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Subjects and methods: In a total of 232 members of a large Omani pedigree, there were 27 diabetics, 17 with impaired fasting glucose, 33 with impaired glucose tolerance (IGT), while the remaining 155 were normoglycaemic. All 232 individuals were genotyped for the following genes: TCF7L2, CDKN2A/B, PPARG, FTO, IGF2BP2, KCNJ11, HHEX, SLC30A8, CDKAL1 and CAPN10. Measured genotype analysis was used to test association between variants of the above genes and T2D, IFG, IGT, fasting and postprandial glucose values, HbA1C, fasting insulin, weight, BMI, waist circumference and body fat (%).
Results: The rs5219 (KCNJ11) gene variant was found to be significantly associated with the combined IFG, IGT and T2D group (p = 0.049, OR = 1.63, age â‰¥40 years) and with HbA1C values (p = 0.012). The rs8050136 (FTO) was found to be associated with T2D (p = 0.041, OR = 1.56, age â‰¥40 years) and with BMI (p = 0.027). Some other gene variants were associated with parameters related to insulin resistance, but did not reach significance level after Bonferroni correction.
Conclusions: This family-based study confirmed the association between KCNJ11 and FTO genes variants with T2D among members of the Omani Arab pedigree. However, in this study we could not detect the effect of some of the well known common variants of T2D susceptibility genes like TCF7L2.
Genetics of Type 2 diabetes
CAPN10, cysteine protease calpain 10; CDKAL1, cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1; CDKN2A/B, cyclin-dependent kinase inhibitor 2A/B; FTO, fat mass and obesity associated protein; GWAS, genome wide association studies; HHEX, hematopoietically expressed homeobox; IFG, impaired fasting glucose; IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; IGT, impaired glucose tolerance; KATP, ATP-sensitive K+ channel; KCNJ11, subunit kir6.2 of the ATP-sensitive potassium channel gene of Î²-cells; MGA , measured genotype analysis; NCBI, National Center for Biotechnology information; OFS, Oman Family Study; PPARG, peroxisome proliferator-activated receptor Î³ gene; SLC30A8, solute carrier family 30/zinc transporter; SNP, single nucleotide polymorphism; S-TDT ,Sibship transmission/ disequilibrium test; T2D, type 2 diabetes; TCF7L2 , transcription factor 7-like 2.
Type 2 diabetes mellitus (T2D) is a worldwide chronic disease. The interaction between multiple genetic and environmental factors plays a major role in the pathogenesis of T2D. A family history of diabetes confers up to a 3-fold increased risk for first-degree relatives to develop the disease [1, 2]. The high prevalence of T2D among specific populations provides further evidence for the contribution of genetic factors in this disease . Different approaches have been used to search for T2D susceptibility genes: candidate gene approach, linkage analysis and genome wide association studies (GWAS). Only a few T2D susceptibility genes were identified using the candidate gene approach and also replicated in GWAS. Variants identified within candidate genes were: Pro12Ala (rs1801282) in peroxisome proliferator-activated receptor Î³ gene (PPARG) , Glu23Lys (rs5219) in the subunit kir6.2 of the ATP-sensitive potassium channel gene of beta cells (KCNJ11)  and a series of polymorphisms and haplotypes (UCSNP-43 or rs3792267; UCSNP-19 or rs3842570 and UCSNP-63 or rs5030952) in the coding region of the cysteine protease calpain 10 (CAPN10) . In 2006, common intronic variants within the transcription factor 7-like 2 (TCF7L2) gene (rs7903146 and rs12255372) were found to hold a strong genetic risk for T2D, which was subsequently confirmed in GWAS studies and replicated in different ethnicities [6-8]. Since 2007, further GWAS identified and confirmed the following as T2D risk genes or loci: solute carrier family 30/zinc transporter (SLC30A8), hematopoietically expressed homeobox (HHEX), cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1), insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), a genomic region between cyclin-dependent kinase inhibitor 2A/B (CDKN2A and CDKN2B) and fat mass and obesity associated protein (FTO) [9-12]. Most of the above identified and replicated gene variants have odds ratios of 1.14 to 1.4 on T2D risk, which is considered to be a fairly modest risk.
Up to date, more than forty common T2D susceptibility gene variants and loci have been identified, but all these variants could only explain ~10-15% of the heritability of T2D; which suggests that more variants remain to be discovered . Common ancient polymorphisms (frequency>5%) are shared by all human populations and account for approximately 90 to 95% of human variation . They were assumed to increase susceptibility to polygenic diseases like T2D but their contribution is modest . Rare large-effect mutations have recently been recognized as causes of many complex diseases [14, 16, 17].
