Genetic variation in exon 17 of INSR is associated with insulin resistance and hyperandrogenemia among lean Indian women with polycystic ovary syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a multigenic disorder, and insulin resistance is one of its hallmark features. Polymorphisms in exon 17 of insulin receptor (INSR) gene are reported to be associated with PCOS. We investigated this association in Indian women and its putative relationship with PCOS associated traits, which has not been explored so far.
In this case control study, the polymorphisms were investigated by direct sequencing in 180 women with PCOS and 144 age matched controls. Clinical, anthropometric, biochemical, and hormonal parameters were also estimated.
The silent C/T polymorphism at His1058 in exon 17 of INSR was found to be present in our study population. The polymorphic genotype (CT+TT) was significantly associated with PCOS in lean women (chi(2)=8.493, df=1, P=0.004). It showed association with higher fasting insulin levels (P=0.02), homeostasis model assessment of insulin resistance (P=0.005), free androgen index (P=0.03), and lower quantitative insulin sensitivity check index (P=0.004) in lean PCOS women. No other novel or known polymorphism was identified in exon 17 in this cohort.
The study shows significant association of C/T polymorphism at His1058 of INSR with PCOS in the lean rather than obese Indian women. Its association with indices of insulin resistance and hyperandrogenemia is also seen in the same group. The findings strengthen the concept that pathogenesis of PCOS is different in lean and obese women.

Full text

CLINICAL STUDY
Genetic variation in exon 17 of INSR is associated with insulin
resistance and hyperandrogenemia among lean Indian women
with polycystic ovary syndrome
Srabani Mukherjee, Nuzhat Shaikh, Sushma Khavale, Gayatri Shinde, Pervin Meherji, Nalini Shah
1
and Anurupa Maitra
Department of Molecular Endocrinology, National Institute for Research in Reproductive Health, Indian Council of Medical Research (ICMR), Jehangir
Merwanji Street, Parel, Mumbai 400012, India, and
1
Seth G S Medical College and KEM Hospital, Parel, Mumbai, 400012, India
(Correspondence should be addressed to S Mukherjee; Email: mukherjees@nirrh.res.in)
Abstract
Objective: Polycystic ovary syndrome (PCOS) is a multigenic disorder, and insulin resistance is one of its
hallmark features. Polymorphisms in exon 17 of insulin receptor (INSR) gene are reported to be
associated with PCOS. We investigated this association in Indian women and its putative relationship
with PCOS associated traits, which has not been explored so far.
Methods: In this case control study, the polymorphisms were investigated by direct sequencing in
180 women with PCOS and 144 age matched controls. Clinical, anthropometric, biochemical, and
hormonal parameters were also estimated.
Results: The silent C/T polymorphism at His1058 in exon 17 of INSR was found to be present in our
study population. The polymorphic genotype (CTCTT) was significantly associated with PCOS in lean
women (c
2
Z8.493, dfZ1, PZ0.004). It showed association with higher fasting insulin levels
(PZ0.02), homeostasis model assessment of insulin resistance (PZ0.005), free androgen index
(PZ0.03), and lower quantitative insulin sensitivity check index (PZ0.004) in lean PCOS women. No
other novel or known polymorphism was identified in exon 17 in this cohort.
Conclusions: The study shows significant association of C/T polymorphism at His1058 of INSR with
PCOS in the lean rather than obese Indian women. Its association with indices of insulin resistance and
hyperandrogenemia is also seen in the same group. The findings strengthen the concept that
pathogenesis of PCOS is different in lean and obese women.
European Journal of Endocrinology 160 855–862
Introduction
Polycystic ovary syndrome (PCOS) is a heterogeneous
disorder with a prevalence of w7% in women of
childbearing age (1). It is the most common cause of
anovulatory infertility with characteristic mani-
festations such as menstrual irregularities; signs of
androgen excess like hirsutism, acne, alopecia; altered
LH:FSH ratio (O2:1) and polycystic ovaries (2–4). The
majority of women with PCOS have insulin resistance,
which plays a key role in its pathogenesis (5, 6).In
addition, it increases the risk of developing glucose
intolerance, type 2 diabetes mellitus, hypertension, and
dyslipidaemia, which can lead to cardiovascular
disorders in later life (7–10). Both obese and lean
women with PCOS manifest insulin resistance (2) and
obesity further contributes an additional component of
insulin resistance (5). Insulin resistance progresses
towards compensatory hyperinsulinemia, which pro-
motes both hyperandrogenemia and anovulation in
PCOS (1, 11).
