Abstract

Background

Type 2 diabetes (T2D) is emerging as an important international health problem. The International Diabetes Federation (IDF) report that 382 million people worldwide are living with diabetes, with a further 316 million with impaired glucose tolerance and therefore at high risk from the disease [1]. Evidence published in the IDF Diabetes Atlas [1] disproves the misconception that diabetes is a disease of the wealthy. Indeed, more than 80 % of people with diabetes live in low and middle-income countries, with emerging hotspots including countries in the Middle East and Sub-Saharan Africa [1]. In Sudan, Awad et al. [2] reported a prevalence of 3.4 % and considered diabetes as the commonest cause of hospital admission and morbidity due to a non- communicable disease. The highest prevalence is reported in northern Sudan [3] and the lowest in the western desert regions [4], with data reported in the IDF Diabetes Atlas [1] suggesting prevalence varying from 6-10 % and 60 % of deaths due to diabetes occurring in people under 60 years of age.

Since the introduction of genome-wide association studies (GWAS) a large number of genes have been shown to be associated with T2D at genome-wide significance (generally accepted to be P≤5x10⁻⁸ [5]), including many that have been shown to have effects across multiple ethnicities [6–19] and in trans-ancestry meta-analysis of large-scale GWAS [20]. Only a few studies have looked at these genes in Arab [16, 18, 19, 21–23] or Sub-Saharan African populations [8, 17]. In this study we looked for association between 14 single nucleotide polymorphisms (SNPs) in 7 of the top GWAS genes (TCF7L2, CDKAL1, HHEX, IGF2BP2, KCNJ11, PPARG and CAPN10) and risk of T2D in Sudan.

Methods

Results

Demographic and clinical profile of cases and controls

Demographic, clinical and biochemical parameters for 225 T2D cases (age range 20 to 89 years) and 129 healthy controls (age range 20 to 85 years) originally recruited into the study are presented in Table 1. As expected, significant differences in clinical traits associated with T2D (systolic blood pressure, fasting glucose, and total cholesterol) were observed between cases and controls. Age and BMI also differed between cases and controls. Since age, sex and BMI are known to be independent risk factors for T2D [1], we adjusted for these in our genetic analyses looking for associations between SNPs and T2D. A total of 186 cases and 106 controls with complete records for age, sex, and BMI were taken forward in these adjusted association analyses as presented below.

Table 1

Clinical and biochemical parameters of type 2 diabetes cases and controls

Trait	Controls	Cases	T testa
	Mean±SD (n)	Mean±SD (n)	P-value
Age (years)	48±15 (118)	58±13 (225)	<0.001*
Systolic BP (mmHg)	122±18 (104)	132±22 (202)	<0.001*
Diastolic BP (mmHg)	75±13 (105)	77±15 (204)	0.231
BMI (kg/m2)	27±6 (107)	29±7 (234)	0.017
Fasting glucose (mg/dl)	105±32 (66)	155±58 (124)	<0.001*
Cholesterol (mg/dl)	198±72 (67)	171±63 (125)	0.012
HDL (mg/dl)	76±43 (59)	69±43 (115)	0.287
LDL (mg/dl)	115±58 (47)	104±60 (95)	0.281
TG (mg/dl)	129±75 (66)	105±68 (125)	0.026

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^aT-Test with Welch’s correction for unequal variance where applicable; *indicates unequal variances. SD=Standard Deviation, n=number of the samples, BP=Blood pressure, BMI=Body Mass Index, HDL=High Density Lipoprotein, LDL=Low Density Lipoproptein, TG=Triglyceride

Characteristic of SNPs in the study population

All SNPs were in Hardy-Weinberg equilibrium in the control sample (p>0.05). Minor allele frequencies (MAF) for SNPs are provided in Table 2, and compared with MAF for Hapmap populations. Two SNPs, PPARG rs1801282 and KCNJ11 rs1800467, were at MAF<0.05 in our population and were removed from the association analyses. These low MAFs were consistent with low MAFs for these SNPs in all three HapMap populations. The remaining 12 SNPs were taken forward in the association analyses. The strict P-value needed to achieve statistical significance taking multiple testing into account is P=0.004 (=0.05/12). However, this is conservative since multiple SNPs within genes showed some degree of linkage disequilibrium, as might be expected given that they were selected as top SNPs for association with T2D in other populations. Therefore, P≤0.007 (=0.05/7 genes) was used as the P-value required to achieve correction for multiple testing.

