Tom Burr

Rationale and cross-sectional study design of the Research on Obesity and type 2 Diabetes among African Migrants: the RODAM study.

Introduction Obesity and type 2 diabetes (T2D) are highly prevalent among African migrants compared with European descent populations. The underlying reasons still remain a puzzle. Gene–environmental interaction is now seen as a potential plausible factor contributing to the high prevalence of obesity and T2D, but has not yet been investigated. The overall aim of the Research on Obesity and Diabetes among African Migrants (RODAM) project is to understand the reasons for the high prevalence of obesity and T2D among sub-Saharan Africans in diaspora by (1) studying the complex interplay between environment (eg, lifestyle), healthcare, biochemical and (epi)genetic factors, and their relative contributions to the high prevalence of obesity and T2D; (2) to identify specific risk factors within these broad categories to guide intervention programmes and (3) to provide a basic knowledge for improving diagnosis and treatment. Methods and analysis RODAM is a multicentre cross-sectional study among homogenous sub-Saharan African participants (ie, Ghanaians) aged >25 years living in rural and urban Ghana, the Netherlands, Germany and the UK (http://rod-am.eu/). Standardised data on the main outcomes, genetic and non-genetic factors are collected in all locations. The aim is to recruit 6250 individuals comprising five subgroups of 1250 individuals from each site. In Ghana, Kumasi and Obuasi (urban stratum) and villages in the Ashanti region (rural stratum) are served as recruitment sites. In Europe, Ghanaian migrants are selected through the municipality or Ghanaian organisations registers. Ethics and dissemination Ethical approval has been obtained in all sites. This paper gives an overview of the rationale, conceptual framework and methods of the study. The differences across locations will allow us to gain insight into genetic and non-genetic factors contributing to the occurrence of obesity and T2D and will inform targeted intervention and prevention programmes, and provide the basis for improving diagnosis and treatment in these populations and beyond.

Epigenome-wide association study in whole blood on type 2 diabetes among sub-Saharan African individuals: findings from the RODAM study.

BACKGROUND Type 2 diabetes (T2D) results from a complex interplay between genetics and the environment. Several epigenome-wide association studies (EWAS) have found DNA methylation loci associated with T2D in European populations. However, data from African populations are lacking. We undertook the first EWAS for T2D among sub-Saharan Africans, aiming at identifying ubiquitous and novel DNA methylation loci associated with T2D. METHODS The Illumina 450k DNA-methylation array was used on whole blood samples of 713 Ghanaian participants (256 with T2D, 457 controls) from the cross-sectional Research on Obesity and Diabetes among African Migrants (RODAM) study. Differentially methylated positions (DMPs) for T2D and HbA1c were identified through linear regression analysis adjusted for age, sex, estimated cell counts, hybridization batch, array position and body mass index (BMI). We also did a candidate analysis of previously reported EWAS loci for T2D in non-African populations, identified through a systematic literature search. RESULTS Four DMPs [cg19693031 (TXNIP), cg04816311 (C7orf50), cg00574958 (CPT1A), cg07988171 (TPM4)] were associated with T2D after correction for inflation by possible systematic biases. The most strongly associated DMP-cg19693031, TXNIP (P = 2.6E-19) -showed hypomethylation in T2D cases compared with controls. Two out of the four DMPs [cg19693031 (TXNIP), cg04816311 (C7orf50)] remained associated with T2D after adjustment for BMI, and one locus [cg07988171 (TPM4)] that has not been reported previously. CONCLUSIONS In this first EWAS for T2D in sub-Saharan Africans, we have identified four DMPs at epigenome-wide level, one of which is novel. These findings provide insight into the epigenetic loci that underlie the burden of T2D in sub-Saharan Africans.

Abstract 2953: Evaluating gene expression in FFPE breast cancer tissues using DASL®

The study of gene expression in conventionally processed tissues is hampered by degradation of mRNA. Expensive, low multiplex, quantitative PCR methods can be unreliable due to the limited template sizes. One way to overcome this problem is to use array-based methods. DASL ® technology relies on random priming for production of cDNA, in concert with universal bead-arrays to allow the detection and relative quantitation of expression of specific gene subsets. Using the Illumina DASL ® Cancer Panel (500 cancer-associated genes on one array), we evaluated the expression of key genes in archival FFPE tissue samples from 80 breast cancer patients with well characterised pathological and clinical features. We first assessed transcript integrity in the samples on the basis of levels of mRNA encoding RPL13A, prior to running the Cancer Panel. A subset of genes of interest was then assessed by qPCR to confirm the relative levels observed using the DASL ® assay. Finally the expression of the same subset of genes was evaluated at the protein level by immunohistochemistry. We were able to predict, with good accuracy based upon RPL13A assays, those samples unsuitable for DASL ® analysis. Furthermore, the results of DASL ® analysis showed good correlation with protein levels, as measured by immunohistochemistry, for a number of key genes including ERBB2 (HER2) and ESR1 (ER). We conclude that DASL ® represents a powerful tool for assessing expression of multiple genes in archival tissue. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2953.

Evaluating gene expression in formalin-fixed, paraffin-embedded breast cancer tissues using DASL®

The study of gene expression in conventionally processed tissues is hampered by degradation of mRNA. Expensive, low-multiplex, quantitative PCR methods can be unreliable due to the limited template sizes. One way to overcome this problem is to use array-based methods. DASL® technology relies on random priming for production of cDNA, in concert with universal bead arrays to allow the detection and relative quantitation of expression of specific gene subsets. Using the Illumina DASL® Cancer Panel (500 cancer-associated genes on one array), we evaluated the expression of key genes in archival formalin-fixed, paraffin-embedded tissue samples from 80 breast cancer patients with well-characterised pathological and clinical features. We first assessed transcript integrity in the samples on the basis of levels of mRNA encoding RPL13A, prior to running the Cancer Panel. A subset of genes of interest was then assessed by quantitative PCR to confirm the relative levels observed using the DASL® assay. Finally the expression of the same subset of genes was evaluated at the protein level by immunohistochemistry. We were able to predict, with good accuracy based upon RPL13A assays, those samples unsuitable for DASL® analysis. Furthermore, the results of DASL® analysis showed good correlation with protein levels, as measured by immunohistochemistry, for a number of key genes including ERBB2 (HER2) and ESR1 (ER). We conclude that DASL® represents a powerful tool for assessing expression of multiple genes in archival tissue.