Familial clustering seen in this syndrome suggests
contribution of a genetic component to its pathogenesis
(12, 13). Over the past decade, a number of candidate
genes involved in steroidogenesis, insulin signaling
pathway, gonadotropin secretion and chronic inflam-
mation have been explored to identify the susceptibility
genes for PCOS (14, 15); however, the results so far have
been inconclusive.
Available evidence suggests insulin resistance in
PCOS could be due to post-binding defects in insulin
signaling (2, 16, 17). Hence, insulin receptor (INSR),
being an integral part of insulin signaling could be a
potential candidate gene. INSR is located on chromo-
some 19 and encompasses 22 exons. Linkage analysis
studies that have found an association of the micro-
satellite marker D19S884 which is located on chromo-
some 19p13.2 and relatively close (1 cM) to INSR with
PCOS, emphasize it to be a candidate gene (18, 19).
Other studies showed that the number and affinity of
insulin receptor is not altered in PCOS but its tyrosine
phosphorylation status and subsequent signaling is
European Journal of Endocrinology (2009) 160 855–862 ISSN 0804-4643
q2009 European Society of Endocrinology DOI: 10.1530/EJE-08-0932
Online version via www.eje-online.org

affected, suggesting the defect may lie in the b-chain
(16, 20). Studies to determine mutations in INSR failed
to find any major variations; however, several poly-
morphisms were identified (21–28). The most frequent
of these were at exon 17, which encodes the partial
tyrosine kinase domain containing the ATP binding
site of INSR, important for its downstream signaling
(21–28). Among these polymorphisms, a C/T SNP
at His1058 in exon 17 has been reported to be
significantly associated with PCOS in two independent
studies in Caucasian and Chinese women (25, 26).
A subsequent study from Korea however, failed to
confirm this association (27). Also, the possible
relationship between this polymorphism and PCOS
associated traits has not been explored so far. In
another study, a novel T/C polymorphism at Cys1008
in exon 17 of INSR was found to be associated with
PCOS and insulin resistance, which is yet to be verified
independently (28). Nevertheless, these studies do
suggest that the genetic variants in exon 17 of INSR
may have an association with PCOS or its pathophy-
siology. Thus, in the present study, we sought to
evaluate the association of genetic variations in exon
17 of INSR with PCOS and its related traits in Indian
women, who are ethnically distinct from the popu-
lations already studied.
Subjects and methods
Subjects
A total of 180 Indian women with PCOS, aged 17–39
years were recruited consecutively from the Infertility
Clinic of the National Institute for Research in
Reproductive Health (NIRRH) as well as the Endo-
crinology Clinic of Seth GS Medical College and KEM
Hospital, Mumbai, India. The diagnosis of PCOS was
defined by ESHRE/ASRM consensus criteria, including
presence of at least two of the following three features:
i) oligomenorrhoea and/or anovulation, ii) clinical
and/or biochemical signs of hyperandrogenemia, and
iii) polycystic ovaries on ultrasound (29). Other related
disorders like non-classical congenital adrenal hyper-
plasia, thyroid dysfunction, androgen secreting tumors,
and hyperprolactinemia were excluded. One hundred
and fifty healthy Indian women, aged 17–38 years, with
regular menstrual cycles and no clinical and/or
biochemical signs of hyperandrogenemia or polycystic
ovaries were carefully selected for the study as the
control group from the general population. On detailed
biochemical evaluation, six control women were
excluded mainly due to abnormal thyroid status. The
women in the control and PCOS groups were genetically
unrelated. Women with diabetes were excluded from the
study. None of the subjects had taken any medication
known to affect carbohydrate and lipid metabolism or
endocrine parameters for at least 3 months prior to
entering the study. Ethical committee approval from
both institutions and written informed consent from all
participants were obtained.
