The FTO rs9939609 “A” allele is associated with impaired fasting glucose and insulin resistance in Emirati population

Accepted Manuscript The FTO rs9939609 “A” allele is associated with impaired fasting glucose and insulin resistance in Emirati population

Maha Saber-Ayad, Shaista Manzoor, Ahmed El Serafi, Ibrahim Mahmoud, Sarah Hammoudeh, Aghila Rani, Salah Abusnana, Nabil Sulaiman PII: DOI: Reference:

S0378-1119(18)31017-5 doi:10.1016/j.gene.2018.09.053 GENE 43255

To appear in:

Gene

Received date: Accepted date:

27 July 2018 26 September 2018

Please cite this article as: Maha Saber-Ayad, Shaista Manzoor, Ahmed El Serafi, Ibrahim Mahmoud, Sarah Hammoudeh, Aghila Rani, Salah Abusnana, Nabil Sulaiman , The FTO rs9939609 “A” allele is associated with impaired fasting glucose and insulin resistance in Emirati population. Gene (2018), doi:10.1016/j.gene.2018.09.053

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ACCEPTED MANUSCRIPT Title: The FTO rs9939609 “A” allele is associated with impaired fasting glucose and insulin resistance in Emirati Population

Authors:

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Maha Saber-Ayad, College of Medicine & Research Institute for Medical and Health Sciences,

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University of Sharjah, UAE, and College of Medicine, Cairo University, Egypt (corresponding)

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Shaista Manzoor, College of Medicine & Research Institute for Medical and Health Sciences, University of Sharjah, UAE.

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Ahmed El Serafi, College of Medicine & Research Institute for Medical and Health Sciences,

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University of Sharjah, UAE, College of Medicine, Suez Canal University, Egypt. Ibrahim Mahmoud, College of Medicine, University of Sharjah, UAE.

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University of Sharjah, UAE.

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Sarah Hammoudeh, College of Medicine & Research Institute for Medical and Health Sciences,

Aghila Rani, Research Institute for Medical and Health Sciences, University of Sharjah, UAE.

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Salah Abusnana, College of Medicine & Research Institute for Medical and Health Sciences,

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University of Sharjah, UAE.

Nabil Sulaiman, College of Medicine & Research Institute for Medical and Health Sciences, University of Sharjah, UAE.

Address: M27-147 College of Medicine, University of Sharjah, 27272, Sharjah, UAE No conflict of interest to be declared Funding: the study was funded by the College of Research and graduate studies, University of Sharjah (#1701090225-P) and Boehringer Ingelheim Student grant. 1

ACCEPTED MANUSCRIPT Acknowledgement: I’d like to acknowledge the kind help of Prof Reyad Obaid, College of Health Science for allowing access to his lab facility and Mr Hisham Siddiq, the laboratory director of Rashid Center for Diabetes and Research for helping in preserving the blood samples, Rahaf Wardeh, Hussein Jabbar, and Ahmed Ashraf for participating in genotyping. Key words: Fat mass and obesity-associated protein gene; FTO; rs9939609; rs9930506; insulin

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resistance; obesity; overweight, metabolic syndrome; Diabetes mellitus; HOMA; Emirati population

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Running title: FTO gene polymorphisms and insulin resistance in Emirati population

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Abstract: 299 words

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Main Body of text: 2930 words

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Tables: 2 Figures: 3

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References: 49

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The authors declare no conflict of interest

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ACCEPTED MANUSCRIPT Abstract: Background: Fat mass and obesity-associated protein gene variants have shown diverse influence on body weight and metabolism across different populations. Overweight, obesity and metabolic

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syndrome are multifactorial major health problems in the UAE and worldwide. Insulin resistance represents the link between overweight and development of metabolic syndrome

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and type 2 diabetes mellitus. We investigated two (FTO) variants in Emirati population, in

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relation to insulin resistance and different parameters of metabolic syndrome.

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Methods:

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We recruited 259 Emiratis through the UAE National Diabetes and Lifestyle Project. Ethical approval was obtained. Besides basic data collection, venous blood samples were collected.

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Fasting blood glucose, Lipid profile, and insulin levels were measured. Genotyping for (FTO)

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rs9939609 (A>T) and rs9930506 (G>A) were performed using real time-PCR. Insulin resistance were identified using HOMA2-IR calculation; with a cut-off point of 1.4 for

Results:

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female and 1.18 for male subjects.

