Research ArticleInsulin Receptor Substrate-1 (IRS-1) Gly927ArgCorrelation with Gestational Diabetes Mellitus in Saudi Women
Khalid Khalaf Alharbi1 Imran Ali Khan1 Zeinab Abotalib2
and Malak Mohammed Al-Hakeem2
1 Department of Clinical Laboratory Sciences College of Applied Medical Sciences King Saud University PO Box 10219Riyadh 11433 Saudi Arabia
2Department of Obstetrics and Gynecology King Khalid University Hospital College of Medicine King Saud UniversityPO Box 60826 Riyadh 11555 Saudi Arabia
Correspondence should be addressed to Imran Ali Khan imkhanksuedusa
Received 1 December 2013 Accepted 10 January 2014 Published 17 February 2014
Academic Editor Isao Usui
Copyright copy 2014 Khalid Khalaf Alharbi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Pregnant women with gestational diabetes mellitus (GDM) and type 2 diabetes mellitus (T2DM) share a common pathophysiologyassociated with similar risk factors Genetic variants used to determine the risk of developing T2DMmight also be associated withthe prevalence of GDM The aim of the present study was to scrutinize the relationship between the G972R polymorphism of theinsulin receptor substrate-1 (IRS-1) gene with GDM in the Saudi female populationThis is a case-control study that monitored 500Saudi women Subjects with GDM (119899 = 200) were compared with non-GDM (119899 = 300) controls We opted to evaluate rs1801278polymorphism in the IRS1 gene which plays a critical role in the insulin-signaling pathway Genotyping was performed with thePolymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method The frequency of the rs1801278polymorphism was significantly higher in women with GDM than in women with non-GDM (for TT + CT versus CC 119875 = 002)Additionally there was a significant increase in the frequency of the Arg-encoding mutant allele from GDM to non-GDM (for Tversus C 119875 = 001) Our results suggest that the rs1801278 polymorphism in the IRS-1 gene is involved in the occurrence of GDMin the Saudi population
1 Introduction
Gestational diabetesmellitus (GDM) is a growing health con-cern that usually appears during the latter half of pregnancyand is characterized by carbohydrate intolerance of variableseverity [1]
The prevalence of GDM which affects 2ndash22 of allpregnancies varies across populations (eg ethnic groups)[2] Risk factors for GDM include obesity advancedmaternalage family history of type 2 diabetes mellitus (T2DM) pasthistory of GDM previous adverse pregnancy outcomes andbelonging to a high-risk ethnic group [3] The genetic back-ground of T2DMmay also be a factor in GDMbecause ampleevidence has demonstrated the presence of T2DM in womenwith GDM Moreover the prevalence of T2DM is relativelyhigher in mothers with GDM after pregnancy [4] Epidemi-ological studies confirmed that the prevalence of GDM is
in direct proportion to the prevalence of T2DM [5] GDMand T2DM share a common genetic background includingglucose intolerance insulin resistance and impaired insulinsecretion However association with similar risk factors andthe genetic variants used to determine the risk of developingT2DMmight also be associated with the prevalence of GDM[6 7]
The insulin receptor substrate- (IRS-) 1 gene located onchromosome 2q36 encodes a member of the IRS proteinsubstrate family IRS-1 is an endogenous substrate of theinsulin receptor plays a crucial role in the insulin signalingpathway and is expressed in insulin-sensitive tissues Severalgenetic polymorphisms of this gene and their effects oninsulin action have already been identified [8] A glycine-to-arginine substitution in codon 972 (Gly972Arg) of the IRS-1gene (rs1801278) has been shown to be associated with a highprevalence of T2DM and GDM due to insulin resistance and
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 146495 5 pageshttpdxdoiorg1011552014146495
2 BioMed Research International
impaired insulin secretion [6 9] The G972R polymorphismof IRS-1 which is located between two potential tyrosinephosphorylation sites involved in binding of the p85 subunitof PI-3 kinase has previously been associated with T2DM[10] However a meta-analysis of 32 studies including 12076cases and 11285 controls did not show the R972 variant to besignificantly associated with T2DM [11]
We examined the IRS-1 G972R polymorphism in 500Saudi women 200GDM cases and 300 non-GDM subjectsThe goal of this study is to determine whether the IRS-1G972R polymorphism affects GDM-afflicted Saudi womenliving in Saudi Arabia In particular our study focusedon the G972R polymorphism since this polymorphism isassociated with T2DM obesity polycystic ovarian syndromeand insulin resistance
2 Materials and Methodology
21 Ethics Informed consent was obtained from each patientinvolved in this study (E-12-675) and the Institutional ReviewBoard of the College of Medicine King Saud University(Riyadh Saudi Arabia) granted ethical approval
22 Sample Collection Five milliliters of venous blood wascollected 3mL of serum was used for biochemical analysisand 2mL was used for molecular analysis
23 Study Population In total 500 pregnant women wereincluded in this study The subjects were from Riyadh SaudiArabia Of these 200 were unrelated patients with GDMwho visited the primary health care outpatient clinic atKing Khalid University Hospital (KKUH) in RiyadhWomendiagnosed with diabetes prior to pregnancy were excludedfrom this study All pregnant women (119899 = 300) exhibitingnormal glucose tolerance (NGT) were selected based on ageand were designated as non-GDMhealthy controls
24 Validation of GDM in Study Participants All the preg-nant women in this study were recruited after they visited theoutpatient clinic at KKUH Initially the Glucose ChallengeTest (GCT) was performed on all the pregnant womenPlasma glucose standards that exceeded 78mmolL wereconsidered GCT-positive Subsequently the Oral GlucoseTolerance Test (OGTT) was performed Before this testpregnant women were advised to fast overnight in additionto three days of an unrestricted diet Fasting plasma sampleswere drawn after 1 2 and 3 h of administration of glucoseto perform the 100 g OGTT test In this study Bhat et al[12] abnormal values were followed (Table 1) If two or morevalues were abnormal the patient was considered GDM-positive Pregnant women with abnormal glucose tolerancetests were included in the study NGT group members wereconsidered non-GDMhealthy controls or normal controls
25 Anthropometric andBiochemicalMeasurements Anthro-pometric measurements were obtained by trained personnelat health care centers Height and bodyweight weremeasuredto the nearest 05 cm or 01 kg Body Mass Index (BMI) was
Table 1 Diagnosis of GDM with a 100 g oral glucose tolerance test
calculated as weightheight2 (kgm2) Subjects with BMI gt30 kgm2 were categorized as obese Fasting and postpran-dial blood biochemical parameters included high densitylipoprotein cholesterol (HDL-C) low density lipoproteincholesterol (LDL-C) triglycerides (TG) and total cholesterol(TC) GCT and OGTT were measured by a commerciallyavailable clinical chemistry kit provided purchased fromKonelab (Espoo Finland)
26 Molecular Analysis Genomic DNA was extracted fromperipheral blood leukocytes using AccuVis DNA extrac-tion kit (AccuVis Bio UAE) DNA samples were stored atminus80∘C Molecular analysis was performed at the facility ofthe Department of Clinical Laboratory Sciences College ofApplied Medical Sciences King Saud University (RiyadhSaudi Arabia)
27 Amino Acid Conversion at Gly972Arg We used PCR-RFLP to amplify and genotype the nucleotide 972 polymor-phism (ie rs1801278) in IRS-1 which is responsible forthe glycine-to-arginine amino acid mutation Amplificationof the fragment was performed with forward primer 51015840-CTTCTGTCAGGTGTCCATCC-31015840 and reverse primer 51015840-TGGCGAGGTGTCCACGTAGC-31015840 which has been pub-lished by Pappa et al [6] Primers were synthesized byBioserve Biotechnology (Hyderabad India) for PCRanalysisDNA was denatured at 95∘C for 5min and amplified by 35cycles at 95∘C for 30 s 62∘C for 30 s 72∘C for 45 s and thefinal extension step at 72∘C for 5min A total volume of 20 120583Lreactionmixture contains 2120583L of each primer (10 pmol) 7120583Lof sterile water and 10 120583L of 2times master mix (MgCl
2 10times Taq
buffer 10 unit of Taq DNA polymerase (Norgen Biotek corpCanada)) and the 1 120583L template DNA used for amplificationof the G972R polymorphism PCR products were digested for2 h with BstNI (CCdarrWGG) (New England Biolabs IpswichMA) at 60∘C (25 120583L of distilled water with 10 units ofenzyme for 15 120583L PCR product and 2 120583L of 10times buffer in afinal volume of 20 120583L) in nondenaturing Polyacrylamide Gelelectrophoresis (PAGE) and visualized after silver stainingThree genotypes could be determined after electrophore-sis G972 homozygotes (1598123 bp) R972 (108815123 bpband) and G972R (159108815123 bp) heterozygotes
28 Analysis Data are expressed as mean plusmn SD Independentsample 119905-test was used to test GDM and non-GDM groupsSignificance was set at 119875 lt 005 Allele and genotypefrequency differences between GDM patients and non-GDM
BioMed Research International 3
Table 2 Demographic characteristics of the pregnant women
All continuous variables represented by mean plusmn standard deviation Independent sample 119905-test is done comparing GDM and non-GDM subjects Categoricalvariables compared using chi-square analysis A 119875 lt 005 was considered significant NA not applicablenot analyzed