McClellan and King  suggested that genetic heterogeneity among a common disease is described at multiple levels of causations. Rare mutations collectively may play an important role in causing the disease and the same gene may harbor many different severe rare mutations. Furthermore, one mutation may lead to different phenotypes in different individuals and mutations in different genes may lead to the same disorder .
Six out of the world's top ten countries with the highest prevalence for T2D, are located in the Middle East and North Africa region . Few studies investigating the genetics of T2D were conducted among Arabs [19-24]. Recently, a large case-control study among Moroccan and Tunisian Arabs investigated the effect of 44 polymorphisms, located at 37 validated European loci and confirmed the association of some of these polymorphisms with T2D among North African Arabs .
In this study we investigated the effect of 15 known common gene polymorphisms (with modest odds ratios) on susceptibility to T2D among Omani Arabs in one of the five pedigrees of Oman Family Study (OFS) . Like the other four pedigrees, this one has maintained homogeneity by extremely high level of inbreeding due to the tradition of consanguineous marriages, mostly between first cousins. It was selected because of a high number of individuals with T2D and abnormal glucose homeostasis among members of the pedigree.
Subjects and methods
This study was carried out on one, large, extended and highly consanguineous Omani pedigree of 232 individuals aged 16 - 80 years; a subset of the OFS [26-28]. Ninety nine participants were 40 years or older of whom 23 had T2D, 13 had IFG and 22 had IGT. The remaining 41 participants were normoglycaemic. All participants went through a standard 75g oral glucose tolerance test. T2D, IGT and IFG were defined according to 2006 World Health Organization (WHO) criteria (Diabetes: fasting plasma glucose â‰¥ 7.0 mmol/l or 2-h postprandial plasma glucose â‰¥ 11.1 mmol/l; IGT: Fasting plasma glucose < 7.0 mmol/l and 2-h postprandial plasma glucose â‰¥7.8 and <11.1 mmol/l; IFG: fasting plasma glucose 6.1 to 6.9 mmol/l and 2-h postprandial plasma glucose < 7.8 mmol/l). Obesity status was defined among participants using the international classification of adult normal weight, overweight and obesity according to BMI (Global database on body mass index), using criteria of the WHO-1995, WHO-2000 and WHO-2004 [Normal range: 18.5-24.99, overweight: 25.00-29.99 and obese â‰¥ 30.00] . Anthropometric and clinical characteristics of all participants are summarized in Table 1.
Participants were informed about the project and written or thumb stamped consents were obtained. The study was approved by the Ethics and Research Committee of the College of Medicine, Sultan Qaboos University, Muscat, Oman.
The 232 participants were genotyped for the following gene variants: TCF7L2 (rs7903146 & rs7901695), CDKN2A/B (rs10811661), PPARG (rs1801282), FTO (rs8050136 & rs9939609), IGF2BP2 (rs4402960), KCNJ11 (rs5219), HHEX (rs1111875), SLC30A8 (rs13266634), CDKAL1 (rs10946398) and CAPN10 (rs41266971, rs2975760, rs3792267 & rs3842570). DNA fragments containing polymorphic sites were amplified and sequenced. Reference sequences were obtained from the NCBI reference sequence database and primers designed using GeneFisher interactive primer design website (http://bibiserv.techfak.uni-bielefeld.de/bibi/Tools_Primer_Design.html) . The selected DNA fragments were amplified using polymerase chain reaction (PCR), and the products were genotyped by Applied Biosystems 3130xl Genetic analyzer (Applied Biosystems, USA). Accuracy was achieved by duplicating ~10 % of the samples. The sequencing results were aligned against reference sequences on the NCBI database and variants identified.
The rs3842570 within intron 6 of the CAPN10 gene contains either 3 repeats of a 32-bp sequence (insertion) or 2 repeats of the 32-bp sequence (deletion). The ins/del was genotyped by amplifying the DNA fragment, which contains the repeats, and separating the PCR products on a 3% agarose gel.
Allele and genotype frequencies of variants were calculated for the full pedigree (n=232) using a maximum-likelihood estimation method and were tested for departures from Hardy-Weinberg equilibrium (Table 2).
Measured genotype analysis (MGA) ; was used as a family-based association method as implemented in the SOLAR program . The association between T2D susceptibility variants, T2D, IFG, IGT and the parameters under study (weight, BMI, waist circumference, body fat (%), fasting and postprandial glucose values, HbA1C and fasting insulin), was tested among all participants within the pedigree. For multiple testing Bonferroni correction was applied and the level of significance was calculated to be pâ‰¤ 0.004.
Beta coefficients and odds ratios were also estimated. To confirm the positive association results, linear regression analysis was done using the whole family and repeated using individuals 40 years and older.