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The study included 259 Emiratis (age range 30-53 yrs, mean 41.76 yrs, 54.4% females), 24.5% are diabetic and 30.8% are hypertensive, with body mass index of 28.4±5.9 and 28.7±5.7 kg/m2 in female and male subjects, respectively. Homozygous A of rs9939609 showed significantly higher fasting glucose compared to other genotypes (p= 0.04) with a trend of higher insulin level and HOMA-2IR. The A/A diabetic patients (n=13) showed significantly higher insulin levels compared to other genotypes. G allele of rs9930506

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ACCEPTED MANUSCRIPT showed a trend of higher fasting glucose and HOMA-2IR, but lower insulin level and HbA1c. No association of genotypes was detected with other components of metabolic syndrome. Conclusion: There is an association of FTO rs9939609 A/A genotype and impaired fasting glucose and

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insulin resistance. Homozygous A genotype diabetic patients may be more vulnerable to blood glucose fluctuation. Focused genotyping can help the health care providers to identify

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high risk groups of both normal population and diabetic patients to intervene accordingly.

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ACCEPTED MANUSCRIPT 1. Introduction: Overweight and obesity are major health problem worldwide, linked to an increased risk of insulin resistance, type 2 diabetes mellitus (DM) and cardiovascular diseases (1). Over the past 20 years, obesity rates have been rising all over the world; including the Emirati

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population; what can be explained by the lifestyle changes in the country (2,3). The UAE national survey estimated the prevalence of overweight and obesity to be 65% in adult

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women, 28% in male adolescents and 40% in female adolescents; the study was done

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between 2009 and 2010 (3). Obesity is considered multifactorial, with complex interaction between the genetic makeup and the environmental factors. By modifying either energy

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expenditure or food intake or both, genetics may account for 40-70% of BMI variation (4).

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Genetic basis of obesity has been thoroughly explored through genome wide association studies (GWAS). The first single nucleotide polymorphism (SNP) associated with increased

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BMI was mapped to the Fat mass and obesity-associated protein gene (FTO) in 2007 (5).

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Although the function of such a protein has not been clearly elucidated, a recent study linked it to insulin secretion in an in-vitro model of pancreatic β-cell (6).

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The FTO single nucleotide polymorphisms have been inconsistently linked to obesity across different populations (7–9). The FTO gene, particularly the rs9939609 (A>T) variant, first

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identified by Frayling et al., is the most well-known gene that is notorious for its role in obesity development (5,10). This SNP was also associated with morbid obesity in a study of 927 Japanese patients (11). Interestingly, it was associated with insulin resistance in another study (12). The intronic single nucleotide variant was found to be in high linkage disequilibrium (LD) with rs1421085, (13), which is related to loss of mitochondrial thermogenesis (14). In addition, individuals carrying the rs9939609 homozygous A genotype have suppression of acyl-ghrelin post meals and increased satiety (15).

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ACCEPTED MANUSCRIPT Another SNP, the rs9930506 (G>A) variant of FTO was reported to be highly associated with BMI, and weight (16). Homozygotes of the rare ‘‘G’’ allele of this SNP experienced additional 1.3 BMI units compared to homozygotes of the common ‘‘A’’ allele (6). Among ethnicities, strong links were found in European Americans and Hispanic Americans (17). Moreover, correlations with obesity were evident in Europeans, ethnic Chinese and Malays in

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Singapore (5).

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In a study of prevalence of type 2 DM in the UAE by Alsafar and her colleagues, 2012, 23%

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of UAE Nationals were diagnosed with diabetes and 30% with prediabetes (18). This was consistent with an older study in 2005 that showed a prevalence of 25% (19). A recent study

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from a close Iraqi population showed a link of rs9939609 to development of type 2 DM (16).