Table 3 Genotype and allele distribution of the IRS1 (G972R) gene polymorphism for GDM and non-GDM
IRS1(rs1801278)
GDM119873 ()
Non-GDM119873 ()
Odds ratioa(95 CI) 119875 value Odds ratiob
(95 CI) 119875 value
119873 200 300CC 189 (945) 295 (983) ReferenceCT 10 (50) 5 (17) 31 (099 92) 006lowast 29 (089 99) 007TT 1 (05) 0 (00) mdash mdashCT + TT 11 (55) 5 (17) 34 (12 101) 002lowast 33 (10 109) 004C 388 (97) 595 (992) ReferenceT 12 (3) 5 (008) 37 (13 105) 001aCrude odds ratio (95 CI) bodds ratio (95 CI) adjusted for age and BMIlowastGenotype and allele frequency distribution with GDM and Non-GDM subjects
subjects were tested by chi-square analysis Odds ratios(ORs) and 95 confidence intervals were calculated bymultiple logistic regression analysis Genotype and haplotypefrequencies were used to identify departures from the Hardy-Weinberg equilibrium (HWE) Statistical analyses were per-formed with SPSS version 190 software A 119875 value of lt005was considered to be statistically significant
3 Results
31 Clinical Characteristics Contrasts between maternaldemographic characteristics of GDM cases and non-GDMcontrols are listed in Table 2 The results illustrate that GDMsubjects were similar to the controls and anthropomet-ric measurements including age weight height and BMIremained the same (119875 gt 005) in the GDM and non-GDMsubjects FBS PPBG GCT OGTT and lipid profile (TC TG
HDL-C and LDL-C) were the biochemical tests performedfor all pregnant women and were found to be significant (119875 lt005) Family history of T2DM and GDM was significantlydifferent between both groups (119875 lt 005) Identified GDMwomen (90) were prescribed a diet to maintain normalglucose values whereas 10 of GDM women were using 4ndash8units of insulin due to the diet failure
32 Molecular Scrutiny for Gly972Arg The distribution ofGly972Arg polymorphism in IRS-1 is provided in Table 3 Weobserved that the frequencies of CC CT and TT genotypes ofthe rs1801278 SNPwere statistically significant IRS1 genotypefrequencies of Gly972Gly Gly972Arg and Arg972Arg were945 5 and 05 in the GDM group and 983 17 and0 in the non-GDM respectively Allele frequencies of Glyand Arg were 97 and 3 for the GDM group and 992
4 BioMed Research International
and 0008 for the non-GDM group respectively The TTgenotype was absent in non-GDM groups The minor allelefrequencies of GDM and non-GDM were 3 and 0008respectively (119875 lt 005) Similarly the frequency of CC CTand TT genotypes of the Gly972Arg SNP was comparableamong the GDM and non-GDM (minor allele frequency119875 = 001 OR = 37 95 CI [13 105]) There were significantdifferences in the frequencies of the genotype distributionsof IRS-1 Gly972Arg between the GDM group and non-GDMgroup (TT + CT versus CC 119875 = 002 OR-34 95 CI [12101]) The odds ratio for any genotype of the Gly972Arg SNPwas significantly correlated with the risk of developing GDM(119875 lt 005) in the Saudi women monitored in this studyGenotype data is associated with HWE
4 Discussion
King Saud University is one of the foremost institute in theKingdom of Saudi Arabia as well as in an Arab countriesinvolved deeply in the research with human and molecularmedical geneticsThis is a case-control study performed with500 pregnant Saudi women including 200 GDM and 300non-GDM cases We investigated the relationship betweenthe IRS-1 G972R gene and GDM in a Saudi populationScreening and identifying GDM are based on biochemicalcriteria with serum sample despite the progress of geneticscreening methods There is no genetic test to identify GDMin women during pregnancy To the best of our knowledgethis is the first study conducted with pregnant Saudi womenIn this study we explored the effects of G972R of theIRS-1 gene variants The results of our study revealed thatthe variant allele Arg972 of the IRS-1 gene is significantlyassociated with GDM
Pregnancy is characterized by peripheral insulin resis-tance which is compensated by an increase in insulin secre-tion to maintain glucose homeostasis [13] GDM represents aheterogeneous disorder with both a genetic and an environ-mental component The prevalence of GDM in Saudi Arabiais 187 [2] According to additional studies women withGDMwere older and of higher parity and had a less favorablereproductive history than did nondiabetic pregnant womenThe independent association between high parity and GDMwas corroborated by previous reports [14] Advanced mater-nal age is independently associated with an increased riskof GDM [2] Several genetic polymorphisms of this genehave already been identified as they affect insulin action[15] The IRS-1 protein is a cytoplasmic molecule expressedin many insulin-sensitive tissues It plays an important rolein regulating the cellular effects of insulin [16] Tyrosinephosphorylation of IRS-1 proteins initiates the downstreameffects of insulin including activation of phosphatidylinositol3-kinase (PI3 K) and translocation of glucose transporter4 [17] The G972R polymorphism has also been associatedwith impaired 120573-cell function in NGT subjects as well aswith reduced insulin content and impaired insulin secretionin isolated human islets [10] Ample evidence suggests thatsusceptibility to GDM has a genetic component Althoughno studies have evaluated the heritability of GDM family
studies indicate that GDM aggregates within families andis associated with a history of T2DM [18] Genetic studiesof T2DM and GDM further suggest that it is a multigenicdisease in which common variants in multiple genes interactwith environmental factors to cause the disease [19 20]Because of the striking parallels between GDM and T2DMit is likely that GDM is also a multigenic disease relatedto T2DM Thus recent studies on the etiology of GDMhave begun to evaluate the role of common polymorphismsin genes involved in T2DM predisposition So far geneticpredisposition to GDM has been reported for variations ingenes involved in insulin resistance and insulin secretionHowever no association could be replicated in differentstudies [21ndash23] These discrepancies could be due to lack ofpower given the small effect size of most common variantsor to ethnic heterogeneity among different populations
Studies conducted by Pappa et al [6] in the Greek popu-lation with 148 women with GDM and 107 control subjectsand the results indicate that the G972R polymorphism of theIRS-1 gene is strongly associated with increased susceptibilityto GDM Our results in the Saudi population are in completeagreement with the results reported by Fallucca et al [13]and Pappa et al [6] which indicate that the polymorphicallele R972 is significantly associated with IRS-1 and may beinvolved in the development of GDM In contrast Shaat etal [10] and Tok et al [24] did not observe any associationbetween the G972R polymorphism and GDM Although noassociation was observed in these studies Tok et al [24]reported that the variant was associated with higher fastingglucose and insulin levels in women with GDM Shaat etal [10] concluded that IRS-1 protein levels are reduced inthe adipose tissue of obese Scandinavian women with GDMOur findings established that homozygosity for the G972Rpolymorphism which was observed only in women withGDM might indicate an increased risk for GDM in Saudiwomen
In our study 90 of the women were following a dietmeant to regularize the levels of certain biochemical mark-ers before pregnancy Maintaining good nutrition duringpregnancy while consuming 2250ndash2500 calories daily isessential Physical exercise is also very important for GDMwomen especially those identified as either overweight orobese Insulin is the only medicationoption for pregnantwomen who develop GDM during pregnancy Insulin doesnot transfer to the baby through the placenta however theoral drug metformin (glyburide) can be used to circumventthis Ten percent of the women in our study used insulinThe biochemical profiles of GDM subjects were significantlyhigher than the biochemical profiles of non-GDM subjects(119875 lt 005)
In conclusion our data indicate that the IRS-1 G972Rvariant may facilitate the development of GDM Howeverthis variant was also associated with the baseline character-istics of the patients with GDM
Conflict of Interests
All the authors declare that there is no conflict of interestsregarding the publication of this paper
BioMed Research International 5
Acknowledgments
The authors would like to extend their sincere appreciationto the Deanship of Scientific Research at King Saud Uni-versity for its funding of this research through the ResearchGroup Project no RGP-VPP-244 The authors are grateful toBenjamin Vinodson for helping with the complete statisticalanalysis The authors are thankful to Mrs Virginia Rubio(Jean) and Mrs Laila H Assobah for helping with thecollection of blood samples
References
[1] C Zhang W Bao Y Rong et al ldquoGenetic variants and the riskof gestational diabetes mellitus a systematic reviewrdquo HumanReproduction Update vol 19 pp 376ndash390 2013
[2] H AWahabi S A Esmaeil A Fayed and R A Alzeidan ldquoGes-tational diabetes mellitus maternal and perinatal outcomes inKing Khalid University Hospital Saudi Arabiardquo The Journalof the Egyptian Public Health Association vol 88 pp 104ndash1082013
[3] H M Georgiou M Lappas G M Georgiou et al ldquoScreeningfor biomarkers predictive of gestational diabetes mellitusrdquo ActaDiabetologica vol 45 no 3 pp 157ndash165 2008