Clinical and laboratory parameters
The anthropometric data – height, weight, body mass
index (BMI), waist, and hip circumference, waist to hip
ratio were obtained from all subjects. Blood samples were
collected between 3–7 days of the menstrual cycle (early
follicular phase) or during amenorrhea and serum was
stored at K80 8C until assayed. After overnight fasting,
each subject underwent an oral glucose tolerance test.
Glucose and insulin levels were measured at 0 min and
2 h after 75 g glucose load. Fasting serum were used to
measure total testosterone, androstenedione, sex hor-
mone binding globulin (SHBG), LH, FSH, TSH, T3, T4,
and prolactin levels. Glucose was measured enzy-
matically by glucose oxidase method. TSH, T3, T4,
FSH, LH, and prolactin were measured by chemi-
luminescence immunoassay (Immulite 1000, Llanberis,
UK). Serum insulin, total testosterone, androstenedione,
and SHBG levels were measured using commercial kits
(DSL-1600 RIA kit, DSL-4000 RIA kit, DSL-3800 RIA
kit and DSL-7400 IRMA kit respectively from Diagnostic
System Laboratories, Webster, TX, USA). Free testoster-
one (active free testosterone in circulation) and bioavail-
able testosterone (free plus weakly bound to albumin)
was calculated from total testosterone and SHBG values
using a web-based calculator (http://www.issam.ch/
freetesto.htm)(30). Free-androgen index (FAI) was
assessed by the following formula: total testosterone
(nmol/l)/SHBG (nmol/l) !100 (31). Insulin resistance
was evaluated by the homeostasis model assessment of
insulin resistance (HOMA-IR) using the following
formula: fasting plasma glucose (mmol/l) !fasting
insulin (mU/ml)/ 22.5) (32). Insulin sensitivity was
assessed by quantitative insulin sensitivity check index
(QUICKI), defined as 1/(log fasting serum insulin
(mU/ml) Clog fasting plasma glucose (mg/dl)) (33).
Obesity was defined as BMI R23 kg/m
2
based on Indian
norms (34), accordingly, subjects were categorized as
lean (BMI !23 kg/m
2
) and obese (BMI R23 kg/m
2
).
Genetic analysis
Genomic DNA was extracted from peripheral whole
blood by QIAamp DNA Blood Mini Kit (Qiagen GmbH).
Exon 17 was amplified using the following primers:
50-CCAAGGATGCTGTGTAGATAAG-30and 50-TCAG-
GAAAGCCAGCCCATGTC-30according to Siegel et al.
with some modifications (25). The amplifications were
performed using 0.5 mg genomic DNA in a final volume
of 50 ml containing 1.5 mM MgCl
2
, 200 mM of each
dNTPs, 10 pmol of each primer and 0.5 U of Taq
Polymerase (Fermentas, Vilnius, Lithuania). After initial
denaturation for 5 min at 94 8C, 35 cycles of amplifica-
tions were carried out at 94 8C for 45 s, 56 8C for 40 s,
856 S Mukherjee and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2009) 160
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and at 72 8C for 1 min with a final extension of 10 min
at 72 8C. The resulting amplified product was of 317 bp,
which was purified by QIAquick Gel Extraction Kit,
(Qiagen GmbH) and subjected to direct sequencing in
3130xl Genetic Analyzer using Big Dye Terminator
Chemistry (Applied Biosystems, Foster City, USA).
Statistical analysis
Univariate comparison of all continuous variables
between the PCOS and control groups were done by
unpaired t-tests. Results are expressed as meanGS.D.
The association between genotype and PCOS was
analyzed using c
2
test. Stratified analysis by obesity
(i.e., lean BMI !23 kg/m
2
and obese BMI R23 kg/m
2
)
was also performed. Variables that were significant at the
univariate level were further assessed in multivariable
analysis. Analysis of covariance (ANCOVA) was used to
compare hormonal profiles between genotypes after
adjusting for age and BMI. Statistical significance was
based on a P!0.05. Statistical analysis was performed
by using SAS version 9.2 software packages.