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Insulin resistance has been established as the major link between obesity and type 2 diabetes (20). Clinically, insulin resistance refers to a state in which a given concentration of insulin is

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associated with a subnormal glucose response (21). Metabolic syndrome is defined as a

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collection of obesity, insulin resistance, hypertension, and dyslipidemia, which represent major cardio-metabolic risk factors (22). The homeostasis model assessment of insulin

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resistance (HOMA-IR) is a simple method to measure insulin resistance (23). Previous studies tried to define the cut-off values of HOMA-IR and the metabolic syndrome in order to

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identify patients at high risk (24). Recently, a simple risk score model was developed to identify UAE Nationals at high risk of having type 2 DM, as a screening tool for the early detection of the disease (25). We expect that assessing specific SNPs can add to defining subjects at risk of insulin resistance and metabolic syndrome. In our current study, we aimed at studying FTO rs9939609 (A>T) and rs9930506 (G>A) variants in Emirati population, in relation to different parameters of metabolic syndrome. 6

ACCEPTED MANUSCRIPT 2. Subjects and Methods 2.1 Subjects A group of 259 Emiratis were recruited within the UAE National Diabetes and Lifestyle Project. All participants gave informed consent. The study was conducted according to the

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protocol approved by the Research and Ethics Committee, University of Sharjah.

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2.2. Methods

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We collected basic clinical data in addition to venous blood samples. Basic laboratory investigations including; fasting blood glucose, HbA1c and Lipid profile were ordered. For

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each subject, weight in kilograms was divided by height in meters squared; to calculate BMI

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in kg/m2.

Diabetes was defined as fasting plasma glucose ≥7 mmol/dL, or glycated hemoglobin of ≥

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6.5%. Prediabetes was defined as diagnosed as fasting plasma glucose from 100 to 125

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mg/dL or glycated hemoglobin of ≥ 5.8 and < 6.5%.

Plasma insulin was measured by radioimmunoassay (Diabetes Research Center, University of

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Washington). We used updated computer models for homeostasis model assessment 2

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(HOMA2) to calculate the indices of insulin resistance (HOMA2-IR) (https://www.dtu.ox.ac.uk/homacalculator/), Relevant values for the calculator includes plasma glucose in the range of 3.5 to 25.0 mmol/L and plasma insulin in the range of 20-400 pmol/L (26).

Genotyping for chromosome 16 (16q12.2a) FTO rs9939609 (A>T) and rs9930506 (A>G) were performed as described in our previous study (27); thorough StepOne Real-Time PCR Systems (ThermoFischer Scientific, USA) using TaqMan® Drug Metabolism Genotyping

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ACCEPTED MANUSCRIPT Assay (Applied Biosystems, USA). We used the on-line tool http://www.oege.org/software/hwe-mr-calc.shtml; to estimate Hardy–Weinberg equilibrium (28). Then the haplotype and allele distribution was calculated. NCBI SNP

Context Sequence

Reference GGTTCCTTGCGACTGCTGTGAATTT[A/T]GTGATGCACTTGGATAGTCTCTGTT

rs9930506

AGGGACACAAAAAGGGACATACTAC[A/G]TGAATTACTAATATCTAAGAAAATA

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rs9939609

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2.3.Statistical Analysis:

We described data in terms of mean ± standard deviation (SD), frequencies (number of cases)

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and percentages when appropriate. Categorical data was compared using Chi square (x2). Fisher Exact test was used instead when the expected frequency is less than 5. The one-way

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analysis of variance (ANOVA) was used for continuous variables. When the data was not

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normally distributed, Kruskal-Wallis Test was used instead of ANOVA. P‐value ≤ 0.05 was considered statistically significant. We used Bonferroni correction to adjust for multiple

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comparisons. All statistical calculations were done using computer programs SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 23 for

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Microsoft Windows.

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ACCEPTED MANUSCRIPT 3. Results: The study included 259 Emiratis (age range 30-53 yrs, mean 41.76 yrs, 54.4% females), 24.5% had Diabetes mellitus and 30.8% were hypertensive. Table (1) shows baseline characteristics of the study group and their FTO rs9939609 (A > T) and rs9930506 (G>A)

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genotypes. The frequency of the major allele, based on Hardy- Weinberg Equilibrium calculation, was 0.56 for rs9939609 and 0.55 for rs9930506. This frequency suggests the

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presence of normal distribution for each of the two alleles.

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To calculate the HOMA-2IR to determine the insulin resistance, we excluded diabetic

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patients (n=63) and subjects with plasma insulin >400 pmol/L (n=11) or <20 pmol/L (n=2). Plasma glucose levels of the remaining subjects ranged from 3.9 - 6.9 mmol/L. The cut-off

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point to determine insulin resistance was defined as 1.18 for male and 1.4 for female subjects (29). No difference among genotypes was observed.