Table 2

Details of SNPs used in this study

Gene	Chr	SNP	bp Location GRCh38	Minor Allele	MAF SDNa	MAF HapMap Populations
					N=129	CEU	HCB	YRI
CAPN10	2	rs2975760	240591746	C	0.07	0.16	0.11	0.01
		rs5030952	240603286	T	0.35	0.05	0.26	0.63
PPARG	3	rs17036314	12335246	C	0.26	0.20	0.22	0.18
		rs1801282	12351626	G	0.02	0.08	0.02	0
IGF2BP2	3	rs4402960	185793899	G	0.46	0.72	0.78	0.46
		rs1470579	185811292	A	0.28	0.72	0.74	0.13
CDKAL1	6	rs9465871	20717024	C	0.34	0.16	0.48	0.61
HHEX	10	rs1111875	92703125	A	0.25	0.42	0.69	0.13
TCF7L2	10	rs7903146	112998590	T	0.30	0.28	0.02	0.26
		rs11196205	113047288	G	0.48	0.58	0.96	0.14
		rs12255372	113049143	T	0.24	0.22	0	0.27
KCNJ11	11	rs5215	17387083	C	0.13	0.39	0.35	0.01
		rs1800467	17387284	G	0.03	0.05	0	0.01
		rs5219	17388025	T	0.08	0.39	0.39	0

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^aMAF – minor allele frequency for Sudan (SDN) healthy controls

Details include minor allele frequencies (MAF) in control subjects (N=129) from the Sudanese (SDN) study compared with CEU, HCB and YRI HapMap populations. Chr=chromosome; bp Location=chromosomal base pair location of the SNP in Genome Reference Consortium Human Build 38 (GRCh38)

Haplotype associations and stepwise logistic regression analysis for SNPs at TCF7L2

Since all three SNPs at TCF7L2 were associated with T2D in our population, the question arises as to whether all SNPs were tagging a single functional variant, or whether multiple main effects occur. SNPs within TCF7L2 were in strong linkage disequilibrium as determined by D' (rs7903146 and rs11196205: D'=0.94; rs7903146 to rs12255372: D'=0.68; rs11196205 to rs12255372: D'=0.80), suggesting that all 3 SNPs may be tagging a single haplotype carrying the risk variant. This was not so strongly supported by linkage disequilibrium as determined by r² (rs7903146 and rs11196205: r²=0.36; rs7903146 to rs12255372: r²=0.35; rs11196205 to rs12255372: r²=0.23) which takes allele frequencies into account. It was of interest too that the minor allele at rs11196205 was associated with protection, whereas for the other two TCF7L2 SNPs the minor alleles were the risk alleles. We therefore carried out haplotype association analyses to see where the strongest associated haplotypes occur. As before (Table 3), the strongest single point association was at rs7903146 (odds ratio 1.70; P _{adj:age/sex/BMI}=0.005). Notably, the associated (odds ratio >1) two point and three point risk haplotypes were on the background of the common C allele at rs11196205 (Table 4), with the strongest association (odds ratio 2.24; P _{adj:age/sex/BMI}=0.0003) comprising the two point haplotype T_C across rs7903146 and rs11196205. In a stepwise logistic regression analysis (Table 5), SNP rs7903146 added significant main effects to rs11196205, whereas the reverse was not true. Similarly, whilst rs7903146 added significant main effects to rs12255372, the reverse was not true. Together these results are consistent with a single main effect for association with T2D in this population that is most strongly tagged by SNP rs7903146.

