nagwan biochem

8/6/2019 Nagwan Biochem

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Study of Single Nucleotide Polymorphism rs13266634 in Zinc Transporter

Solute Carrier Family 30, Member 8 (SLC30A8) Gene in Type 2 Diabetes

Patients Resident in Ismailia City

Taher I. EL-Serafi, Samir M. Abdel-Moneium, Nagwan A. Sabek and Marwa

M. Hosny

Department of Medical Biochemistry, Faculty of Medicine, Suez Canal University

Abstract

Background: Several genome-wide association studies identified a strong

association of SLC30A8 with type 2 diabetes in individuals of European ancestry.

The effect of the association of rs13266634 with type 2 diabetes or related

glycemic traits has not been fully extended to non-European populations, and a

comprehensive examination of common variants in the gene has not yet been

carried out in our population.

Objective:The aim of the present study was to investigate the association among

the polymorphisms of SLC30A8, and the risk of T2DM and to determine the

presence and frequency of single nucleotide polymorphism (SNP) rs13266634 in

SLC30A8 gene in T2D patients resident in Ismailia city.

Design: SLC30A8 SNP was genotyped using real time PCR allelic discrimination

TaqMan assay.A case control study was conducted in 68 casesof type 2 diabetes

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(51 women and 17 men) and 29 controlsubjects (13 women and 16 men) from out-

patients diabetic clinic of Suez Canal hospital and age and gender were matched.

The SNP rs13266634 was evaluated in SLC30A8 C > T genotype.

Results: The genotypes of control subjects were 27 (93%, C/T), 1 normal

homozygot (3.5% C/C) and 1 mutant homozygous (3.5%, T/T). In diabetic

subjects, there were 58 subjects carriers of heterozygous (C/T, 85%), 6 normal

homozygots (C/C, 9%) and 4 mutant homozygous (T/T, 6%). There was

significant difference in fasting blood glucose levels in control subjects compared

to diabetic subjects' P


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summary, the data in this study support substantial associations between the

common SLC30A8 polymorphisms in gene and the risk of type 2 diabetes.

Conclusions: Our results may provide evidence that SLC30A8 is a susceptible

locus for type 2 diabetes in our population, and its variant can influence insulin

secretion.

Keywords: SNP, Single Nucleotide Polymorphism, Solute Carrier Family 30,

Member 8 (SLC30A8).

Introduction

T2D represents 90-95% of the population with diabetes. According to the World

Health Organization(1), the number of patients with T2D in Egypt was 2,623,000

and in 2030, it is expected to increase to 6,726,000.It has reached epidemic

proportions and currently affects about 170 million people worldwide, with the

figure projected to be more than double by 2030(2).

Diabetes is a leading cause of both mortality and early disability, it is the leading

cause of blindness among working age adults, of end-stage renal disease, and of

nontraumatic limb amputations. Diabetes increases the risk of cardiac, cerebral and

peripheral vascular disease two to seven fold(3). T2D has a strong genetic

component. Major genes that predispose to this disorder have yet to be identified,

but it is clear that the disease is polygenic and multifactorial(4). T2D is due to

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variations within several genetic loci that confer increased susceptibility towards

the environmental challenges such as high-caloric fat and carbohydrate-enriched

diets and sedentary lifestyle with markedly reduced physical activity(5).

Linkage studies, candidate-gene approaches, and genome-wide association studies

identified single nucleotide polymorphisms (SNPs) within currently up to ten

genes which associate with an increased T2D risk. Most T2D risk loci are

supposed to contribute to -cell dysfunction(6). Zinc transporter 8, a member of the

zinc transporter family, is coded by solute carrier family 30 member 8 gene

(SLC30A8) on chromosome 8q24.11. The encoded protein has 369 amino acid

residues, with six transmembrane domains and a histidine-rich loop between

transmembrane domains IV and V, like other family members(7).

It has been reported that SLC30A8 is expressed predominantly in pancreatic cells

and transports zinc from cytoplasm into insulin secretary vesicles(8), in which

insulin is stored as a solid hexamer bound with two Zn2 ions before secretion(9).

Zinc plays an important role in all processes of insulin trafficking, i.e. synthesis,

storage, and secretion(8). The variations in SLC30A8 may affect zinc accumulation

in insulin granules and hence influence insulin stability and insulin trafficking.

Glucose stimulated insulin secretion is enhanced in insulinoma (INS-1) cells

transfected with SLC30A8 in a high glucose challenge(10).