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Homozygous A of rs9939609 (A>T) showed statistically significant higher fasting glucose

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(Impaired fasting glucose –IFG) compared to A/T and T/T genotypes (p= 0.04). There is a trend of having higher insulin level and HOMA-2IR in subjects with this minor allele, but

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lower HbA1c (mean of 6.0%, median 5.5%, mode 5.3 %), Figure (1).

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Diabetic patients with rs9939609 A/A genotype (n=13) showed significantly higher insulin levels compared to other genotypes (161.17±25.3 in A/A vs 112.83±20.8 in other genotypes, respectively). However, FBG among all genotypes was comparable (10.7±2.2 mmol/L in A/A vs10.1±2.4 mmol/L in other genotypes, respectively). Glycated hemoglobin levels were also not significantly different (7.59±2.6 in A/A vs 8.2±2.9% in other genotypes, respectively).

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ACCEPTED MANUSCRIPT For rs9930506 (G>A), G allele showed a trend of higher fasting glucose and HOMA-2IR, but lower insulin level in comparison to the wild genotype. The subjects were further stratified according to concordance of the two alleles of both genes. The eight out of the expected nine haplotypes were detected in our sample with variable

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frequencies. While 39% of the population carried the haplotype AT/AG, less than 1% (two subjects) had TT/GG. The genotype AA/AA was not found in our cohort.

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Subjects with TT/AG and TT/GG (14 and 2 subjects, respectively) had lower glucose level

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compared to other groups, whereas the latter group had a significantly lower glycated

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hemoglobin (p= 0.01), whereas insulin level, HOMA-2IR and lipid profile had no significant difference among genotypes, fasting blood glucose, Figure (2). Number of diabetics and pre-

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diabetics did not show significant difference in subgroups of combined genotypes, Figure (3). There was no statistically significant difference of BMI among genotypes of either rs9939609

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(A>T) or rs9930506 (G>A); with BMI>27 kg/m2 in all groups. There is a trend of higher

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BMI in association with A allele of rs9939609 and G allele of rs9930506. However, differences are not statistically significant. We further classified the study population as

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having normal BMI (<25 kg/m2), over weight (25-29.9 kg/m2) or obese (>30 kg/m2). There

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was no significant difference among genotypes of either SNP. No difference was observed in lipid profile among different genotypes, Table (2).

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ACCEPTED MANUSCRIPT 4. Discussion: Prevalence of metabolic syndrome and cardiovascular disease are expected to rise along with the global obesity epidemic. There should be a greater emphasis on effective early weight-management to reduce risk in pre-symptomatic individuals with overweight and obesity. The metabolic syndrome is a condition characterized by a special constellation of reversible major risk factors for

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cardiovascular disease and type 2 diabetes including dyslipidaemia, high blood pressure and high

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fasting plasma glucose, which may or may not co-exist with a number of inflammatory markers and

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prothrombotic state (30). The significance of this clustering of metabolic abnormalities, particularly in overweight individuals was first clarified by Reaven (20).

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The metabolic syndrome is strongly linked to a lifestyle characterized by an easy access to unlimited

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supply of high caloric, low nutrient-dense, foods and physical inactivity (31). Metabolic syndrome now affects 30–40% of people by age 65, is driven mainly by adult weight gain,

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and by interplay of genetic and epigenetic factors. Early intervention by reducing weigh, by diet and

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exercise, with or without, anti-obesity drugs, substantially lowers all metabolic syndrome components, and risk of type 2 diabetes and cardiovascular disease. Bariatric surgery offers an

(32).

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alternative treatment for those with BMI ≥ 40 or 35–40 kg/m2 with other significant co-morbidity

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In our study, homozygous A of rs9939609 (A>T) showed statistically significant higher fasting glucose (Impaired fasting glucose –IFG) with a trend of higher insulin and HOMA2IR, but not with higher BMI. In concordance to our study, Grunnet et al showed that FTO rs9939609 A-allele was associated with elevated fasting blood glucose and plasma insulin and hepatic insulin resistance. However, it does not influence the mRNA expression of FTO or a set of key nuclear or mitochondrial encoded genes in skeletal muscle during rest (33).