[4] S J Chon S Y Kim N R Cho D L Min Y J Hwang andM Mamura ldquoAssociation of variants in PPAR1205742 IGF2BP2 andKCNQ1 with a susceptibility to gestational diabetes mellitus ina Korean populationrdquo Yonsei Medical Journal vol 54 pp 352ndash357 2013
[5] Y Wang L Chen R Horswell et al ldquoRacial differences in theassociation between gestational diabetes mellitus and risk oftype 2 diabetesrdquo Journal ofWomenrsquos Health vol 21 pp 628ndash6332012
[6] K I Pappa M Gazouli K Economou et al ldquoGestationaldiabetes mellitus shares polymorphisms of genes associatedwith insulin resistance and type 2 diabetes in the GreekpopulationrdquoGynecological Endocrinology vol 27 no 4 pp 267ndash272 2011
[7] Y M Cho T H Kim S Lim et al ldquoType 2 diabetes-associatedgenetic variants discovered in the recent genome-wide associ-ation studies are related to gestational diabetes mellitus in theKorean populationrdquo Diabetologia vol 52 no 2 pp 253ndash2612009
[8] Y Tang X Han X Sun et al ldquoAssociation study of a commonvariant near IRS1 with type 2 diabetes mellitus in Chinese Hanpopulationrdquo Endocrine vol 43 pp 84ndash91 2013
[9] F Mousavinasab T Tahtinen J Jokelainen et al ldquoCommonpolymorphisms in the PPAR1205742 and IRS-1 genes and their inter-action influence serum adiponectin concentration in youngFinnish menrdquo Molecular Genetics and Metabolism vol 84 no4 pp 344ndash348 2005
[10] N Shaat M Ekelund A Lernmark et al ldquoAssociation ofthe E23K polymorphism in the KCNJ11 gene with gestationaldiabetes mellitusrdquo Diabetologia vol 48 no 12 pp 2544ndash25512005
[11] E Morini S Prudente E Succurro et al ldquoIRS1 G972R poly-morphism and type 2 diabetes a paradigm for the difficultascertainment of the contribution to disease susceptibility oflsquolow-frequency-low-riskrsquo variantsrdquo Diabetologia vol 52 no 9pp 1852ndash1857 2009
[12] M Bhat K N Ramesha S P Sarma C V Sowmini and SGanesh Kumar ldquoDeterminants of gestational diabetes mellitus
a case control study in a district tertiary care hospital in southIndiardquo International Journal of Diabetes inDeveloping Countriesvol 30 no 2 pp 91ndash96 2010
[13] F Fallucca M G Dalfra E Sciullo et al ldquoPolymorphisms ofinsulin receptor substrate 1 and 1205733-adrenergic receptor genesin gestational diabetes and normal pregnancyrdquoMetabolism vol55 no 11 pp 1451ndash1456 2006
[14] S M Shahida M A Islam S Begum M A Hossain and MS Azam ldquoMaternal outcome of grand multiparardquoMymensinghMedical Journal vol 20 no 3 pp 381ndash385 2011
[15] K Almind C Bjorbaek H Vestergaard T Hansen S Echwaldand O Pedersen ldquoAminoacid polymorphisms of insulin recep-tor substrate-1 in non-insulin-dependent diabetesmellitusrdquoTheLancet vol 342 no 8875 pp 828ndash832 1993
[16] X J Sun P Rothenberg C R Kahn et al ldquoStructure ofthe insulin receptor substrate IRS-1 defines a unique signaltransduction proteinrdquoNature vol 352 no 6330 pp 73ndash77 1991
[17] Y Kaburagi T Yamauchi R Yamamoto-Honda et al ldquoThemechanism of insulin-induced signal transductionmediated bythe insulin receptor substrate familyrdquo Endocrine Journal vol 46pp S25ndashS34 1999
[18] M A Williams C Qiu J C Dempsey and D A LuthyldquoFamilial aggregation of type 2 diabetes and chronic hyperten-sion in women with gestational diabetes mellitusrdquo Journal ofReproductiveMedicine for theObstetrician andGynecologist vol48 no 12 pp 955ndash962 2003
[19] L Groop ldquoPathogenesis of type 2 diabetes the relative con-tribution of insulin resistance and impaired insulin secretionrdquoInternational Journal of Clinical Practice vol 113 pp 3ndash13 2000
[20] L Groop and V Lyssenko ldquoGenes and type 2 diabetes mellitusrdquoCurrent Diabetes Reports vol 8 no 3 pp 192ndash197 2008
[21] N Shaat M Ekelund A Lernmark et al ldquoGenotypic andphenotypic differences between Arabian and Scandinavianwomen with gestational diabetes mellitusrdquoDiabetologia vol 47no 5 pp 878ndash884 2004
[22] M Alevizaki LThalassinou S I Grigorakis et al ldquoStudy of theTrp64Arg polymorphism ofthe 1205733-adrenergic receptor in greekwomen with gestational diabetesrdquo Diabetes Care vol 23 no 8pp 1079ndash1083 2000
[23] H Leipold M Knoefler C Gruber A Huber P Haslingerand C Worda ldquoPeroxisome proliferator-activated receptorgamma coactivator-1alpha gene variations are not associatedwith gestational diabetes mellitusrdquo Journal of the Society forGynecologic Investigation vol 13 pp 104ndash107 2006
[24] E C Tok D Ertunc O Bilgin E M Erdal M Kaplanogluand S Dilek ldquoAssociation of insulin receptor substrate-1 G972Rvariant with baseline characteristics of the patients with ges-tational diabetes mellitusrdquo American Journal of Obstetrics ampGynecology vol 194 pp 868ndash872 2006
impaired insulin secretion [6 9] The G972R polymorphismof IRS-1 which is located between two potential tyrosinephosphorylation sites involved in binding of the p85 subunitof PI-3 kinase has previously been associated with T2DM[10] However a meta-analysis of 32 studies including 12076cases and 11285 controls did not show the R972 variant to besignificantly associated with T2DM [11]
We examined the IRS-1 G972R polymorphism in 500Saudi women 200GDM cases and 300 non-GDM subjectsThe goal of this study is to determine whether the IRS-1G972R polymorphism affects GDM-afflicted Saudi womenliving in Saudi Arabia In particular our study focusedon the G972R polymorphism since this polymorphism isassociated with T2DM obesity polycystic ovarian syndromeand insulin resistance
2 Materials and Methodology
21 Ethics Informed consent was obtained from each patientinvolved in this study (E-12-675) and the Institutional ReviewBoard of the College of Medicine King Saud University(Riyadh Saudi Arabia) granted ethical approval
22 Sample Collection Five milliliters of venous blood wascollected 3mL of serum was used for biochemical analysisand 2mL was used for molecular analysis
23 Study Population In total 500 pregnant women wereincluded in this study The subjects were from Riyadh SaudiArabia Of these 200 were unrelated patients with GDMwho visited the primary health care outpatient clinic atKing Khalid University Hospital (KKUH) in RiyadhWomendiagnosed with diabetes prior to pregnancy were excludedfrom this study All pregnant women (119899 = 300) exhibitingnormal glucose tolerance (NGT) were selected based on ageand were designated as non-GDMhealthy controls
24 Validation of GDM in Study Participants All the preg-nant women in this study were recruited after they visited theoutpatient clinic at KKUH Initially the Glucose ChallengeTest (GCT) was performed on all the pregnant womenPlasma glucose standards that exceeded 78mmolL wereconsidered GCT-positive Subsequently the Oral GlucoseTolerance Test (OGTT) was performed Before this testpregnant women were advised to fast overnight in additionto three days of an unrestricted diet Fasting plasma sampleswere drawn after 1 2 and 3 h of administration of glucoseto perform the 100 g OGTT test In this study Bhat et al[12] abnormal values were followed (Table 1) If two or morevalues were abnormal the patient was considered GDM-positive Pregnant women with abnormal glucose tolerancetests were included in the study NGT group members wereconsidered non-GDMhealthy controls or normal controls
25 Anthropometric andBiochemicalMeasurements Anthro-pometric measurements were obtained by trained personnelat health care centers Height and bodyweight weremeasuredto the nearest 05 cm or 01 kg Body Mass Index (BMI) was
Table 1 Diagnosis of GDM with a 100 g oral glucose tolerance test
calculated as weightheight2 (kgm2) Subjects with BMI gt30 kgm2 were categorized as obese Fasting and postpran-dial blood biochemical parameters included high densitylipoprotein cholesterol (HDL-C) low density lipoproteincholesterol (LDL-C) triglycerides (TG) and total cholesterol(TC) GCT and OGTT were measured by a commerciallyavailable clinical chemistry kit provided purchased fromKonelab (Espoo Finland)
26 Molecular Analysis Genomic DNA was extracted fromperipheral blood leukocytes using AccuVis DNA extrac-tion kit (AccuVis Bio UAE) DNA samples were stored atminus80∘C Molecular analysis was performed at the facility ofthe Department of Clinical Laboratory Sciences College ofApplied Medical Sciences King Saud University (RiyadhSaudi Arabia)
27 Amino Acid Conversion at Gly972Arg We used PCR-RFLP to amplify and genotype the nucleotide 972 polymor-phism (ie rs1801278) in IRS-1 which is responsible forthe glycine-to-arginine amino acid mutation Amplificationof the fragment was performed with forward primer 51015840-CTTCTGTCAGGTGTCCATCC-31015840 and reverse primer 51015840-TGGCGAGGTGTCCACGTAGC-31015840 which has been pub-lished by Pappa et al [6] Primers were synthesized byBioserve Biotechnology (Hyderabad India) for PCRanalysisDNA was denatured at 95∘C for 5min and amplified by 35cycles at 95∘C for 30 s 62∘C for 30 s 72∘C for 45 s and thefinal extension step at 72∘C for 5min A total volume of 20 120583Lreactionmixture contains 2120583L of each primer (10 pmol) 7120583Lof sterile water and 10 120583L of 2times master mix (MgCl
2 10times Taq