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Researchers have identified SNP rs13266634, a nonsynonymous Arg (Arginine)325

Trp (Tryptophan)325 variant in the pancreatic beta-cell-specific zinc transporter

SLC30A8(8 ,11). Beneficial antioxidant effects of zinc supplementation have been

found in people with type 2 diabetes(12). Excessive apoptosis of pancreatic beta

cells has been associated with diabetes(13). Zinc depletion by itself is a well-known

inducer of apoptosis(14), thereby participating in decreased in beta cell mass. In

addition, some studies suggest that cells with deprived zinc stores are less able to

defend themselves against oxidative injuries, underlining the antioxidant properties

of zinc. Therefore, enhancing the capacity of beta cells to store zinc may help to

protect the pancreas against zinc depletion and/or oxidative stress frequently

observed in diabetes(10).

In the light of these novel findings, this study aimed to determine the presence and

the frequency of the SNP rs13266634, variant in the pancreatic beta-cell-specific

zinc transporter SLC30A8 and the association among the polymorphisms of

SLC30A8, and the risk of T2DM in our population resident in Ismailia city.

Subjects and Methods

Subjects: casecontrol study of 68 cases of type 2 diabetes (51 women and 17

men) aged 436 years and 29 controlsubjects (13 women and 16men) aged 42 6

years, attending out patient's diabetic clinic of Suez Canal hospital. Diagnosis of

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type 2 diabetes mellitus was based on clinical and laboratory criteria (1). Inclusion

criteria were as follows: 1) BMI (weight in kilograms divided by the square of

height in meters)


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NanoDrop (ND)-1000 Spectrophotometer V3.1.0 (NanoDrop Tech., Inc.

Wilmington, DE USA.) and stored at -20C.

Genotyping of SNP (rs13266634) SLC30A8: SLC30A8 SNP was genotyped using

real time PCR allelic discrimination TaqMan assay (Applied Biosystems,

California, USA). Each assay includes non-labeled forward and reverse primers

along with two fluorescent TaqMan oligonucleotide probes (allele 1-specific probe

labeled with VIC fluorophore, allele 2 specific probe labeled with FAM (6-

carboxy- fluoroescein fluorophore). The VIC and FAM reporter dyes are

covalently attached to 5' terminal base of the two probes and the non fluorescent

quencher dye is attached near the 3' ends. The probes are capable of differentially

binding to the amplicons generated during PCR reactions contained 20 ng of

sample DNA, 12.5l of TaqMan Universal PCR master mix, and 1.25 l of

TaqMan SNP genotyping assay mix. Appropriate negative controls were also run.

Real- time PCR was performed on an ABI Prism 7000 Sequence Detection System

(Applied Biosystems, Foster City, USA) using the following condition: 95C for

10 minutes, and then 40 cycles of amplification (92C denaturation for 15 seconds,

60C annealing/extension for 60 seconds) according to manufacture's protocol and

the discrepancy rate on duplicate genotyping was 0-1%.

Statistical analysis: Descriptive results of continuous variables are expressed as

mean SD. Before statistical analysis, normal distribution and homogeneity of the

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variables were tested. Comparison of variables between groups of subjects was

performed using the Students t test. We used Statistical Package for Social

Science SPSS for WINDOWS software (version 11.0; SPSS Inc, Chicago, IL) for

all statistical analyses. We evaluate the influence of selected SNPs on continuous

variables by using the Bonferroni test to adjust for covariates such as age, sex, and

BMI. Each variable was examined for normal distribution; mean values are

presented on untransformed and unadjusted variables. Statistical analysis was

performed with paired samples t-test. A 2-tailed value of P < 0.05 was considered

significant. All analyses were repeated after sequentially adding sex, current age,

and BMI as covariates.

Results

Genotyping and allelic frequencies among diabetic patients and controls subjects in

the association study were summarized in Table (I). To investigate the association

of SLC30A8 with risk of T2DM in our populations, real time PCR allelic

discrimination TaqMan assay was used to determine the distribution of allele and

genotype frequency of this variant. We conducted a casecontrol study of 68 cases

of type 2 diabetes (mean age, 426 years; 51 women and 17 men) and 29 control

subjects (mean age, 436 years; 13 women and 16 men) from out patient's diabetic

clinic of Suez Canal hospital. The genotypes were consistent with Hardy-Weinberg

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equilibrium, the frequencies of C and T alleles for control subjects were (50% and

50%) respectively, and for diabetic subjects were (51% and 49%) respectively.

The genotype distributions of SNP (rs13266634) SLC30A8 in control subjects

were (3.5%C/C, 93% C/T and 3.5%T/T) and (9% C/C, 85% C/T & 6% T/T) for

diabetic subjects (Table I).

Clinical and biochemical characteristics: The clinical and biochemical

characteristics of all subjects in the association study were summarized in Table 2.