Table 4

The effects of multiple SNPs at TCF7L2 on T2D risk in the Sudanese population

Allele/Haplotype	Frequency	rs7903146	rs11196205	rs12255372
T	0.398	OR=1.79; P=0.005
C	0.567		OR=1.56; P=0.018
T	0.33			OR=1.60; P=0.021
TC	0.377	OR=2.24; P =0.0003
CT	0.294		OR=1.86; P=0.005
TCT	0.236	OR=1.86; P=0.010

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Analysis undertaken under an additive logistic regression model (adjusted for age, sex, and BMI) to look for associations between haplotypes for the 3 SNPs at TCF7L2 and risk of T2D in the Sudanese population. Bold indicates the most significant haplotype association

Table 5

The effects of multiple SNPs at TCF7L2 on T2D risk in the Sudanese population

Null model	Alternative model	OR	P-value
rs7903146	rs7903146+rs11196205	0.98	0.938
rs7903146	rs7903146+rs12255372	1.22	0.437
rs11196205	rs11196205+rs7903146	2.04	0.010
rs11196205	rs11196205+rs12255372	1.53	0.078
rs12255372	rs12255372+rs7903146	1.61	0.052
rs12255372	rs12255372+rs11196205	0.77	0.219

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Stepwise logistic regression analysis to determine whether SNPs at TCF7L2 contribute separate main effects. A significant test comparing null and alternative models indicates that the marker added under the alternative model is contributing a separate main effect from the marker considered under the null hypothesis. Bold indicates nominal P<0.05

Discussion

In the present study, we investigated a possible association between T2D in Sudan and SNPs from 6 genes (PPARG, IGF2BP2, CDKAL1, HHEX, TCF7L2, and KCNJ11) that had achieved genome-wide significance in multiple GWAS undertaken across different ethnicities [31], in addition to CAPN10 which had been shown to be associated with T2D in a large study of Caucasians [26] and in Tunisia [27]. Of these, we found significant evidence for association between T2D and SNPs at TCF7L2 in our Sudanese population, and suggestive evidence for associations at SNPs in CAPN10 and HHEX. Given the limitations of our sample size, we cannot discount the possibility that further associations with SNPs in more of these genes would be found in this population if larger sample sizes were employed. However, relatively few studies have determined the role of common global T2D associated genetic variants in Sub-Saharan Africa, and to our knowledge this is the first study to provide evidence for associations between T2D and these important diabetes genes in a Sudanese population. Thus our study contributes to the growing need to replicate associations observed in GWAS carried out predominantly in Caucasian populations.

CAPN10 is of some interest as the first T2D gene to have been identified in a genome wide linkage scan [32], followed by positional cloning [33]. Subsequent large-scale meta-analysis of 13,628 subjects found only a modest effect size (odds ratio 1.15; 95 % confidence intervals 1.07-1.23; P=0.0002) for allele T at SNP rs2975760 as a risk factor for T2D [26], and no GWAS have reported P<1x10⁻⁵ for association between T2D and CAPN10 as currently recorded in the NCBI Catalog of published GWAS [31]. Some evidence for associations with larger effect sizes (odds ratios 1.35 to 1.61) were recently reported for SNPs at CAPN10 and risk of T2D in a Tunisian Arab population [27]. Here we show that the T allele at SNP rs2975760 was also associated with a larger effect size (odds ratio 2.38; 95 % confidence interval 1.09-5.26; P=0.03) in the Sudanese study population following adjustment for age. Further work is required to determine whether this is indicative of population-specific roles for polymorphisms at CAPN10 as risk factors for T2D in African populations.