buffer 10 unit of Taq DNA polymerase (Norgen Biotek corpCanada)) and the 1 120583L template DNA used for amplificationof the G972R polymorphism PCR products were digested for2 h with BstNI (CCdarrWGG) (New England Biolabs IpswichMA) at 60∘C (25 120583L of distilled water with 10 units ofenzyme for 15 120583L PCR product and 2 120583L of 10times buffer in afinal volume of 20 120583L) in nondenaturing Polyacrylamide Gelelectrophoresis (PAGE) and visualized after silver stainingThree genotypes could be determined after electrophore-sis G972 homozygotes (1598123 bp) R972 (108815123 bpband) and G972R (159108815123 bp) heterozygotes
28 Analysis Data are expressed as mean plusmn SD Independentsample 119905-test was used to test GDM and non-GDM groupsSignificance was set at 119875 lt 005 Allele and genotypefrequency differences between GDM patients and non-GDM
BioMed Research International 3
Table 2 Demographic characteristics of the pregnant women
All continuous variables represented by mean plusmn standard deviation Independent sample 119905-test is done comparing GDM and non-GDM subjects Categoricalvariables compared using chi-square analysis A 119875 lt 005 was considered significant NA not applicablenot analyzed
Table 3 Genotype and allele distribution of the IRS1 (G972R) gene polymorphism for GDM and non-GDM
IRS1(rs1801278)
GDM119873 ()
Non-GDM119873 ()
Odds ratioa(95 CI) 119875 value Odds ratiob
(95 CI) 119875 value
119873 200 300CC 189 (945) 295 (983) ReferenceCT 10 (50) 5 (17) 31 (099 92) 006lowast 29 (089 99) 007TT 1 (05) 0 (00) mdash mdashCT + TT 11 (55) 5 (17) 34 (12 101) 002lowast 33 (10 109) 004C 388 (97) 595 (992) ReferenceT 12 (3) 5 (008) 37 (13 105) 001aCrude odds ratio (95 CI) bodds ratio (95 CI) adjusted for age and BMIlowastGenotype and allele frequency distribution with GDM and Non-GDM subjects
subjects were tested by chi-square analysis Odds ratios(ORs) and 95 confidence intervals were calculated bymultiple logistic regression analysis Genotype and haplotypefrequencies were used to identify departures from the Hardy-Weinberg equilibrium (HWE) Statistical analyses were per-formed with SPSS version 190 software A 119875 value of lt005was considered to be statistically significant
3 Results
31 Clinical Characteristics Contrasts between maternaldemographic characteristics of GDM cases and non-GDMcontrols are listed in Table 2 The results illustrate that GDMsubjects were similar to the controls and anthropomet-ric measurements including age weight height and BMIremained the same (119875 gt 005) in the GDM and non-GDMsubjects FBS PPBG GCT OGTT and lipid profile (TC TG
HDL-C and LDL-C) were the biochemical tests performedfor all pregnant women and were found to be significant (119875 lt005) Family history of T2DM and GDM was significantlydifferent between both groups (119875 lt 005) Identified GDMwomen (90) were prescribed a diet to maintain normalglucose values whereas 10 of GDM women were using 4ndash8units of insulin due to the diet failure
32 Molecular Scrutiny for Gly972Arg The distribution ofGly972Arg polymorphism in IRS-1 is provided in Table 3 Weobserved that the frequencies of CC CT and TT genotypes ofthe rs1801278 SNPwere statistically significant IRS1 genotypefrequencies of Gly972Gly Gly972Arg and Arg972Arg were945 5 and 05 in the GDM group and 983 17 and0 in the non-GDM respectively Allele frequencies of Glyand Arg were 97 and 3 for the GDM group and 992
4 BioMed Research International
and 0008 for the non-GDM group respectively The TTgenotype was absent in non-GDM groups The minor allelefrequencies of GDM and non-GDM were 3 and 0008respectively (119875 lt 005) Similarly the frequency of CC CTand TT genotypes of the Gly972Arg SNP was comparableamong the GDM and non-GDM (minor allele frequency119875 = 001 OR = 37 95 CI [13 105]) There were significantdifferences in the frequencies of the genotype distributionsof IRS-1 Gly972Arg between the GDM group and non-GDMgroup (TT + CT versus CC 119875 = 002 OR-34 95 CI [12101]) The odds ratio for any genotype of the Gly972Arg SNPwas significantly correlated with the risk of developing GDM(119875 lt 005) in the Saudi women monitored in this studyGenotype data is associated with HWE
4 Discussion
King Saud University is one of the foremost institute in theKingdom of Saudi Arabia as well as in an Arab countriesinvolved deeply in the research with human and molecularmedical geneticsThis is a case-control study performed with500 pregnant Saudi women including 200 GDM and 300non-GDM cases We investigated the relationship betweenthe IRS-1 G972R gene and GDM in a Saudi populationScreening and identifying GDM are based on biochemicalcriteria with serum sample despite the progress of geneticscreening methods There is no genetic test to identify GDMin women during pregnancy To the best of our knowledgethis is the first study conducted with pregnant Saudi womenIn this study we explored the effects of G972R of theIRS-1 gene variants The results of our study revealed thatthe variant allele Arg972 of the IRS-1 gene is significantlyassociated with GDM
Pregnancy is characterized by peripheral insulin resis-tance which is compensated by an increase in insulin secre-tion to maintain glucose homeostasis [13] GDM represents aheterogeneous disorder with both a genetic and an environ-mental component The prevalence of GDM in Saudi Arabiais 187 [2] According to additional studies women withGDMwere older and of higher parity and had a less favorablereproductive history than did nondiabetic pregnant womenThe independent association between high parity and GDMwas corroborated by previous reports [14] Advanced mater-nal age is independently associated with an increased riskof GDM [2] Several genetic polymorphisms of this genehave already been identified as they affect insulin action[15] The IRS-1 protein is a cytoplasmic molecule expressedin many insulin-sensitive tissues It plays an important rolein regulating the cellular effects of insulin [16] Tyrosinephosphorylation of IRS-1 proteins initiates the downstreameffects of insulin including activation of phosphatidylinositol3-kinase (PI3 K) and translocation of glucose transporter4 [17] The G972R polymorphism has also been associatedwith impaired 120573-cell function in NGT subjects as well aswith reduced insulin content and impaired insulin secretionin isolated human islets [10] Ample evidence suggests thatsusceptibility to GDM has a genetic component Althoughno studies have evaluated the heritability of GDM family
studies indicate that GDM aggregates within families andis associated with a history of T2DM [18] Genetic studiesof T2DM and GDM further suggest that it is a multigenicdisease in which common variants in multiple genes interactwith environmental factors to cause the disease [19 20]Because of the striking parallels between GDM and T2DMit is likely that GDM is also a multigenic disease relatedto T2DM Thus recent studies on the etiology of GDMhave begun to evaluate the role of common polymorphismsin genes involved in T2DM predisposition So far geneticpredisposition to GDM has been reported for variations ingenes involved in insulin resistance and insulin secretionHowever no association could be replicated in differentstudies [21ndash23] These discrepancies could be due to lack ofpower given the small effect size of most common variantsor to ethnic heterogeneity among different populations
Studies conducted by Pappa et al [6] in the Greek popu-lation with 148 women with GDM and 107 control subjectsand the results indicate that the G972R polymorphism of theIRS-1 gene is strongly associated with increased susceptibilityto GDM Our results in the Saudi population are in completeagreement with the results reported by Fallucca et al [13]and Pappa et al [6] which indicate that the polymorphicallele R972 is significantly associated with IRS-1 and may beinvolved in the development of GDM In contrast Shaat etal [10] and Tok et al [24] did not observe any associationbetween the G972R polymorphism and GDM Although noassociation was observed in these studies Tok et al [24]reported that the variant was associated with higher fastingglucose and insulin levels in women with GDM Shaat etal [10] concluded that IRS-1 protein levels are reduced inthe adipose tissue of obese Scandinavian women with GDMOur findings established that homozygosity for the G972Rpolymorphism which was observed only in women withGDM might indicate an increased risk for GDM in Saudiwomen
In our study 90 of the women were following a dietmeant to regularize the levels of certain biochemical mark-ers before pregnancy Maintaining good nutrition duringpregnancy while consuming 2250ndash2500 calories daily isessential Physical exercise is also very important for GDMwomen especially those identified as either overweight orobese Insulin is the only medicationoption for pregnantwomen who develop GDM during pregnancy Insulin doesnot transfer to the baby through the placenta however theoral drug metformin (glyburide) can be used to circumventthis Ten percent of the women in our study used insulinThe biochemical profiles of GDM subjects were significantlyhigher than the biochemical profiles of non-GDM subjects(119875 lt 005)
In conclusion our data indicate that the IRS-1 G972Rvariant may facilitate the development of GDM Howeverthis variant was also associated with the baseline character-istics of the patients with GDM
Conflict of Interests
All the authors declare that there is no conflict of interestsregarding the publication of this paper
BioMed Research International 5
Acknowledgments