To confirm results, the analysis was repeated using sibship transmission/ disequilibrium test (S-TDT), which is a family based association for disease trait. S-TDT compares the marker genotypes in affected and unaffected offspring using the PLINK-1.07 software. This is a free, open-source whole genome association analysis toolset , (http://pngu.mgh.harvard.edu/purcell/plink/).
Anthropometric and biochemical characteristics of all participants are summarized in Table 1. One third (34%) of the participants in the combined IFG, IGT and T2D group were obese in comparison to 22.5 % of the normoglycaemic group.
The frequencies of the genotypes and variant alleles of the tested polymorphisms in all 232 individuals are summarized in Table 2. There were no significant deviations in the proportions of all genotypes tested from the Hardy-Weinberg equilibrium; except for the CAPN10 gene variant rs3842570 (Table 2).
Using MGA analysis for all 232 individuals in the pedigree, the combined group with abnormal glucose homeostasis (IFG, IGT and T2D) was found to be significantly (p<0.05) associated with rs5219 in the KCNJ11 gene (p=0.004, OR= 1.64).
Repeating the analysis using individuals who are older than 40 years old (n= 99), confirmed the significant association between (IFG, IGT and T2D) as a group (n=58) and rs5219 (p=0.049, OR=1.63). In addition, T2D group (n=23, age â‰¥ 40 years) was found to be associated with the FTO variant rs8050136 (p=0.041, OR=1.56), (Table 3). However, none of the known gene polymorphisms such as TCF7L2 [rs7903146 & rs7901695], CDKN2A/B [rs10811661], IGF2BP2 [rs4402960], PPARG [rs1801282], SLC30A8 [rs13266634], CDKAL1 [rs10946398], CAPN10 [rs41266971, rs2975760, rs3792267, rs3842570] and HHEX [rs1111875] were found to be associated with T2D in this Omani pedigree.
Analysis was repeated using Sib-TDT and results confirmed the association of rs5219 (KCNJ11) and rs41266971 (CAPN10) variants with the combined T2D, IFG and IGT group (p=0.047, p=0.035, respectively), [Data not shown].
For the association between the measured parameters and gene variants, Bonferroni correction was applied for multiple testing. None of the above tested parameters reached the significance level of p â‰¤ 0.004 (Table 4).
In this family-based T2D association study, we investigated the association of 15 common gene variants with susceptibility to T2D in one, large, extended and highly consanguineous Omani Arab pedigree. Selection of variants was based on earlier GWAS studies that investigated T2D extensively and showed significant association of those variants with modest odds ratios [3-12, 34-36].
Of the 15 known common gene variants, only KCNJ11 and FTO genes variants were found to be significantly associated with T2D among members of the Omani Arab pedigree. KCNJ11 gene variant was also associated with [IFG and IGT] group (p â‰¤ 0.05). Some other gene variants were associated with parameters related to insulin resistance, but did not reach significance level after Bonferroni correction. In this study we could not replicate the association of other gene variants (TCF7L2 [rs7903146 & rs7901695], CDKN2A/B [rs10811661], IGF2BP2 [rs4402960], PPARG [rs1801282], SLC30A8 [rs13266634], CDKAL1 [rs10946398], CAPN10 [rs41266971, rs2975760, rs3792267, rs3842570], HHEX [rs1111875]), with susceptibility to T2D reported previously in other populations [3, 5, 6, 8-11].
Our results for KCNJ11 (rs5219) gene variant association with T2D and other abnormal glucose homoeostasis parameters is in agreement with previously published findings in other ethnic groups [4, 10, 37]. KCNJ11 gene was reported to be associated with impaired insulin secretion and also have other effects on glucose metabolism . In pancreatic beta-cells, the ATP-sensitive K+ channel (KATP) is a key component of the insulin secretary mechanism and it is a complex of two subunits: kir6.2 and SUR1 . The rs5219 is located within the N-terminal of the kir6.2 subunit of KATP and can cause spontaneous hyperactivity of pancreatic beta-cells and reduced sensitivity of KATP channels to ATP, resulting in impaired insulin secretion .
The findings of this study also supported the involvement of FTO (rs8050136) gene variant in the pathogenesis of T2D (p= 0.041, OR=1.56, age â‰¥ 40 years) among members of the Omani Arab pedigree. However, the FTO (rs9939609) gene variant did not reach significant statistical level (p=0.051, age â‰¥ 40 years). T2D GWAS detected strong associations of common gene variants at the FTO locus and T2D risk; and the risk of T2D could be explained by the effect on BMI . Obesity, with increased fatty acids concentrations, is an important predictor and cause of T2D. Free fatty acids cause defective glucose metabolism through development of insulin resistance . Lipids accumulation also has a direct lipotoxic effect on Î²-cells, leading to reduced insulin secretion and apoptosis .