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ACCEPTED MANUSCRIPT The Genetic Investigation of ANthropometric Traits (GIANT) consortium reported 97 loci to be associated with BMI by a meta-analysis on around 340,000 individuals; including more than 120 studies, using either genome-wide association studies (GWAS) or metabochip. These loci accounted for only 2.7% of the variation in BMI, and it was estimated much as around 20% of BMI variation can be accounted for by common genetic variation. The A

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allele of both rs9939609 and rs9930506 showed highly significant association with high

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BMI in the database of combined all ancestary subjects and in male and female subgroups

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(34). In our study, BMI showed no significant difference among different genotypes. A recent study on Emirati population showed that the minor allele A of the rs9939609 has a

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significant association with increasing BMI values in females (p=0.048), (35), with

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increasing risks of other comorbidities such as hypertension, poorer glycemic control, and higher triglycerides. In addition, physical activity was found to decrease the effect of the "A/A" genotype on the predisposition to higher BMI values. Our study is an attempt to

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explore the association of this SNP with insulin resistance among other features of metabolic syndrome (35). However, our study did not find such association with rs9939609 or rs9930506. This may be explained by the different study population and the group size in

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both studies. The mean age in our study population is higher than the study of Kahn et al,

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(35), who detected a significant interaction between FTO rs9939609 A/A genotype and physical activity. Accordingly, A/A subjects in our study are likely to be less physically active with higher BMI. In our study, A/A genotype showed a favorable lipid profile; with a trend of higher HDL, lower LDL and TG compared to other genotypes.

previous investigations suggested that the effect of FTO intron 1 SNPs on adiposity phenotypes, shows genetic heterogeneity dependent on ethnicity (36). This SNP is in linkage disequilibrium with rs1421085 (T> C), which may lead to obesity through disruption of

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ACCEPTED MANUSCRIPT AR1D5B- mediated repression of IRX3 and IRX5. This leads to a shift from browning to whitening programs in the mitochondria and to loss of mitochondrial thermogenesis (14).

Moreover, in the multiethnic Insulin Resistance Atherosclerosis Study cohort analysis of FTO SNPs, there was an association of FTO intron 1 variants with BMI in non-Hispanic Whites

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and Hispanic Americans, but not in African Americans, suggesting that the effect of FTO variants on adiposity phenotypes shows genetic heterogeneity dependent on ethnicity (36).

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This denotes the importance of studying different populations to draw specific practical and

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clinically significant conclusions relevant for each.

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The association between FTO and obesity is significantly linked to rs1421085 T>C SNP (14) Many SNPs appear to be co-inherited in the FTO gene. The three FTO SNPs, rs1421085,

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rs17817449, and rs9939609, are in strong linkage disequilibrium (pairwise r2>0.97). Many FTO SNPs were identified in the first intron of the gene that associated with both BMI and

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type-2 diabetes. As they all correlated in studies with each other, many investigators chose to

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examine one of the SNPs (rs9939609) in detail. It has been also shown in a large study to be associated with type-2 diabetes; with odds ratio of 1.55 for the homozygous A (37).

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In most studies, the gulf countries are under-represented (e.g in the Hap Map and 1000 Genome projects). In a recent investigation, genome wide association studies on Emirati

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population have identified multiple SNPs associated with high BMI in FTO, and others, whereas DM type 2 was strongly associated with TCF7L2 and MC4R, KCNK3 and RARB loci (38). In our study, we showed an association of overweight and obesity with insulin resistance through focusing on 2 SNPs of FTO. Interestingly, A/A genotype diabetic patients showed significantly higher insulin level compared to other genotypes; with comparable FBG and HbA1c among all genotypes. We assume that this group of diabetic patients are likely to develop fluctuation in the blood

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ACCEPTED MANUSCRIPT glucose level. Among diabetic patients, this group should be more closely monitored and prescribed more flexible treatment regimens according to their life style to avoid possible hidden hypoglycemic episodes. We also studied rs9930506 (G>A) SNP that previously showed a strong association with high BMI. In our study, rs9930506 G allele showed a trend of higher fasting glucose and HOMA-

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2IR, but lower insulin level. Homozygotes of the rare ‘‘G’’ allele of this SNP experienced

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additional 1.3 BMI units compared to homozygotes of the common ‘‘A’’ allele. Among