The authors would like to extend their sincere appreciationto the Deanship of Scientific Research at King Saud Uni-versity for its funding of this research through the ResearchGroup Project no RGP-VPP-244 The authors are grateful toBenjamin Vinodson for helping with the complete statisticalanalysis The authors are thankful to Mrs Virginia Rubio(Jean) and Mrs Laila H Assobah for helping with thecollection of blood samples
References
[1] C Zhang W Bao Y Rong et al ldquoGenetic variants and the riskof gestational diabetes mellitus a systematic reviewrdquo HumanReproduction Update vol 19 pp 376ndash390 2013
[2] H AWahabi S A Esmaeil A Fayed and R A Alzeidan ldquoGes-tational diabetes mellitus maternal and perinatal outcomes inKing Khalid University Hospital Saudi Arabiardquo The Journalof the Egyptian Public Health Association vol 88 pp 104ndash1082013
[3] H M Georgiou M Lappas G M Georgiou et al ldquoScreeningfor biomarkers predictive of gestational diabetes mellitusrdquo ActaDiabetologica vol 45 no 3 pp 157ndash165 2008
[4] S J Chon S Y Kim N R Cho D L Min Y J Hwang andM Mamura ldquoAssociation of variants in PPAR1205742 IGF2BP2 andKCNQ1 with a susceptibility to gestational diabetes mellitus ina Korean populationrdquo Yonsei Medical Journal vol 54 pp 352ndash357 2013
[5] Y Wang L Chen R Horswell et al ldquoRacial differences in theassociation between gestational diabetes mellitus and risk oftype 2 diabetesrdquo Journal ofWomenrsquos Health vol 21 pp 628ndash6332012
[6] K I Pappa M Gazouli K Economou et al ldquoGestationaldiabetes mellitus shares polymorphisms of genes associatedwith insulin resistance and type 2 diabetes in the GreekpopulationrdquoGynecological Endocrinology vol 27 no 4 pp 267ndash272 2011
[7] Y M Cho T H Kim S Lim et al ldquoType 2 diabetes-associatedgenetic variants discovered in the recent genome-wide associ-ation studies are related to gestational diabetes mellitus in theKorean populationrdquo Diabetologia vol 52 no 2 pp 253ndash2612009
[8] Y Tang X Han X Sun et al ldquoAssociation study of a commonvariant near IRS1 with type 2 diabetes mellitus in Chinese Hanpopulationrdquo Endocrine vol 43 pp 84ndash91 2013
[9] F Mousavinasab T Tahtinen J Jokelainen et al ldquoCommonpolymorphisms in the PPAR1205742 and IRS-1 genes and their inter-action influence serum adiponectin concentration in youngFinnish menrdquo Molecular Genetics and Metabolism vol 84 no4 pp 344ndash348 2005
[10] N Shaat M Ekelund A Lernmark et al ldquoAssociation ofthe E23K polymorphism in the KCNJ11 gene with gestationaldiabetes mellitusrdquo Diabetologia vol 48 no 12 pp 2544ndash25512005
[11] E Morini S Prudente E Succurro et al ldquoIRS1 G972R poly-morphism and type 2 diabetes a paradigm for the difficultascertainment of the contribution to disease susceptibility oflsquolow-frequency-low-riskrsquo variantsrdquo Diabetologia vol 52 no 9pp 1852ndash1857 2009
[12] M Bhat K N Ramesha S P Sarma C V Sowmini and SGanesh Kumar ldquoDeterminants of gestational diabetes mellitus
a case control study in a district tertiary care hospital in southIndiardquo International Journal of Diabetes inDeveloping Countriesvol 30 no 2 pp 91ndash96 2010
[13] F Fallucca M G Dalfra E Sciullo et al ldquoPolymorphisms ofinsulin receptor substrate 1 and 1205733-adrenergic receptor genesin gestational diabetes and normal pregnancyrdquoMetabolism vol55 no 11 pp 1451ndash1456 2006
[14] S M Shahida M A Islam S Begum M A Hossain and MS Azam ldquoMaternal outcome of grand multiparardquoMymensinghMedical Journal vol 20 no 3 pp 381ndash385 2011
[15] K Almind C Bjorbaek H Vestergaard T Hansen S Echwaldand O Pedersen ldquoAminoacid polymorphisms of insulin recep-tor substrate-1 in non-insulin-dependent diabetesmellitusrdquoTheLancet vol 342 no 8875 pp 828ndash832 1993
[16] X J Sun P Rothenberg C R Kahn et al ldquoStructure ofthe insulin receptor substrate IRS-1 defines a unique signaltransduction proteinrdquoNature vol 352 no 6330 pp 73ndash77 1991
[17] Y Kaburagi T Yamauchi R Yamamoto-Honda et al ldquoThemechanism of insulin-induced signal transductionmediated bythe insulin receptor substrate familyrdquo Endocrine Journal vol 46pp S25ndashS34 1999
[18] M A Williams C Qiu J C Dempsey and D A LuthyldquoFamilial aggregation of type 2 diabetes and chronic hyperten-sion in women with gestational diabetes mellitusrdquo Journal ofReproductiveMedicine for theObstetrician andGynecologist vol48 no 12 pp 955ndash962 2003
[19] L Groop ldquoPathogenesis of type 2 diabetes the relative con-tribution of insulin resistance and impaired insulin secretionrdquoInternational Journal of Clinical Practice vol 113 pp 3ndash13 2000
[20] L Groop and V Lyssenko ldquoGenes and type 2 diabetes mellitusrdquoCurrent Diabetes Reports vol 8 no 3 pp 192ndash197 2008
[21] N Shaat M Ekelund A Lernmark et al ldquoGenotypic andphenotypic differences between Arabian and Scandinavianwomen with gestational diabetes mellitusrdquoDiabetologia vol 47no 5 pp 878ndash884 2004
[22] M Alevizaki LThalassinou S I Grigorakis et al ldquoStudy of theTrp64Arg polymorphism ofthe 1205733-adrenergic receptor in greekwomen with gestational diabetesrdquo Diabetes Care vol 23 no 8pp 1079ndash1083 2000
[23] H Leipold M Knoefler C Gruber A Huber P Haslingerand C Worda ldquoPeroxisome proliferator-activated receptorgamma coactivator-1alpha gene variations are not associatedwith gestational diabetes mellitusrdquo Journal of the Society forGynecologic Investigation vol 13 pp 104ndash107 2006
[24] E C Tok D Ertunc O Bilgin E M Erdal M Kaplanogluand S Dilek ldquoAssociation of insulin receptor substrate-1 G972Rvariant with baseline characteristics of the patients with ges-tational diabetes mellitusrdquo American Journal of Obstetrics ampGynecology vol 194 pp 868ndash872 2006
All continuous variables represented by mean plusmn standard deviation Independent sample 119905-test is done comparing GDM and non-GDM subjects Categoricalvariables compared using chi-square analysis A 119875 lt 005 was considered significant NA not applicablenot analyzed
Table 3 Genotype and allele distribution of the IRS1 (G972R) gene polymorphism for GDM and non-GDM
IRS1(rs1801278)
GDM119873 ()
Non-GDM119873 ()
Odds ratioa(95 CI) 119875 value Odds ratiob
(95 CI) 119875 value
119873 200 300CC 189 (945) 295 (983) ReferenceCT 10 (50) 5 (17) 31 (099 92) 006lowast 29 (089 99) 007TT 1 (05) 0 (00) mdash mdashCT + TT 11 (55) 5 (17) 34 (12 101) 002lowast 33 (10 109) 004C 388 (97) 595 (992) ReferenceT 12 (3) 5 (008) 37 (13 105) 001aCrude odds ratio (95 CI) bodds ratio (95 CI) adjusted for age and BMIlowastGenotype and allele frequency distribution with GDM and Non-GDM subjects
subjects were tested by chi-square analysis Odds ratios(ORs) and 95 confidence intervals were calculated bymultiple logistic regression analysis Genotype and haplotypefrequencies were used to identify departures from the Hardy-Weinberg equilibrium (HWE) Statistical analyses were per-formed with SPSS version 190 software A 119875 value of lt005was considered to be statistically significant
3 Results
31 Clinical Characteristics Contrasts between maternaldemographic characteristics of GDM cases and non-GDMcontrols are listed in Table 2 The results illustrate that GDMsubjects were similar to the controls and anthropomet-ric measurements including age weight height and BMIremained the same (119875 gt 005) in the GDM and non-GDMsubjects FBS PPBG GCT OGTT and lipid profile (TC TG
HDL-C and LDL-C) were the biochemical tests performedfor all pregnant women and were found to be significant (119875 lt005) Family history of T2DM and GDM was significantlydifferent between both groups (119875 lt 005) Identified GDMwomen (90) were prescribed a diet to maintain normalglucose values whereas 10 of GDM women were using 4ndash8units of insulin due to the diet failure
32 Molecular Scrutiny for Gly972Arg The distribution ofGly972Arg polymorphism in IRS-1 is provided in Table 3 Weobserved that the frequencies of CC CT and TT genotypes ofthe rs1801278 SNPwere statistically significant IRS1 genotypefrequencies of Gly972Gly Gly972Arg and Arg972Arg were945 5 and 05 in the GDM group and 983 17 and0 in the non-GDM respectively Allele frequencies of Glyand Arg were 97 and 3 for the GDM group and 992
4 BioMed Research International
and 0008 for the non-GDM group respectively The TTgenotype was absent in non-GDM groups The minor allelefrequencies of GDM and non-GDM were 3 and 0008respectively (119875 lt 005) Similarly the frequency of CC CTand TT genotypes of the Gly972Arg SNP was comparableamong the GDM and non-GDM (minor allele frequency119875 = 001 OR = 37 95 CI [13 105]) There were significantdifferences in the frequencies of the genotype distributionsof IRS-1 Gly972Arg between the GDM group and non-GDMgroup (TT + CT versus CC 119875 = 002 OR-34 95 CI [12101]) The odds ratio for any genotype of the Gly972Arg SNPwas significantly correlated with the risk of developing GDM(119875 lt 005) in the Saudi women monitored in this studyGenotype data is associated with HWE
4 Discussion
King Saud University is one of the foremost institute in theKingdom of Saudi Arabia as well as in an Arab countriesinvolved deeply in the research with human and molecularmedical geneticsThis is a case-control study performed with500 pregnant Saudi women including 200 GDM and 300non-GDM cases We investigated the relationship betweenthe IRS-1 G972R gene and GDM in a Saudi populationScreening and identifying GDM are based on biochemicalcriteria with serum sample despite the progress of geneticscreening methods There is no genetic test to identify GDMin women during pregnancy To the best of our knowledgethis is the first study conducted with pregnant Saudi womenIn this study we explored the effects of G972R of theIRS-1 gene variants The results of our study revealed thatthe variant allele Arg972 of the IRS-1 gene is significantlyassociated with GDM