Results
The clinical, anthropometric, and hormonal parameters
of control and PCOS women per se and as per obesity
status have been depicted in Table 1. The majority of the
women with PCOS were oligomenorrhoeic (55%,
99/180), whereas 38.9% (70/180) were amenorrhoeic
and the rest 6.1% (11/180) were regularly cycling. As
regards clinical signs of hyperandrogenism, 53.3%
(96/180) of PCOS women presented with hirsutism,
36.1% (65/180) with acne and 16% (29/180) with
mild alopecia.
The control and PCOS women were of similar mean
age but did differ significantly with respect to
anthropometric parameters (P!0.05) (Table 1). The
PCOS women had significantly higher levels of fasting
and 2 h glucose and fasting insulin compared with
controls, although withinnormalrange.Insulin
resistance index like HOMA-IR (P!0.0001) was
elevated and insulin sensitivity as measured by QUICKI
(vs P!0.0001) was low in PCOS women (Table 1).
Even after classifying control and PCOS women in lean
and obese groups, the BMI, 2 h glucose level, fasting
insulin level, and HOMA-IR remained higher, whereas
QUICKI was lower in both PCOS groups compared with
controls. Acanthosis nigricans, a sign of insulin
resistance, was present in 23.3% of PCOS women
(42/180).
Women with PCOS were more hyperandrogenic
exhibiting higher levels of total, free, and bioavailable
testosterone, androstenedione, and increased FAI values
(P!0.0001). The difference between these hormones
was more evident in the obese rather than in the lean
group (Table 1). SHBG levels were significantly lower
in obese PCOS women (P!0.0001), which is common
in obesity and insulin resistance state (35),with
concurrent high free and bioavailable testosterone
(P!0.0001) and FAI (P!0.0001).
Analysis of exon 17 of INSR by direct sequencing
revealed presence of one silent polymorphism, a C/T
SNP at His1058 site. No other known or novel
polymorphism in this region was detected in this
cohort. The genotype distributions of this SNP were in
Hardy–Weinberg equilibrium. In our study cohort,
52.8% (76/144) of controls and 43.9% (79/180) of
PCOS were CC; 38.9% (56/144) of controls and 42.8%
(77/180) of PCOS were CT and 8.3% (12/144) controls
and 13.3% (24/180) PCOS women were TT. The
frequencies of CT and TT genotypes were relatively
high but not significantly different in PCOS women
compared with controls (c
2
Z3.416, dfZ2, PZ0.181).
PCOS women are frequently obese which also
contributes to insulin resistance. To understand the
relationship of this INSR polymorphism with obesity,
the distribution of these genotypes in lean and obese
groups were evaluated. The frequency of CTCTT
genotype was significantly higher in lean PCOS group
(69.3%, 52/75) compared with lean control group
(46.4%, 39/84) (c
2
Z8.493, dfZ1, PZ0.004)
(Table 1). As the number of subjects with TT genotype
was low in some subgroups, CT and TT genotypes were
combined together as CTCTT genotype.
To understand the influence of this polymorphism on
PCOS related traits, indices of insulin resistance and
hyperandrogenemia in both lean and obese groups of
PCOS and controls were analyzed (Tables 2 and 3). The
carriers of CC genotype did not differ significantly from
polymorphic (CTCTT) genotype in lean control, obese
control, and also in obese PCOS group, in terms of waist
circumference, BMI, fasting glucose levels, fasting
insulin levels and insulin resistance indices (HOMA-IR
and QUICKI). Additionally, total testosterone levels, free
and bioavailable testosterone, androstenedione, and FAI
were also similar within these groups. Interestingly, the
lean PCOS subgroup, with polymorphic genotype
showed a significantly higher BMI (20.48G1.77 vs
19.15G2.07 kg/m
2
,PZ0.005) and waist circumfer-
ence (73.24G6.43 vs 69.89G5.64 cm, PZ0.03)
compared with CC genotype. Furthermore, the indices
of insulin resistance were significantly different between
women with polymorphic and wildtype genotypes in the
same group, showing higher fasting insulin levels
(11.43G5.22 vs 8.32G2.70 uIU/ml, PZ0.009) and
HOMA-IR (2.40G1.10 vs 1.64G0.54,PZ0.002) and
lower QUICKI values (0.340G0.021 vs 0.357G0.018,
PZ0.002) in the polymorphic subgroup (Table 2). Also
regarding androgen parameters, these women with
polymorphic genotype exhibited elevated free testoster-
one (22.81G9.91 vs 17.95G6.87 pmol/l, PZ0.04),
bioavailable testosterone (0.54G0.23 vs 0.42G
0.16 nmol/l, PZ0.04), and FAI (2.94G1.50 vs
2.16G0.82, PZ0.02; Table 2). The variables that
Insulin receptor gene polymorphism in PCOS 857EUROPEAN JOURNAL OF ENDOCRINOLOGY (2009) 160
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Table 1 Clinical and hormonal profile of control and polycystic ovary syndrome (PCOS) women.
