on personal and family history. Criteria for excluding
type 1 diabetes included an absence of first-degree relatives with type 1 diabetes and
an interval of ≥1 year between diagnosis and institution of regular insulin therapy. In
addition, in all cohorts evidence for autoimmunity to islet antigens was sought by
measurement of titres of antibodies to glutamic acid decarboxylase (anti-GAD). All
cases are GAD-antibody negative.
1A) Warren 2 Sibpair Probands (W2SP)
The W2SP cases are all members of the 573 sibpair probands described in
detail in Wiltshire et al, 2001. These cases were therefore ascertained on the basis that
they came from families which included, at minimum, a sib pair with T2D. The 573
families are derived from a larger collection of 843 sibship pedigrees collected during
1995-1998. Age at diagnosis, of both members of the index sib pair, was initially
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restricted to the age range 35–75 years and subsequently was narrowed to 35–70
years, with 97.6% of families meeting the latter criterion. Pedigrees either reporting
bilineal inheritance (both parents diabetic) or having a high proportion of affected
individuals within large sibships were excluded from collection. The attrition from
843 to 573 pedigrees is based on a number of factors (described in detail in Wiltshire
et al, 2001) which included availability of DNA (at the time genotyping commenced),
DNA quantities, GAD-negativity, non-mendelization (based on genome-wide
linkage genotyping) and half-sibships.
Details of the subject characteristics are given in main table 1 of the present
paper. This cohort is diagnosed at a mean age of 55.0 years and has a mean BMI of
28.4 kgm-2. They were clearly enriched for family history as they all had at least one
diabetic sibling.
1B) Warren 2 Cases (W2C)
The Warren 2 case sample studied for this gene comprises 1586 T2D cases
ascertained under the same ethnicity and diagnostic criteria described above.
However, the inclusion criteria for this cohort stipulated no minimum requirements
for family history, though age at diagnosis was restricted to 35 to 65 years. This
cohort includes 1126 individuals previously described (Weedon et al, 2004) along
with a further 460 individuals subsequently collected under exactly the same
recruitment strategy.
The details of the W2C are shown in table 1 of the present paper. The mean
age at diagnosis is 51.4 years, and the mean BMI 31.5 kgm-2. Unlike the W2SP, the
W2C were not recruited on the basis of family history. However, 44% had at least
one parent with T2D.
1C) Warren 2 Trios Probands (W2TP)
The W2TP sample represents the offspring probands from the Warren 2 trio
(parent-offspring triad) collection. A total of 390 complete trios were available for
study (though two have been eliminated due to repeated markers being inconsistent
with the stated relationships.) All trios probands were collected using the same
diagnostic criteria and exclusions for type 2 diabetes described above except that, to
aid recruitment (given the difficulties of finding parents for a late-onset disease), a
lower limit for age of diagnosis (25 years) was used. Probands that had clinical
features of monogenic forms of diabetes particularly MODY and maternally
inherited diabetes and deafness (MIDD) were excluded and if there was any doubt,
sequencing of the MODY genes and testing for the mt3243 mutations was performed.
Since the aim of this collection was to allow family-based association analyses
(avoiding concerns about population stratification), this collection does include 38
trios in which any of the proband, parents or grandparents are of non British/Irish
European origin. Such trios are included in the family-based analysis, but the
probands from such trios are not included in any of the case-control analyses
(leaving 350 W2TP for most of the analyses). A detailed description of the first 150
trios is provided in Frayling et al, 1999.
The W2TP samples represent T2D subjects enriched for early diagnosis (mean
40.3 years) given the need to have both parents alive. The early diagnosis is the likely
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explanation of why these probands were enriched for both obesity (mean BMI 32.3
kgm-2) and for family history of type 2 diabetes with 58% of trios having one or more
diabetic parent, although neither feature was an explicit requirement for recruitment.
The specific advantage of this resource is that it allows family-based association
analyses to be performed. It is important to note that the main case-control analyses
reported in the present paper did not include the W2TP samples: this was to allow
the family-based association analysis in the Warren 2 trios to be entirely independent
of the case-control analysis.
Collectively, these resources represent one of the largest collections of T2D
cases available worldwide. Because of the various ascertainment effects it is likely
that they are significantly enriched for disease susceptibility genes (which is an
advantage for gene discovery efforts), but that estimates of epidemiological
parameters (such as population attributable risk) are likely to be upwardly biased
(particularly in the W2SP and W2TP).
T2D Familial Resources
2A) Warren 2 Sibpair Collection
To establish whether or not the TCF7L2 variants were responsible for the
chromosome 10q linkage signal previously reported in our UK samples (Wiltshire et
al, 2001), we typed all members of the 573 sibship pedigrees on which that linkage
study was based. The criteria for recruitment are described above (see 1A) and in the
original manuscript (Wiltshire et al, 2001). These 573 pedigrees included a total of
1406 ascertained individuals.
2B) Warren 2 Trios collection
Family-based association analysis was conducted in the 388 trios described
under section 1C (see also, Frayling et al 1999).
Control Resources
We used two complementary UK-national control samples for the purposes
of this study. Both have been widely used in genetic studies in the UK without any
problems with stratification. Only UK subjects of European descent are included in
the analyses reported.
3A) Human Random Control resource (HRC)
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/PC
R/Product_Lines/Human_Genomic_DNA.html
The Human Random Control (HRC) DNA samples represent a control
population of 480 UK blood donors of European descent. These are a panel of
commercially available DNAs which we have supplemented with an additional 70
samples from the same source (European Collection of Cell Cultures [ECACC]) to
provide a resource of 550 Random British Control samples we define as “HRC+”. The
HRC DNA is extracted from lymphoblastoid cell lines derived by Epstein Barr Virus.
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No phenotype data are available for this resource, although sample sex has been
determined in-house using a gender-specific PCR method.
3B) 1958 British Birth Cohort (58BC)
2088 samples were made available from the Medical Research
Council/Wellcome Trust-funded 1958 British birth cohort DNA resource. This
represents a national birth cohort made up of subjects born in the same week in
March 1958 (Power & Elliot, 2006). They have been the subject of extensive
prospective analysis, culminating with a further round of data gathering in 2003-2005
at which samples were taken for DNA extraction and cell-line generation. The 2088
samples to which we have access represent the first batch of samples arising out this
process: they have been made widely available to serve as a common UK national
control sample. Of the 2088 samples, 2024 are designated as “White” and these form
the basis of our sample in the present paper. Though extensive phenotypic
information has been collected on these individuals, access to all but a minimum set
(gender, UK region of origin) requires specific dedicated approval from the
Oversight Committee.
As will be clear from the above, all controls have been ascertained without
explicit reference to phenotype. Inevitably, this means that some of the controls will
be destined to develop diabetes (and some may be diabetic already). However,
misclassification is likely to be modest, should not generate a bias away from the
null, and will, in most feasible situations, be associated with only a small loss of
power compared with the use of hypernormal controls (Colhoun et al, 2003).
Overall Validation
The value of these samples, their appropriateness for the task in hand, and
confidence that they are reasonably free from misdiagnosis, is confirmed by the fact
that associations with all well-established T2D susceptibility variants (replicated by
many centres) have been seen using these samples (including those in PPARG
[Zeggini et al, 2005]; KCNJ11 [Gloyn et al, 2003]; HNF4A [Weedon et al, 2004] and
CAPN10 [Weedon et al, 2003]).
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