Pregnancy is characterized by peripheral insulin resis-tance which is compensated by an increase in insulin secre-tion to maintain glucose homeostasis [13] GDM represents aheterogeneous disorder with both a genetic and an environ-mental component The prevalence of GDM in Saudi Arabiais 187 [2] According to additional studies women withGDMwere older and of higher parity and had a less favorablereproductive history than did nondiabetic pregnant womenThe independent association between high parity and GDMwas corroborated by previous reports [14] Advanced mater-nal age is independently associated with an increased riskof GDM [2] Several genetic polymorphisms of this genehave already been identified as they affect insulin action[15] The IRS-1 protein is a cytoplasmic molecule expressedin many insulin-sensitive tissues It plays an important rolein regulating the cellular effects of insulin [16] Tyrosinephosphorylation of IRS-1 proteins initiates the downstreameffects of insulin including activation of phosphatidylinositol3-kinase (PI3 K) and translocation of glucose transporter4 [17] The G972R polymorphism has also been associatedwith impaired 120573-cell function in NGT subjects as well aswith reduced insulin content and impaired insulin secretionin isolated human islets [10] Ample evidence suggests thatsusceptibility to GDM has a genetic component Althoughno studies have evaluated the heritability of GDM family
studies indicate that GDM aggregates within families andis associated with a history of T2DM [18] Genetic studiesof T2DM and GDM further suggest that it is a multigenicdisease in which common variants in multiple genes interactwith environmental factors to cause the disease [19 20]Because of the striking parallels between GDM and T2DMit is likely that GDM is also a multigenic disease relatedto T2DM Thus recent studies on the etiology of GDMhave begun to evaluate the role of common polymorphismsin genes involved in T2DM predisposition So far geneticpredisposition to GDM has been reported for variations ingenes involved in insulin resistance and insulin secretionHowever no association could be replicated in differentstudies [21ndash23] These discrepancies could be due to lack ofpower given the small effect size of most common variantsor to ethnic heterogeneity among different populations
Studies conducted by Pappa et al [6] in the Greek popu-lation with 148 women with GDM and 107 control subjectsand the results indicate that the G972R polymorphism of theIRS-1 gene is strongly associated with increased susceptibilityto GDM Our results in the Saudi population are in completeagreement with the results reported by Fallucca et al [13]and Pappa et al [6] which indicate that the polymorphicallele R972 is significantly associated with IRS-1 and may beinvolved in the development of GDM In contrast Shaat etal [10] and Tok et al [24] did not observe any associationbetween the G972R polymorphism and GDM Although noassociation was observed in these studies Tok et al [24]reported that the variant was associated with higher fastingglucose and insulin levels in women with GDM Shaat etal [10] concluded that IRS-1 protein levels are reduced inthe adipose tissue of obese Scandinavian women with GDMOur findings established that homozygosity for the G972Rpolymorphism which was observed only in women withGDM might indicate an increased risk for GDM in Saudiwomen
In our study 90 of the women were following a dietmeant to regularize the levels of certain biochemical mark-ers before pregnancy Maintaining good nutrition duringpregnancy while consuming 2250ndash2500 calories daily isessential Physical exercise is also very important for GDMwomen especially those identified as either overweight orobese Insulin is the only medicationoption for pregnantwomen who develop GDM during pregnancy Insulin doesnot transfer to the baby through the placenta however theoral drug metformin (glyburide) can be used to circumventthis Ten percent of the women in our study used insulinThe biochemical profiles of GDM subjects were significantlyhigher than the biochemical profiles of non-GDM subjects(119875 lt 005)
In conclusion our data indicate that the IRS-1 G972Rvariant may facilitate the development of GDM Howeverthis variant was also associated with the baseline character-istics of the patients with GDM
Conflict of Interests
All the authors declare that there is no conflict of interestsregarding the publication of this paper
BioMed Research International 5
Acknowledgments
The authors would like to extend their sincere appreciationto the Deanship of Scientific Research at King Saud Uni-versity for its funding of this research through the ResearchGroup Project no RGP-VPP-244 The authors are grateful toBenjamin Vinodson for helping with the complete statisticalanalysis The authors are thankful to Mrs Virginia Rubio(Jean) and Mrs Laila H Assobah for helping with thecollection of blood samples
References
[1] C Zhang W Bao Y Rong et al ldquoGenetic variants and the riskof gestational diabetes mellitus a systematic reviewrdquo HumanReproduction Update vol 19 pp 376ndash390 2013
[2] H AWahabi S A Esmaeil A Fayed and R A Alzeidan ldquoGes-tational diabetes mellitus maternal and perinatal outcomes inKing Khalid University Hospital Saudi Arabiardquo The Journalof the Egyptian Public Health Association vol 88 pp 104ndash1082013
[3] H M Georgiou M Lappas G M Georgiou et al ldquoScreeningfor biomarkers predictive of gestational diabetes mellitusrdquo ActaDiabetologica vol 45 no 3 pp 157ndash165 2008
[4] S J Chon S Y Kim N R Cho D L Min Y J Hwang andM Mamura ldquoAssociation of variants in PPAR1205742 IGF2BP2 andKCNQ1 with a susceptibility to gestational diabetes mellitus ina Korean populationrdquo Yonsei Medical Journal vol 54 pp 352ndash357 2013
[5] Y Wang L Chen R Horswell et al ldquoRacial differences in theassociation between gestational diabetes mellitus and risk oftype 2 diabetesrdquo Journal ofWomenrsquos Health vol 21 pp 628ndash6332012
[6] K I Pappa M Gazouli K Economou et al ldquoGestationaldiabetes mellitus shares polymorphisms of genes associatedwith insulin resistance and type 2 diabetes in the GreekpopulationrdquoGynecological Endocrinology vol 27 no 4 pp 267ndash272 2011
[7] Y M Cho T H Kim S Lim et al ldquoType 2 diabetes-associatedgenetic variants discovered in the recent genome-wide associ-ation studies are related to gestational diabetes mellitus in theKorean populationrdquo Diabetologia vol 52 no 2 pp 253ndash2612009
[8] Y Tang X Han X Sun et al ldquoAssociation study of a commonvariant near IRS1 with type 2 diabetes mellitus in Chinese Hanpopulationrdquo Endocrine vol 43 pp 84ndash91 2013
[9] F Mousavinasab T Tahtinen J Jokelainen et al ldquoCommonpolymorphisms in the PPAR1205742 and IRS-1 genes and their inter-action influence serum adiponectin concentration in youngFinnish menrdquo Molecular Genetics and Metabolism vol 84 no4 pp 344ndash348 2005
[10] N Shaat M Ekelund A Lernmark et al ldquoAssociation ofthe E23K polymorphism in the KCNJ11 gene with gestationaldiabetes mellitusrdquo Diabetologia vol 48 no 12 pp 2544ndash25512005
[11] E Morini S Prudente E Succurro et al ldquoIRS1 G972R poly-morphism and type 2 diabetes a paradigm for the difficultascertainment of the contribution to disease susceptibility oflsquolow-frequency-low-riskrsquo variantsrdquo Diabetologia vol 52 no 9pp 1852ndash1857 2009
[12] M Bhat K N Ramesha S P Sarma C V Sowmini and SGanesh Kumar ldquoDeterminants of gestational diabetes mellitus
a case control study in a district tertiary care hospital in southIndiardquo International Journal of Diabetes inDeveloping Countriesvol 30 no 2 pp 91ndash96 2010
[13] F Fallucca M G Dalfra E Sciullo et al ldquoPolymorphisms ofinsulin receptor substrate 1 and 1205733-adrenergic receptor genesin gestational diabetes and normal pregnancyrdquoMetabolism vol55 no 11 pp 1451ndash1456 2006
[14] S M Shahida M A Islam S Begum M A Hossain and MS Azam ldquoMaternal outcome of grand multiparardquoMymensinghMedical Journal vol 20 no 3 pp 381ndash385 2011
[15] K Almind C Bjorbaek H Vestergaard T Hansen S Echwaldand O Pedersen ldquoAminoacid polymorphisms of insulin recep-tor substrate-1 in non-insulin-dependent diabetesmellitusrdquoTheLancet vol 342 no 8875 pp 828ndash832 1993
[16] X J Sun P Rothenberg C R Kahn et al ldquoStructure ofthe insulin receptor substrate IRS-1 defines a unique signaltransduction proteinrdquoNature vol 352 no 6330 pp 73ndash77 1991
[17] Y Kaburagi T Yamauchi R Yamamoto-Honda et al ldquoThemechanism of insulin-induced signal transductionmediated bythe insulin receptor substrate familyrdquo Endocrine Journal vol 46pp S25ndashS34 1999
[18] M A Williams C Qiu J C Dempsey and D A LuthyldquoFamilial aggregation of type 2 diabetes and chronic hyperten-sion in women with gestational diabetes mellitusrdquo Journal ofReproductiveMedicine for theObstetrician andGynecologist vol48 no 12 pp 955ndash962 2003
[19] L Groop ldquoPathogenesis of type 2 diabetes the relative con-tribution of insulin resistance and impaired insulin secretionrdquoInternational Journal of Clinical Practice vol 113 pp 3ndash13 2000