The association at HHEX is also of interest given prior strong candidacy of this gene as a risk factor for T2D in multiple other populations [8, 9, 20], in this case well supported by results of multiple GWAS including trans-ancestry studies recorded in the NCBI Catalog of published GWAS [31]. HHEX belongs to a large family of transcription factors that are distinguished by a 60 amino acid conserved DNA-binding homeodomain. It is expressed in the anterior visceral endoderm during early development and in some adult tissues of endodermal origin, including liver and thyroid. In humans HHEX is associated with decreased insulin secretion in response to oral glucose stimulation, while knockout mouse studies demonstrate that the gene is a regulator of glucose-stimulated insulin secretion [34]. HHEX is located in a region of chromosome 10 that harbors several genes involved in beta-cell function or development, including the TCF7L2 gene. However, the two genes are located some 10 Mb apart, and their effects were independent in our study.

Associations observed between polymorphisms at TCF7L2 and T2D were the most strongly supported in our Sudanese study population. This is in keeping with GWAS and replication studies providing evidence for a role for this gene as a risk factor for T2D across multiple ethnicities [10–12, 14, 20, 31, 35], including a growing number of studies in African Americans [15], Sub-Saharan African [8, 17, 36] and Arab [16, 19, 21, 23, 25, 37–39] populations. Indeed, TCF7L2 was once described as the biggest story in diabetes since HLA [40]. Exceptions include studies in Pima Indians [41] and an Australian Aboriginal population [42]. This may relate to sample size, and/or to the observation across several studies that TCF7L2 variants are associated with reduced BMI in diabetes cases (but not in controls) [40]. Hence, in a population where T2D is predominantly associated with high BMI, the association with TCF7L2 might not be so readily observed. In Emiratis, for example, association between TCF7L2 and T2D was only observed in a non-obese case group and not in obese diabetics [38]. In our Sudanese study, our sample size was too small to stratify by BMI, but our association between TCF7L2 variants and T2D was robust to adjustment for BMI.

TCF7L2 encodes a high-mobility group box-containing transcription factor. Collective evidence (reviewed [43]) supports the hypothesis that WNT/β-catenin signaling via TCF7L2 regulates Glucagon-like peptide 1 effects in pancreatic β-cells by transcriptionally regulating its receptor. In humans, the risk T allele at rs7903146 (the SNP most strongly associated with T2D in our Sudanese population) strongly predicted future T2D in two independent cohorts, and was associated with impaired insulin secretion, incretin effects, and an enhanced rate of hepatic glucose production [44]. TCF7L2 expression in human islets was increased 5-fold in T2D, particularly in carriers of the TT genotype. Overexpression of TCF7L2 in human islets reduced glucose-stimulated insulin secretion. These authors conclude that the increased risk of T2D conferred by variants in TCF7L2 involves the enteroinsular axis, enhanced expression of the gene in islets, and impaired insulin secretion [44].

Conclusions

In this study we found significant evidence for association between T2D and SNPs at TCF7L2 in our Sudanese population, and suggestive evidence for associations at SNPs in CAPN10 and HHEX. The latter require confirmation in studies of larger sample sizes in Sudan, and we cannot exclude the possibility of associations with other genes examined here. Given its strong global associations with T2D, the demonstration of association between TCF7L2 and T2D in this Sudanese population is important. The cumulative evidence for associations between TCF7L2 polymorphisms across multiple ethnicities, together with growing knowledge of WNT/TCF7L2 signaling in β-cells, make it an attractive target for development of novel therapies for diabetes [43]. Our research demonstrates that T2D patients in Sudan would likely benefit from this translation of genetic research to the clinic.

Acknowledgements

Abbreviations

Footnotes

Contributor Information

Amir T. Ibrahim, Email: moc.liamtoh@_86gatrima.

Ayman Hussain, Email: moc.liamg@niassuhnamya.

Mohamed A. M. Salih, Email: moc.liamtoh@65hilasm.

Omima Abdeen Ibrahim, Email: moc.oohay@amimoneedba.

Sarra E Jamieson, Email: ua.gro.sdiknohtelet@noseimaj.arras.

Muntaser E. Ibrahim, Email: gro.dnei@miharbim.

Jenefer M. Blackwell, Email: ua.ude.awu.rhci@llewkcalbj.

Hiba S. Mohamed, Phone: 00249912605221, Email: gro.dnei@halasabih.

References