2.1.1 Participants and samples
2.1.1.1 Northeast (NE) British Caucasian cohort
Anonymised DNA and RNA samples were available for 187 healthy adults. Five samples were excluded from analysis on the basis of inconsistencies between the genotyping results obtained in the DNA and cDNA samples, and five samples subsequently found to be duplicates were also excluded. Of the 177 included samples, 152 were collected through a sub-study of the People of the British Isles
study in 2007274, and 25 were control samples collected for a study investigating the
genetics of colon cancer (the CAPP study)275. 50% were male and the median age
was 63 years (range 25-101, lower quartile 51, upper quartile 69).
The People of the British Isles study recruited healthy adults from different regions to investigate genetic variation within the UK. To be eligible, at least three of their grandparents had to be born within a 30-40 mile radius of one another, and ideally be from a rural population. Of the samples used in this study, 7 were from Cumbria, 1 from Lancashire, and the remainder from northeast England (Northumberland, Tyneside or County Durham).
The CAPP study controls were collected in 2006 as controls for a genetic study
investigating colon cancer275 and comprised four healthy adult staff members at the
Institute of Human Genetics, and 21 phenotypically unaffected adults who had undergone screening for cystic fibrosis or haemochromatosis carrier status.
Informed consent was obtained from all participants and the studies were approved by the Newcastle and North Tyneside Local Research Ethics Committee.
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2.1.1.2 South African (SA) cohort
DNA and RNA samples were collected from 310 healthy adult volunteers at a blood donor clinic at the University of the Western Cape, Cape Town, South Africa. Samples were anonymised, but limited demographic data including age, gender, and self-reported ethnicity were collected. Informed consent was obtained from all participants and the study was approved by the University of Cape Town Faculty of Health Sciences Research Ethics Committee.
The self-reported ethnicity of the SA cohort was: 200 Cape mixed-ancestry; 67 African black; 19 Indian; 10 white; 4 other/unknown. 42% were male, with median age 20 years (range 17-60, lower quartile 19, upper quartile 23).
2.1.1.3 MLH1 validation samples
For the purpose of training and optimisation of AEI techniques, initial validation work was carried out to assess AEI in stored RNA samples using MLH1, as pilot studies investigating AEI in this gene had been previously performed. This validation work was performed using five heterozygous samples in which AEI had been previously assessed: two healthy volunteers without imbalance and three patients known to have imbalance of MLH1 expression who were recruited for the CAPP study. Informed consent was obtained from all participants and the studies were approved by the Newcastle and North Tyneside Local Research Ethics Committee.
2.1.1.4 HTO cohort
DNA was available for this cohort comprising of 1425 members of 248 British Caucasian families ascertained through hypertensive probands and phenotyped for a
quantitative genetic study of cardiovascular risk factors from 1993-200111, 156, 276.
Sample ascertainment
A detailed description of this series and the ascertainment strategy has been published
previously276. Families were selected through a proband with essential hypertension
whose systolic and diastolic BP were in the top 5% of the population distribution (defined as daytime ambulatory BP >140/90mmHg; three clinic BP measurements
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>160/95mmHg; or treatment with at least two antihypertensive medications). Secondary hypertension was excluded using the screening protocol applied in the hypertension clinic. Families were required to consist of at least three siblings (including the proband) clinically assessable for BP if DNA from a parent of the sibship was available, or at least four siblings if no parental DNA was available. Qualifying sibships could be in the generation of the proband, or the offspring. There was no requirement for additional members of the family to be hypertensive, but where additional members of the sibship were found to have hypertension (using the same criteria), families were extended and the spouses and offspring of hypertensive members also collected. The majority (64%) of the individuals in the family
collection therefore have BP within the conventionally accepted “normal range”, and the family collection includes some extended families, though most are nuclear families. The median family size was 5 people, 60% of families comprising between 4 and 6 genotyped and phenotyped members. 71% of families were 2-generation and 29% were 3-generation. 84% of families had an assessable sibship in the generation of the proband, while 16% of families consisted of a proband and their nuclear family (spouse and children over 18 years) only. Informed consent was obtained from all participants and the study was approved by the Central Oxford Research Ethics Committee and Newcastle and North Tyneside Local Research Ethics Committee.