[20] L Groop and V Lyssenko ldquoGenes and type 2 diabetes mellitusrdquoCurrent Diabetes Reports vol 8 no 3 pp 192ndash197 2008
[21] N Shaat M Ekelund A Lernmark et al ldquoGenotypic andphenotypic differences between Arabian and Scandinavianwomen with gestational diabetes mellitusrdquoDiabetologia vol 47no 5 pp 878ndash884 2004
[22] M Alevizaki LThalassinou S I Grigorakis et al ldquoStudy of theTrp64Arg polymorphism ofthe 1205733-adrenergic receptor in greekwomen with gestational diabetesrdquo Diabetes Care vol 23 no 8pp 1079ndash1083 2000
[23] H Leipold M Knoefler C Gruber A Huber P Haslingerand C Worda ldquoPeroxisome proliferator-activated receptorgamma coactivator-1alpha gene variations are not associatedwith gestational diabetes mellitusrdquo Journal of the Society forGynecologic Investigation vol 13 pp 104ndash107 2006
[24] E C Tok D Ertunc O Bilgin E M Erdal M Kaplanogluand S Dilek ldquoAssociation of insulin receptor substrate-1 G972Rvariant with baseline characteristics of the patients with ges-tational diabetes mellitusrdquo American Journal of Obstetrics ampGynecology vol 194 pp 868ndash872 2006
and 0008 for the non-GDM group respectively The TTgenotype was absent in non-GDM groups The minor allelefrequencies of GDM and non-GDM were 3 and 0008respectively (119875 lt 005) Similarly the frequency of CC CTand TT genotypes of the Gly972Arg SNP was comparableamong the GDM and non-GDM (minor allele frequency119875 = 001 OR = 37 95 CI [13 105]) There were significantdifferences in the frequencies of the genotype distributionsof IRS-1 Gly972Arg between the GDM group and non-GDMgroup (TT + CT versus CC 119875 = 002 OR-34 95 CI [12101]) The odds ratio for any genotype of the Gly972Arg SNPwas significantly correlated with the risk of developing GDM(119875 lt 005) in the Saudi women monitored in this studyGenotype data is associated with HWE
4 Discussion
King Saud University is one of the foremost institute in theKingdom of Saudi Arabia as well as in an Arab countriesinvolved deeply in the research with human and molecularmedical geneticsThis is a case-control study performed with500 pregnant Saudi women including 200 GDM and 300non-GDM cases We investigated the relationship betweenthe IRS-1 G972R gene and GDM in a Saudi populationScreening and identifying GDM are based on biochemicalcriteria with serum sample despite the progress of geneticscreening methods There is no genetic test to identify GDMin women during pregnancy To the best of our knowledgethis is the first study conducted with pregnant Saudi womenIn this study we explored the effects of G972R of theIRS-1 gene variants The results of our study revealed thatthe variant allele Arg972 of the IRS-1 gene is significantlyassociated with GDM
Pregnancy is characterized by peripheral insulin resis-tance which is compensated by an increase in insulin secre-tion to maintain glucose homeostasis [13] GDM represents aheterogeneous disorder with both a genetic and an environ-mental component The prevalence of GDM in Saudi Arabiais 187 [2] According to additional studies women withGDMwere older and of higher parity and had a less favorablereproductive history than did nondiabetic pregnant womenThe independent association between high parity and GDMwas corroborated by previous reports [14] Advanced mater-nal age is independently associated with an increased riskof GDM [2] Several genetic polymorphisms of this genehave already been identified as they affect insulin action[15] The IRS-1 protein is a cytoplasmic molecule expressedin many insulin-sensitive tissues It plays an important rolein regulating the cellular effects of insulin [16] Tyrosinephosphorylation of IRS-1 proteins initiates the downstreameffects of insulin including activation of phosphatidylinositol3-kinase (PI3 K) and translocation of glucose transporter4 [17] The G972R polymorphism has also been associatedwith impaired 120573-cell function in NGT subjects as well aswith reduced insulin content and impaired insulin secretionin isolated human islets [10] Ample evidence suggests thatsusceptibility to GDM has a genetic component Althoughno studies have evaluated the heritability of GDM family
studies indicate that GDM aggregates within families andis associated with a history of T2DM [18] Genetic studiesof T2DM and GDM further suggest that it is a multigenicdisease in which common variants in multiple genes interactwith environmental factors to cause the disease [19 20]Because of the striking parallels between GDM and T2DMit is likely that GDM is also a multigenic disease relatedto T2DM Thus recent studies on the etiology of GDMhave begun to evaluate the role of common polymorphismsin genes involved in T2DM predisposition So far geneticpredisposition to GDM has been reported for variations ingenes involved in insulin resistance and insulin secretionHowever no association could be replicated in differentstudies [21ndash23] These discrepancies could be due to lack ofpower given the small effect size of most common variantsor to ethnic heterogeneity among different populations
Studies conducted by Pappa et al [6] in the Greek popu-lation with 148 women with GDM and 107 control subjectsand the results indicate that the G972R polymorphism of theIRS-1 gene is strongly associated with increased susceptibilityto GDM Our results in the Saudi population are in completeagreement with the results reported by Fallucca et al [13]and Pappa et al [6] which indicate that the polymorphicallele R972 is significantly associated with IRS-1 and may beinvolved in the development of GDM In contrast Shaat etal [10] and Tok et al [24] did not observe any associationbetween the G972R polymorphism and GDM Although noassociation was observed in these studies Tok et al [24]reported that the variant was associated with higher fastingglucose and insulin levels in women with GDM Shaat etal [10] concluded that IRS-1 protein levels are reduced inthe adipose tissue of obese Scandinavian women with GDMOur findings established that homozygosity for the G972Rpolymorphism which was observed only in women withGDM might indicate an increased risk for GDM in Saudiwomen
In our study 90 of the women were following a dietmeant to regularize the levels of certain biochemical mark-ers before pregnancy Maintaining good nutrition duringpregnancy while consuming 2250ndash2500 calories daily isessential Physical exercise is also very important for GDMwomen especially those identified as either overweight orobese Insulin is the only medicationoption for pregnantwomen who develop GDM during pregnancy Insulin doesnot transfer to the baby through the placenta however theoral drug metformin (glyburide) can be used to circumventthis Ten percent of the women in our study used insulinThe biochemical profiles of GDM subjects were significantlyhigher than the biochemical profiles of non-GDM subjects(119875 lt 005)
In conclusion our data indicate that the IRS-1 G972Rvariant may facilitate the development of GDM Howeverthis variant was also associated with the baseline character-istics of the patients with GDM
Conflict of Interests
All the authors declare that there is no conflict of interestsregarding the publication of this paper
BioMed Research International 5
Acknowledgments
The authors would like to extend their sincere appreciationto the Deanship of Scientific Research at King Saud Uni-versity for its funding of this research through the ResearchGroup Project no RGP-VPP-244 The authors are grateful toBenjamin Vinodson for helping with the complete statisticalanalysis The authors are thankful to Mrs Virginia Rubio(Jean) and Mrs Laila H Assobah for helping with thecollection of blood samples
References
[1] C Zhang W Bao Y Rong et al ldquoGenetic variants and the riskof gestational diabetes mellitus a systematic reviewrdquo HumanReproduction Update vol 19 pp 376ndash390 2013
[2] H AWahabi S A Esmaeil A Fayed and R A Alzeidan ldquoGes-tational diabetes mellitus maternal and perinatal outcomes inKing Khalid University Hospital Saudi Arabiardquo The Journalof the Egyptian Public Health Association vol 88 pp 104ndash1082013
[3] H M Georgiou M Lappas G M Georgiou et al ldquoScreeningfor biomarkers predictive of gestational diabetes mellitusrdquo ActaDiabetologica vol 45 no 3 pp 157ndash165 2008
[4] S J Chon S Y Kim N R Cho D L Min Y J Hwang andM Mamura ldquoAssociation of variants in PPAR1205742 IGF2BP2 andKCNQ1 with a susceptibility to gestational diabetes mellitus ina Korean populationrdquo Yonsei Medical Journal vol 54 pp 352ndash357 2013
[5] Y Wang L Chen R Horswell et al ldquoRacial differences in theassociation between gestational diabetes mellitus and risk oftype 2 diabetesrdquo Journal ofWomenrsquos Health vol 21 pp 628ndash6332012
[6] K I Pappa M Gazouli K Economou et al ldquoGestationaldiabetes mellitus shares polymorphisms of genes associatedwith insulin resistance and type 2 diabetes in the GreekpopulationrdquoGynecological Endocrinology vol 27 no 4 pp 267ndash272 2011
[7] Y M Cho T H Kim S Lim et al ldquoType 2 diabetes-associatedgenetic variants discovered in the recent genome-wide associ-ation studies are related to gestational diabetes mellitus in theKorean populationrdquo Diabetologia vol 52 no 2 pp 253ndash2612009
[8] Y Tang X Han X Sun et al ldquoAssociation study of a commonvariant near IRS1 with type 2 diabetes mellitus in Chinese Hanpopulationrdquo Endocrine vol 43 pp 84ndash91 2013