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ethnicities, strong links were found in European Americans and Hispanic Americans (39). Moreover, correlations with obesity were evident in Europeans, ethnic Chinese and Malays in

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Singapore (17). However, we did not find such correlation in our study. On the other hand, homozygous carriers of A allele of rs9939609 showed an elevated hepatic insulin resistance

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in 46 young men underwent a hyper-insulinemic euglycemic clamp, (33). Previous studies showed that rs9930506 (A;A) had decreased levels of methylated peroxisome proliferator-

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activated receptor-gamma co-activator 1alpha gene (PPARGC1A) in comparison with other

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genotypes, suggesting a potential role of in metabolic programming (40). The same SNP was associated with overweight in a study on elderly Spanish cohort with cardiorenal metabolic

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syndrome (41).

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We used HOMA2-IR to detect insulin resistance. As we used the HOMA calculator, we eliminated diabetic patients and outlier values from the analysis, as previously discussed in the methods. The HOMA Calculator provides quick and easy access to the HOMA2 model (42). We used cutoff points reported in a close population (29). It has been suggested that HOMA2-IR is rather more accurate representation of metabolic process than HOMA1-IR, as it considers variation in both hepatic and peripheral glucose resistance and include renal glucose losses (42), (43). Insulin resistance is also variable in different organs and could be detected very early in skeletal muscle and liver, but much later in adipose tissue (44). There is 14

ACCEPTED MANUSCRIPT a significant variability of insulin levels among different population (45). Recently, some FTO variants have been recently linked to diabetic nephropathy (46) The aim of identifying population subgroups at risk of developing diabetes is to take action for prevention and delaying occurrence of the disease. In some previous studies, early screening helped reducing overall costs for the healthcare system (47). However, Screening

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for DM and pre-DM was not consistently cost effective in clinical practice (48). Effective

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prediabetic management includes lifestyle interventions of diet and exercise, and use of

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metformin in subjects less than 60 years of age (49). If appropriately implemented, such

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simple measures can prevent or at least delay the major health problem of diabetes mellitus.

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5. Conclusion:

There is an association of FTO rs9939609 A/A genotype and impaired fasting glucose and insulin resistance, but not with increased BMI in Emirati population. This population

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subgroup should be directed to extra measures of life style modification, more frequent screening and close clinical monitoring to prevent risks of metabolic syndrome and occurrence of diabetes. On the other hand, A/A genotype diabetic patients showed

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significantly higher insulin levels compared to other genotypes. Those patients require close

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glycemic monitoring and flexible treatment regimens, as they are more vulnerable to blood glucose fluctuation.

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ACCEPTED MANUSCRIPT Abbreviations:

FTO : Fat mass and obesity-associated protein gene DM: Diabetes mellitus HOMA-IR: homeostasis model assessment of insulin resistance

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BMI: Body Mass Index LDL: low density lipoprotein

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HDL: high density lipoprotein

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TG: triglycerides

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ACCEPTED MANUSCRIPT Table (1): Participants Characteristics by gender Male

(n=141)

(n=118)

38.3 (10.6)

44.6 (13.4)

28.4 (5.9) 37 (26.2) 38 (36.2) 102 (72.3)

28.7 (5.7) 26 (22.4) 23 (24.7) 82 (70.7)

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Age, Mean (SD), years BMI, Mean (SD), (kg/m2) Diabetes, n (%) Hypertension, n (%) Abnormal lipid profile, n (%)

Female

FTO rs9939609 (A>T), n (%) A/A A/T T/T

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29 (20.6) 67 (47.5) 45 (31.9)

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FTO rs9930506 (G>A), n (%)* A/A A/G G/G

43 (30.9) 65 (46.8) 31 (22.3)

24 (20.3) 57 (48.3) 37 (31.4)

35 (29.9) 57 (48.7) 25 (21.4)

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SD = Standard deviation, * 3 cases were not genotyped (2 females and 1 male).

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Table (2): Body mass index (BMI), kg/m2, according to FTO genotype. Genotypes

BMI, Mean (SD)

28.4 (5.1)

A/T

29.4 (6.5)

T/T

27.3 (5.0)

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A/A

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FTO rs9939609 (A>T)

FTO rs9930506 (G>A)

27.6 (5.0)

A/G

29.0 (6.7)

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A/A

28.9 (5.1)

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G/G

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Figure 1

Figure 2

Figure 3