There was no significant differences in age and sex, but there was statistical

significant differences in fasting blood glucose levels were observed between

diabetic subjects comparing to control subjects (215 87 mg/dl vs. 97 7 mg/dl,

p < 0.05), there was statistical significant differences in BMI were observed

between diabetic subjects comparing to control subjects (30 6 vs. 27 3.0 kg/m2

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(III), there were higher non statistical significant differences in lipid profile

(triglyceride, cholesterol and LDL-c levels between diabetic subjects carrier C/C

and C/T genotype. Also, there was statistically significantly a difference in

triglyceride levels between diabetic subject's carrier C/C and diabetic subject's

carrier C/T genotype (13735 mg/dl vs. 11034 mg/dl; P< 0.05); as shown in table

(IV).

Table (I): Genotyping and allelic frequency of SNP (rs13266634) SLC30A8 in T2D patients

and control subjects.Genotyping Allelic frequencyCC CT TT No. C T No.

Control 1(3.5%)

27(93%)

1(3.5%)

29100%

14.5(50%)

14.5(50%)

29100%

Diabetic 6(9%)

58(85%)

4(6%)

68100%

35(51%)

33(49%)

68100%

Table (II): Clinical characteristic and biochemical parameters of genotype of SNP(rs13266634) SLC30A8 between control and diabetic subjects.

Controls SubjectsNo.(29) MeanSD Diabetic patientsNo. (68) MeanSD P

Sex (male/female) 16/13 17/51 NsAge (years) 426 436 NsBMI(kg/m2) 273 306 0.04*FBG (mg/dl) 977 21587 0.001*Cholesterol (mg/dl) 21953 24239 0.211TG (mg/dl) 9027 11452 0.101HDL-C (mg/dl) 8718 5812 0.001*LDL-C (mg/dl) 13536 14246 0.351

Data presented as means SD, Abbreviations: Ns: Not significant, BMI: Body mass index,

FBG: Fasting blood glucose, TG: Triglyceride, HDL-c: High density lipoprotein cholesterol,LDL-C: Low density lipoprotein cholesterol.*P value 0.05 was significant *.

Table (III): Association of clinical characteristic and metabolic parameters with genotypeof SNP (rs13266634) SLC30A8 between control and diabetic subjects C/T genotype.

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Control C/T genotype Diabetic C/T genotype PMean SD Mean SD

No. 27 58Age (years) 42.66 42.96 NSBMI (kg/m2) 273.5 306 0.08

FBG (mg/dl) 987 20681 0.001*Cholesterol (mg/dl) 22150 24539 0.280TG (mg/dl) 8926 11034 0.108HDL-C (mg/dl) 8818.5 57.911 0.001*LDL-C (mg/dl) 13736.5 141.245.8 0.460

Table (IV): Association of clinical characteristic and metabolic parameters with genotypeof SNP SLC30A8 between C/T and C/C genotype diabetic subjects.

Diabetic C/C genotype Diabetic C/T genotype PMean SD Mean SD

No. 6 58Age (years) 472 42.96 NsBMI (kg/m2) 306 306 0.598FBG (mg/dl) 250102 20681 0.3372hrs PPBG (mg/dl) 25492 237113 0.421Cholesterol (mg/dl) 24548 22150 0.776TG (mg/dl) 13735 11034 0.001*HDL-C (mg/dl) 62.513.5 57.911 0.781LDL-C (mg/dl) 15648 141.245.8 0.790

G R O U P

H

D

L

Figure (1): Relation between the mean of HDL-C (mg/dl) between the control subjects anddiabetic patients. There was statistical significant difference between them P 0.05.

Discussion

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Control Diabetic


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Chimienti et al(10) demonstrated that the ZnT-8 protein is expressed only in

pancreatic beta cells in vivo. In cultured cells, overexpression of ZnT-8 augmented

total cellular zinc content, thus protecting cells from zinc depletion and enhanced

insulin secretion under hyperglycemic conditions, suggesting that ZnT-8 has a

major role in the insulin secretion pathway. Triggering ZnT-8 expression and/or

activity may be an interesting approach in the treatment of type 2 diabetes.

The identification of a new gene for polygenic T2DM was recently achieved by

genome-wide SNP assays. Among several SNPs associated with susceptibility to

T2DM, polymorphisms in SLC30A8, hematopoietically expressed homeobox

(HHEX), cyclin-dependent kinase (CDKN2A/B), insulin-like growth factor 2

mRNA-binding protein 2 (IGF2BP2), fat mass and obesity associated (FTO),were

first reported by several studies performed in European and Asian populations(19- 24).

These genetic variants have been repetitively confirmed by multiple studies in

various populations (25- 28). Nevertheless, the effects of genetic variants on the risk

of T2DM are somewhat inconsistent among different ethnic groups.