BP measurement
The BP measurement protocol has been previously described276. BP was measured
using ambulatory monitoring for a period of 24 hours in all subjects willing to undergo monitoring, using the A&D TM2421 monitor. Three readings were taken with the patient in a relaxed seated position at the start of the monitoring period. Simultaneous auscultation was carried out by a trained observer, to confirm
satisfactory (within 5mmHg) agreement between the monitor and auscultatory values; if this criterion was not met, the cuff was repositioned until satisfactory agreement was obtained. The three readings which had satisfactory agreement between the monitor and the observer in the final cuff position are referred to as “clinic readings”. The monitor was programmed to record blood pressure every half-hour during the daytime and every hour during the night, and a recording was considered of satisfactory technical quality if at least 20 daytime ambulatory data points were
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available for analysis. Patients also recorded the time they went to bed and rose in the morning to enable individualised calculation of the “daytime” and “night-time” periods. Mean values for systolic and diastolic blood pressures for the clinic, daytime and night-time periods were analysed for association with genotypes.
Echocardiography and CIMT measurement
Echocardiography and CIMT measurement was performed in a subset of individuals
between 1999 and 2001, as previously described11. Carotid artery ultrasonography
was performed in 953 individuals by two sonographers, with all measurements made by a single observer. The right and left common carotid arteries were scanned using a 7.5 MHz linear array transducer (HP Sonos 5500) and measurements were made from the far wall of the distal 10mm of the common carotid artery. Images were recorded for later offline analysis using computerised edge-detection software with manual editing. Mean and maximal CIMT measurements were performed at end-diastole on each side, and the average reading from these was calculated.
Trans-thoracic echocardiography was performed in 904 individuals according to a standard protocol in which 2D imaging, M-mode imaging and Doppler studies were performed. Left ventricular (LV) wall and cavity measurements were performed by a single observer from the standard M-mode images. LV mass (in grams) was
estimated from M-mode images as recommended by the American Society of
Echocardiography guidelines277, using the formula described by Devereux et al278.
Measurements of LV mass were corrected for body surface area as described by Levy
et al in the Framingham heart study cohort279. Fractional shortening was calculated as 100(LVIDs+LVIDd)/LVIDd, and LV ejection fraction was estimated according to the
formula described by Teichholz et al280. The E/A ratio was calculated using standard
methodology from pulse-wave Doppler estimates of mitral valve inflow velocity281.
Additional phenotyping
A full clinical history was taken, which included the subject’s medical history and lifestyle factors including consumption of alcohol and tobacco, and habitual physical exercise. Anthropometric data including height, weight and waist and hip
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phenotypes relevant to cardiovascular disease, using commercially-available assays. These included: plasma levels of total cholesterol, interleukin-6 (IL-6), tumour
necrosis factor α (TNF-α), C-reactive protein (CRP)282, and leptin283.
2.1.1.5 Congenital heart disease cohort
The congenital heart disease cohort comprised 888 probands with congenital heart disease collected from two studies, the CHANGE study (Congenital Hearts: A National Gene/Environment study) and the FCH study (Freeman Congenital Heart
Disease study)284.
The multi-centre CHANGE study included Caucasian patients with tetralogy of Fallot (TOF), or the related conditions pulmonary stenosis/VSD and double outlet right
ventricle284. Blood or saliva samples were obtained from patients at centres in
Newcastle, Leeds, Bristol and Liverpool. Patients with recognised causes for TOF were excluded (including known chromosome 22q11 deletion syndrome, other malformation syndromes, known chromosomal abnormalities, developmental delay and learning difficulties, and known maternal exposure to significant teratogens during pregnancy). 444 TOF samples were available for analysis.
The FCH study included Caucasian patients with other forms of congenital heart disease recruited at the Freeman Hospital in Newcastle. Blood or saliva samples and phenotypic information were obtained from all patients. 444 non-TOF congenital heart disease samples were available for analysis.
Informed consent was obtained from all patients or their parents/guardians and the studies were approved by the regional ethics committees.
2.1.1.6 Cumbria control cohort
The Cumbria control cohort comprised 1,089 Caucasian women of childbearing age
from the North Cumbria Community Genetics Project (NCCGP)285 for whom usable
DNA was available. Between 1996 and 2003 cord blood samples were collected from infants born consecutively at West Cumberland Hospital, and from 1999 to 2003
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samples was used for the analysis. Informed consent was obtained from all individuals and the studies were approved by the regional ethics committee.
2.1.2 Labware
All experiments were performed using standard sterile nuclease-free plasticware from recognised laboratory suppliers. Barrier pipette tips were used for all pre-PCR
preparation steps, which were performed in designated laminar-flow hoods.