Variables
All women Lean women (nZ159) Obese women (nZ165)
Control (nZ144) PCOS (nZ180) PControl (nZ84) PCOS (nZ75) PControl (nZ60) PCOS (nZ105) P
Age (years) 24.94G5.46 24.82G5.26 0.84 23.42G4.8 23.28G4.25 0.85 27.12G5.58 25.92G5.64 0.19
Body weight (kg) 53.42G10.44 58.59G14.04 0.0003 47.00G7.23 47.05G6.10 1 62.71G6.90 66.98G12.04 0.01
BMI (kg/m
2
) 22.16G4.11 25.01G5.63 !0.0001 19.31G2.34 20.07G1.95 0.03 26.20G2.27 28.53G4.67 0.0004
Waist (cm) 76.36G9.05 82.02G12.02 !0.0001 71.73G7.5 72.21G6.35 0.72 82.86G6.40 89.02G10.09 !0.0001
WHR 0.80G0.05 0.82G0.05 !0.0001 0.78G0.05 0.80G0.05 0.06 0.81G0.04 0.83G0.05 0.0006
FBS (mg/dl) 83.36G7.96 86.70G10.47 0.002 82.94G7.26 85.11G9.46 0.11 83.97G8.88 87.83G11.03 0.02
Two-hour glucose (mg/dl) 92.80G14.98 103.5G21.72 !0.0001 90.69G14.22 96.72G18.97 0.02 95.80G15.63 108.3G22.35 0.0002
Fasting insulin (uIU/ml) 8.96G3.32 13.68G7.26 !0.0001 8.15G2.89 10.54G4.81 0.0002 10.10G3.57 15.92G7.88 !0.0001
HOMA-IR 1.84G0.70 2.84G1.60 !0.0001 1.66G0.59 2.18G1.02 0.0001 2.09G0.75 3.31G1.75 !0.0001
QUICKI 0.353G0.021 0.335G0.026 !0.0001 0.358G0.021 0.345G0.022 0.0003 0.346G0.020 0.327G0.026 !0.0001
FSH (mU/ml) 6.63G1.86 6.56G1.98 0.68 6.78G2.17 6.41G2.08 0.28 6.67G1.34 6.67G1.96 1
LH (mU/ml) 4.93G1.83 9.93G4.57 !0.0001 5.19G1.96 10.95G4.63 !0.0001 4.56G1.57 9.20G4.41 !0.0001
LH: FSH 0.76G0.31 1.66G0.90 !0.0001 0.79G0.29 1.85G0.91 !0.0001 0.73G0.34 1.52G0.86 !0.0001
TT (ng/dl) 49.11G14.52 74.79G31.18 !0.0001 48.55G15.94 68.97G25.59 !0.0001 49.92G12.29 78.94G34.14 !0.0001
Bio-T (nmol/l) 0.35G0.13 0.69G0.39 !0.0001 0.33G0.13 0.50G0.22 !0.0001 0.37G0.12 0.82G0.43 !0.0001
FT (pmol/l) 14.37G5.48 29.41G16.54 !0.0001 13.78G5.52 21.46G9.34 !0.0001 15.20G5.36 35.08G18.18 !0.0001
FAI 1.82G0.76 4.20G2.96 !0.0001 1.75G0.78 2.73G1.38 !0.0001 1.91G0.71 5.24G3.32 !0.0001
SHBG (nmol/l) 102.4G35.10 79.48G39.52 !0.0001 105.7G34.11 97.1G32.56 0.11 99.03G36.73 66.86G39.34 !0.0001
Androstenedione (ng/ml) 1.05G0.56 2.26G1.23 !0.0001 1.09G0.54 2.04G1.04 !0.0001 1.01G0.56 2.36G1.17 !0.0001
Genotype (% of CC) 52.8 43.9 0.11* 53.6 30.7 0.004* 51.7 53.3 0.87*
Data are given as meanGS.D., BMI, body mass index; WHR, waist-to-hip ratio; FBS, fasting glucose; HOMA-IR, homeostasis model assessment for insulin resistance; QUICKI, quantitative insulin sensitivity
check index; TT, total testosterone; FT, free testosterone; Bio-T, bioavailable testosterone; SHBG, sex hormone binding globulin and FAI, free androgen index. Comparison was done by unpaired t-test. P!0.05
is statistically significant. Genotype is presented as percent of CC cases in that category. *The P-values for genotypes are from c
2
-test.