[9] F Mousavinasab T Tahtinen J Jokelainen et al ldquoCommonpolymorphisms in the PPAR1205742 and IRS-1 genes and their inter-action influence serum adiponectin concentration in youngFinnish menrdquo Molecular Genetics and Metabolism vol 84 no4 pp 344ndash348 2005
[10] N Shaat M Ekelund A Lernmark et al ldquoAssociation ofthe E23K polymorphism in the KCNJ11 gene with gestationaldiabetes mellitusrdquo Diabetologia vol 48 no 12 pp 2544ndash25512005
[11] E Morini S Prudente E Succurro et al ldquoIRS1 G972R poly-morphism and type 2 diabetes a paradigm for the difficultascertainment of the contribution to disease susceptibility oflsquolow-frequency-low-riskrsquo variantsrdquo Diabetologia vol 52 no 9pp 1852ndash1857 2009
[12] M Bhat K N Ramesha S P Sarma C V Sowmini and SGanesh Kumar ldquoDeterminants of gestational diabetes mellitus
a case control study in a district tertiary care hospital in southIndiardquo International Journal of Diabetes inDeveloping Countriesvol 30 no 2 pp 91ndash96 2010
[13] F Fallucca M G Dalfra E Sciullo et al ldquoPolymorphisms ofinsulin receptor substrate 1 and 1205733-adrenergic receptor genesin gestational diabetes and normal pregnancyrdquoMetabolism vol55 no 11 pp 1451ndash1456 2006
[14] S M Shahida M A Islam S Begum M A Hossain and MS Azam ldquoMaternal outcome of grand multiparardquoMymensinghMedical Journal vol 20 no 3 pp 381ndash385 2011
[15] K Almind C Bjorbaek H Vestergaard T Hansen S Echwaldand O Pedersen ldquoAminoacid polymorphisms of insulin recep-tor substrate-1 in non-insulin-dependent diabetesmellitusrdquoTheLancet vol 342 no 8875 pp 828ndash832 1993
[16] X J Sun P Rothenberg C R Kahn et al ldquoStructure ofthe insulin receptor substrate IRS-1 defines a unique signaltransduction proteinrdquoNature vol 352 no 6330 pp 73ndash77 1991
[17] Y Kaburagi T Yamauchi R Yamamoto-Honda et al ldquoThemechanism of insulin-induced signal transductionmediated bythe insulin receptor substrate familyrdquo Endocrine Journal vol 46pp S25ndashS34 1999
[18] M A Williams C Qiu J C Dempsey and D A LuthyldquoFamilial aggregation of type 2 diabetes and chronic hyperten-sion in women with gestational diabetes mellitusrdquo Journal ofReproductiveMedicine for theObstetrician andGynecologist vol48 no 12 pp 955ndash962 2003
[19] L Groop ldquoPathogenesis of type 2 diabetes the relative con-tribution of insulin resistance and impaired insulin secretionrdquoInternational Journal of Clinical Practice vol 113 pp 3ndash13 2000
[20] L Groop and V Lyssenko ldquoGenes and type 2 diabetes mellitusrdquoCurrent Diabetes Reports vol 8 no 3 pp 192ndash197 2008
[21] N Shaat M Ekelund A Lernmark et al ldquoGenotypic andphenotypic differences between Arabian and Scandinavianwomen with gestational diabetes mellitusrdquoDiabetologia vol 47no 5 pp 878ndash884 2004
[22] M Alevizaki LThalassinou S I Grigorakis et al ldquoStudy of theTrp64Arg polymorphism ofthe 1205733-adrenergic receptor in greekwomen with gestational diabetesrdquo Diabetes Care vol 23 no 8pp 1079ndash1083 2000
[23] H Leipold M Knoefler C Gruber A Huber P Haslingerand C Worda ldquoPeroxisome proliferator-activated receptorgamma coactivator-1alpha gene variations are not associatedwith gestational diabetes mellitusrdquo Journal of the Society forGynecologic Investigation vol 13 pp 104ndash107 2006
[24] E C Tok D Ertunc O Bilgin E M Erdal M Kaplanogluand S Dilek ldquoAssociation of insulin receptor substrate-1 G972Rvariant with baseline characteristics of the patients with ges-tational diabetes mellitusrdquo American Journal of Obstetrics ampGynecology vol 194 pp 868ndash872 2006
The authors would like to extend their sincere appreciationto the Deanship of Scientific Research at King Saud Uni-versity for its funding of this research through the ResearchGroup Project no RGP-VPP-244 The authors are grateful toBenjamin Vinodson for helping with the complete statisticalanalysis The authors are thankful to Mrs Virginia Rubio(Jean) and Mrs Laila H Assobah for helping with thecollection of blood samples
References
[1] C Zhang W Bao Y Rong et al ldquoGenetic variants and the riskof gestational diabetes mellitus a systematic reviewrdquo HumanReproduction Update vol 19 pp 376ndash390 2013
[2] H AWahabi S A Esmaeil A Fayed and R A Alzeidan ldquoGes-tational diabetes mellitus maternal and perinatal outcomes inKing Khalid University Hospital Saudi Arabiardquo The Journalof the Egyptian Public Health Association vol 88 pp 104ndash1082013
[3] H M Georgiou M Lappas G M Georgiou et al ldquoScreeningfor biomarkers predictive of gestational diabetes mellitusrdquo ActaDiabetologica vol 45 no 3 pp 157ndash165 2008
[4] S J Chon S Y Kim N R Cho D L Min Y J Hwang andM Mamura ldquoAssociation of variants in PPAR1205742 IGF2BP2 andKCNQ1 with a susceptibility to gestational diabetes mellitus ina Korean populationrdquo Yonsei Medical Journal vol 54 pp 352ndash357 2013
[5] Y Wang L Chen R Horswell et al ldquoRacial differences in theassociation between gestational diabetes mellitus and risk oftype 2 diabetesrdquo Journal ofWomenrsquos Health vol 21 pp 628ndash6332012
[6] K I Pappa M Gazouli K Economou et al ldquoGestationaldiabetes mellitus shares polymorphisms of genes associatedwith insulin resistance and type 2 diabetes in the GreekpopulationrdquoGynecological Endocrinology vol 27 no 4 pp 267ndash272 2011
[7] Y M Cho T H Kim S Lim et al ldquoType 2 diabetes-associatedgenetic variants discovered in the recent genome-wide associ-ation studies are related to gestational diabetes mellitus in theKorean populationrdquo Diabetologia vol 52 no 2 pp 253ndash2612009
[8] Y Tang X Han X Sun et al ldquoAssociation study of a commonvariant near IRS1 with type 2 diabetes mellitus in Chinese Hanpopulationrdquo Endocrine vol 43 pp 84ndash91 2013
[9] F Mousavinasab T Tahtinen J Jokelainen et al ldquoCommonpolymorphisms in the PPAR1205742 and IRS-1 genes and their inter-action influence serum adiponectin concentration in youngFinnish menrdquo Molecular Genetics and Metabolism vol 84 no4 pp 344ndash348 2005
[10] N Shaat M Ekelund A Lernmark et al ldquoAssociation ofthe E23K polymorphism in the KCNJ11 gene with gestationaldiabetes mellitusrdquo Diabetologia vol 48 no 12 pp 2544ndash25512005
[11] E Morini S Prudente E Succurro et al ldquoIRS1 G972R poly-morphism and type 2 diabetes a paradigm for the difficultascertainment of the contribution to disease susceptibility oflsquolow-frequency-low-riskrsquo variantsrdquo Diabetologia vol 52 no 9pp 1852ndash1857 2009
[12] M Bhat K N Ramesha S P Sarma C V Sowmini and SGanesh Kumar ldquoDeterminants of gestational diabetes mellitus
a case control study in a district tertiary care hospital in southIndiardquo International Journal of Diabetes inDeveloping Countriesvol 30 no 2 pp 91ndash96 2010
[13] F Fallucca M G Dalfra E Sciullo et al ldquoPolymorphisms ofinsulin receptor substrate 1 and 1205733-adrenergic receptor genesin gestational diabetes and normal pregnancyrdquoMetabolism vol55 no 11 pp 1451ndash1456 2006
[14] S M Shahida M A Islam S Begum M A Hossain and MS Azam ldquoMaternal outcome of grand multiparardquoMymensinghMedical Journal vol 20 no 3 pp 381ndash385 2011
[15] K Almind C Bjorbaek H Vestergaard T Hansen S Echwaldand O Pedersen ldquoAminoacid polymorphisms of insulin recep-tor substrate-1 in non-insulin-dependent diabetesmellitusrdquoTheLancet vol 342 no 8875 pp 828ndash832 1993
[16] X J Sun P Rothenberg C R Kahn et al ldquoStructure ofthe insulin receptor substrate IRS-1 defines a unique signaltransduction proteinrdquoNature vol 352 no 6330 pp 73ndash77 1991
[17] Y Kaburagi T Yamauchi R Yamamoto-Honda et al ldquoThemechanism of insulin-induced signal transductionmediated bythe insulin receptor substrate familyrdquo Endocrine Journal vol 46pp S25ndashS34 1999
[18] M A Williams C Qiu J C Dempsey and D A LuthyldquoFamilial aggregation of type 2 diabetes and chronic hyperten-sion in women with gestational diabetes mellitusrdquo Journal ofReproductiveMedicine for theObstetrician andGynecologist vol48 no 12 pp 955ndash962 2003
[19] L Groop ldquoPathogenesis of type 2 diabetes the relative con-tribution of insulin resistance and impaired insulin secretionrdquoInternational Journal of Clinical Practice vol 113 pp 3ndash13 2000
[20] L Groop and V Lyssenko ldquoGenes and type 2 diabetes mellitusrdquoCurrent Diabetes Reports vol 8 no 3 pp 192ndash197 2008
[21] N Shaat M Ekelund A Lernmark et al ldquoGenotypic andphenotypic differences between Arabian and Scandinavianwomen with gestational diabetes mellitusrdquoDiabetologia vol 47no 5 pp 878ndash884 2004
[22] M Alevizaki LThalassinou S I Grigorakis et al ldquoStudy of theTrp64Arg polymorphism ofthe 1205733-adrenergic receptor in greekwomen with gestational diabetesrdquo Diabetes Care vol 23 no 8pp 1079ndash1083 2000
[23] H Leipold M Knoefler C Gruber A Huber P Haslingerand C Worda ldquoPeroxisome proliferator-activated receptorgamma coactivator-1alpha gene variations are not associatedwith gestational diabetes mellitusrdquo Journal of the Society forGynecologic Investigation vol 13 pp 104ndash107 2006
[24] E C Tok D Ertunc O Bilgin E M Erdal M Kaplanogluand S Dilek ldquoAssociation of insulin receptor substrate-1 G972Rvariant with baseline characteristics of the patients with ges-tational diabetes mellitusrdquo American Journal of Obstetrics ampGynecology vol 194 pp 868ndash872 2006