Previous studies testing the association between common gene variants and susceptibility to T2D among Arabs were not consistent. Our results agree with that among Saudis and Emiratis but disagree with that among Tunisians and Moroccans. The rs5219 (KCNJ11) gene variant showed significant association with T2D, IGT and IFG among members of the Omani Arab pedigree (p= 0.049, OR= 1.63), similar to that among Saudi Arabs (p=0.0001, OR=1.7) . In contrast, this variant showed no association with T2D among Tunisian and Moroccan Arabs [20, 25]. Furthermore, the rs7903146 (TCF7L2) gene variant showed no association with T2D among members of the Omani Arab pedigree, similar to that reported among Saudi and Emirati Arabs [19, 23]. In contrast, the association was confirmed by studies performed among other Arab countries (Tunisia, Morocco, Palestine) and Iran [20, 21, 25, 42]. This also indicates that Arabs are not a homogeneous group and may differ in their susceptibility to T2D.
This study found no association of the rs4402960 (IGF2BP2) gene variant with susceptibility to T2D, but previous studies among Lebanese, Moroccan and Tunisian Arabs confirmed it [24, 25].
In this family-based association study, the effect of SNP-44, -43, -19 and rs41266971 (CAPN10) on susceptibility to T2D was investigated. No statistical significant association was confirmed with T2D, IGT and IFG. However, the G allele, rather than the A allele, of the SNP-43 was found to be associated with a slight increase in postprandial glucose value (p= 0.047), but after correction for multiple testing, that effect could not be confirmed. Horikawa et al. (2000) were the first to report the association of homozygosity of G allele of SNP-43 with T2D . In contrast, Kifagi et al (2008), investigated the effect of UCSNP-43, -19 and -63 on susceptibility to T2D and found that the A allele of the SNP-43 is associated with T2D . A recent large case-control study among Tunisian Arabs examined SNP-43, -19 and -63, and confirmed the association of SNP-19 with susceptibility to T2D. Zaharna et al (2010), investigated the association of four CAPN10 gene variants (SNP-44, -43, -19 and -63) with susceptibility to T2D among Gaza Arabs, and found that the C-allele in SNP-44 to be associated with increased risk for T2D .
We have demonstrated that a small number of individuals in an extended pedigree can be used as a model to replicate results of large GWAS case-control studies. Most of the previously identified variants by GWAS studies have had very modest odds ratios in the range of 1.14-1.4. A large number of participants were required to detect these small effects. In contrast, the small number of participants in this family-based Omani study, yielded significant results that could match some of the large case-control random association studies and was able to confirm the association of some common gene variants, with modest odds ratios, on diabetes risk. If one extends the study to include further pedigrees of the same size, this could improve the statistical power of the study and detect further associations.
This small family based study confirmed the involvement of KCNJ11 (rs5219) and FTO (rs8050136) genes variants in the pathogenesis of T2D among members of an Omani Arab pedigree. However, this study was not able to detect the effect of some of the well known common variants of T2D susceptibility genes like TCF7L2. Our findings among members of the Omani Arab pedigree are similar to those among Arabian Gulf Saudi and Emirati Arabs but different from that among North African Tunisian and Moroccan Arabs, which indicates that Arabs may differ in their susceptibility to T2D.
This is a preliminary study investigating the genetics of T2D within an extended pedigree of Omani Arabs. Extending this preliminary study to further pedigrees will give more statistical power. Family based studies are suited to replicate results from GWAS and can be used to detect rare alleles of susceptibility genes of common diseases.
Disclosure of interest
The authors declare that they have no conflicts of interest concerning this article. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We are grateful to Sultan Qaboos University, Muscat, Oman for the PhD grant to SS. We thank Dr. George Khaukha, Mrs. Hameeda Al Barwany, Mr. Mohammed Al Kindi, Mr. Mohammed Al Tobi and Mr. AbdulRahim Al Abri for their support. We also thank Ms. Nassra Al Maani for her contribution in genotyping the DNA samples. We are indebted to all subjects who participated to this study.
This project was funded by The Research Council (TRC), Muscat, Oman (RC/MED/BIOC/10/01).
SS participated in study design, genotyping and data collection, analyses and drafting of manuscript, and approved the draft. HM participated in study design, helped revise the manuscript. JD, Al-YS and Al-BS participated in study design. RS helped in data management, analysis. AGC and VSV did the statistical analysis, revised the manuscript and approved it. RB was the principal investigator, participated in study design, supervised the study, helped to revise the manuscript, and approved it. The manuscript was finally approved by all authors.