858 S Mukherjee and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2009) 160
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showed significant association with genotype in uni-
variate analysis were further evaluated by ANCOVA
model, in the lean PCOS group. In multivariate analysis
after adjusting for age and BMI, the difference in fasting
insulin (PZ0.02), HOMA-IR (PZ0.005), QUICKI
(PZ0.004) and FAI (PZ0.03) between polymorphic
and wildtype genotype remained significant, whereas
free and bioavailable testosterone were marginally
significant (Table 2).
Discussion
To the best of our knowledge, this is the first study from
India, reporting the presence of the C/T polymorphism
at His1058 in exon 17 of INSR. The polymorphism
showed a strong association with PCOS in lean women.
Detailed analysis also revealed an association of this
polymorphism with indices of insulin resistance (fasting
insulin levels, HOMA-IR, and QUICKI) and hyperan-
drogenemia (FAI) in the same subgroup. It may be
mentioned here that our study cohort is larger than the
other published reports which examined exon 17
polymorphism of INSR in PCOS women.
It is well established that insulin resistance, PCOS,
and obesity are interrelated (36, 37). Interestingly,
stratifying the study cohort into lean and obese groups,
the prevalence of polymorphic genotype was signi-
ficantly higher in the lean PCOS group compared with
lean control, obese PCOS as well as obese control
groups. The analysis clearly establishes an association
Table 2 Insulin resistance indices and hormonal profile of lean and obese women with polycystic ovary syndrome (PCOS) according to
genotype.
Variables
Lean PCOS (nZ75) Obese PCOS (nZ105)
CC (nZ23) CTCTT (nZ52) P*P
†
CC (nZ56) CT CTT (nZ49) P*
Age (years) 21.87G4.15 23.90G4.18 0.06 25.63G5.50 26.27G5.82 0.56
Waist (cm) 69.89G5.64 73.24G6.43 0.03 0.43 88.60G9.90 89.50G10.38 0.65
BMI (kg/m
2
) 19.15G2.07 20.48G1.77 0.005 28.79G5.22 28.23G3.97 0.54
FBS (mg/dl) 84.30G6.77 85.26G10.47 0.68 87.22G11.47 88.53G10.58 0.54
Two-hour glucose (mg/dl) 93.04G7.50 98.36G19.34 0.26 107.0G20.04 109.9G24.85 0.51
Fasting insulin (uIU/ml) 8.32G2.7 11.43G5.22 0.009 0.02 15.96G8.53 15.87G7.15 0.95
HOMA-IR 1.64G0.54 2.40G1.10 0.002 0.005 3.20G1.73 3.43G1.80 0.52
QUICKI 0.357G0.018 0.340G0.021 0.002 0.004 0.328G0.029 0.325G0.023 0.56
TT (ng/dl) 64.96G26.36 70.11G25.48 0.43 79.20G34.71 78.65G33.83 0.94
Bio-T (nmol/l) 0.42G0.16 0.54G0.23 0.04 0.057 0.82G0.41 0.83G0.45 0.85
FT (pmol/l) 17.95G6.87 22.81G9.91 0.04 0.056 34.77G17.35 35.44G19.27 0.85
FAI 2.16G0.82 2.94G1.50 0.02 0.03 5.16G3.06 5.38G3.63 0.73
SHBG (nmol/l) 107.3G29.21 92.42G32.94 0.07 68.52G40.59 64.96G38.20 0.65
Androstenedione (ng/ml) 1.92G0.81 2.07G1.14 0.57 2.33G1.13 2.38G1.24 0.80
Data are given as mean GS.D. BMI, body mass index; FBS, fasting glucose; HOMA-IR, homeostasis model assessment for insulin resistance; QUICKI,
quantitative insulin sensitivity check index; TT, total testosterone; FT, free testosterone, Bio-T, bioavailable testosterone; SHBG, sex hormone binding globulin
and FAI, free androgen index. *P-values are from unpaired t-test. The significant variables at the univariate level were further assessed in an ANCOVA model.