Our result showed that there were increase in prevalence of C/T genotype in

studied subjects, the frequencies of C and T alleles at SNP (rs13266634) SLC30A8

for control subjects were (50% and 50%) respectively, and for diabetic subjects

were (51% and 49%) respectively. The genotype distributions of SNP

(rs13266634) SLC30A8 C > T in control subjects were (3.5% C/C, 93% C/T &

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3.5% T/T) and (9% C/C, 85 % C/T & 6% T/T) for diabetic subjects. According to

our results, the risk allele frequencies of this SNP were significantly different

between our subjects and other ethnic groups. The frequency of risk allele C in

rs13266634 for diabetic subjects in our study (51%) was lower than those reported

in European or African populations (69.9 and 97.1%) respectively(20,26). These

significant differences in risk allele frequencies across ethnic groups indicate that

the genetic variations of T2DM susceptibility genes are diversely distributed

among different populations.

In our study, SNP in SLC30A8 gene showed a significant association with

T2DM.The present data demonstrate that the C/Tgenotype is associatedwith risk

of type 2 diabetes as we found that diabetic subjects carrying the C/Tgenotype

exhibited higher statistical significant of biochemical parameters; fasting blood

glucose, and HDL-c and higher non significant levels of triglyceride, cholesterol

and LDL-c than control subjects carryingthe C/Tallele. These results were similar

to the findings of another study(29) which reported a nominal association between

rs13266634, a nonsynonymous SNP in SLC30A8, and T2DM in a Japanese

population, while Horikoshi et al(25) reported that there was no significant

association in another Japanese population.

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Our results found that this SNP was associated with the risk of type 2 diabetes in

our population, showing similar effect sizes to those in Europeans, although the

risk allele frequencies of most of this SNP was different between populations(28).

The SLC30A8 C allele might not only cause a decrease in insulin release, but may

also lead to decreased beta cell mass via increased apoptosis. In addition,

overexpression of SLC30A8 in insulinoma cells has been shown to increase

glucose-stimulated insulin secretion Therefore, stimulating ZnT-8 production

and/or activity may potentially be a novel approach in the treatment of type 2

diabetes patients, in whom zinc depletion is likely to participate in both acute and

chronic beta cell dysfunction(10).

In 2007, four genome-wide association scans for T2D in European populations

have been published. The first was performed in a French population of lean T2D

patients with familial history of diabetes, and the three subsequent scans were

performed in the UK, Wellcome Trust Case Control Consortium (WTCCC),

showed P value=0.020(30); and two Finnish/Swedish populations, Diabetes Genetics

Initiative (DGI), showed P value=0.047 (20) and Finland-United States

Investigation of NIDDN Genetics (FUSION), showed P value=7.0 X 10-5(11). All

have found a strong evidence for linkage of T2D to SNP rs13266634, variant in the

pancreatic beta-cell-specific zinc transporter SLC30A8 (Solute Carrier family 30,

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member8), to a region on chromosome 8 at the position 118,253,964bp (base

pairs), with risk allele/non risk allele (C/T) cytosine/thymine.

Variant within a novel genetic locus SLC30A8 was reported with other three novel

genetic loci to be more frequent in subjects with T2D than in healthy controls. The

major allele of the SLC30A8 SNP rs13266634 is associated with reduced insulin

secretion stimulated by orally or intravenously administered glucose, but not with

insulin resistance by genotyping of 921 non-diabetic German subjects for the

reported candidate SNP, which is located in the final exon of SLC30A8 gene on

chromosome 8, and it encodes for protein that was shown to be required for insulin

maturation/storage. Therefore, the major allele of type 2 diabetes candidate SNP

within that genetic locus represents crucial allele for -cell dysfunction and, thus,

might confer increased susceptibility of -cells towards adverse environmental

factor(6).

The SLC30A8 gene encodes ZnT-8, a novel member of the cation diffusion

facilitator family, exclusively expressed in pancreatic beta cells(10). ZnT-8 is

thought to be a key protein for insulin secretion by regulating the homeostasis of

zinc, which is known as an essential metal ion for insulin storage and secretion into

intracellular vesicles(31). Because the rs13266634 SNP causes an amino acid change

(R325 W) in the intracellular C-terminus of the ZnT-8 protein, this single

nucleotide substitution might act as a gain-of-function mutation that increases

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protein translation efficiency or modifies the posttranslational structure, thereby

enhancing the beta cell secretory functions.

In conclusion, genetic variants of SLC30A8 loci may be associated with type 2

diabetes and altered glucose-stimulated insulin secretion. Therefore, the major

alleles of candidate SNPs within these loci represent crucial alleles for -cell

dysfunction. Further studies need to be conducted to examine whether these risk

variants predict the development of type 2 diabetes later in life.

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Correspondence to:???????Department of Medical Biochemistry,Faculty of Medicine,Suez Canal UniversityE-mail:Author title: El-Serafi et al.Short cut Title: ?????

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