†
P-values are from ANCOVA after adjusting for age and BMI. P!0.05 is statistically significant.
Table 3 Insulin resistance indices and hormonal profile of lean and obese control women according to genotype.
Variables
Lean control (nZ84) Obese control (nZ60)
CC (nZ45) CT CTT (nZ39) PCC (nZ31) CTCTT (nZ29) P
Age (years) 22.65G5.08 23.93G4.61 0.49 26.42G5.34 27.70G5.78 0.37
Waist (cm) 71.33G8.06 72.03G6.08 0.67 83.18G5.41 83.23G8.37 0.98
BMI (kg/m
2
) 19.45G2.33 19.14G2.37 0.54 26.37G2.27 26.13G2.32 0.69
FBS (mg/dl) 82.43G7.99 83.88G6.31 0.36 84. 00G9.22 83.43G9.07 0.80
Two-hour glucose (mg/dl) 91.24G13.35 89.45G15.45 0.58 97.03G16.89 95.37G14. 51 0.66
Fasting insulin (uIU/ml) 8.00G2.62 8.37G3.14 0.55 9.98G3.61 10.15G3.54 0.85
HOMA-IR 1.62G0.52 1.73G0.66 0.38 2.12G0.83 2. 05G0.67 0.73
QUICKI 0.358G0.019 0.357G0.021 0.70 0.347G0.020 0.346G0.018 0.79
TT (ng/dl) 48.67G16.63 48.38G15.18 0.93 47.77G11.89 51.93G12.14 0.18
Bio-T (nmol/l) 0.32G0.13 0.37G0.13 0.54 0.35G0.10 0.39G0.13 0.23
FT (pmol/l) 13. 21G5.50 14.40G5.61 0.32 14.06G4.85 16.29G5.54 0.10
FAI 1.66G0.72 1.85G0.84 0.26 1.83G0.65 1.99G0.76 0.37
SHBG (nmol/l) 110.0G34.70 101.5G33.43 0.25 98.87G39.47 98.93G28.34 1.0
Androstenedione (ng/ml) 1.12G0.60 1.05G0.53 0.60 0.96G0.52 1.05G0.60 0.55
Data are given as mean GS.D. BMI, body mass index; FBS, fasting glucose; HOMA-IR, homeostasis model assessment for insulin resistance; QUICKI,
quantitative insulin sensitivity check index; TT, total testosterone; FT, free testosterone, Bio-T, bioavailable testosterone; SHBG, sex hormone binding globulin;
FAI, free androgen index. Comparison was done by unpaired t-test. P!0.05 is statistically significant.
Insulin receptor gene polymorphism in PCOS 859EUROPEAN JOURNAL OF ENDOCRINOLOGY (2009) 160
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of the polymorphic allele with PCOS in lean women,
thus corroborating the previous findings in Caucasian
and Chinese women (25, 26). A significantly higher
frequency of CTCTT genotype was reported in lean
Caucasian PCOS women compared with lean controls
(47 vs 29%, PZ0.03) (25). Similarly, prevalence of
polymorphic allele was more in non-obese Chinese
PCOS women compared with obese PCOS women (52.2
vs 25.5%, P!0.01) (26). In our study, the frequency of
polymorphic genotype in lean PCOS (69.3%) was
higher than the frequencies reported earlier, which
may be attributed to ethnic variation. A similar Korean
study had not found any such association (27).
It is known that insulin resistance is present in PCOS
women irrespective of obesity (38, 39,Mukherjee
unpublished observations). However, in the present
study, an association of this polymorphism in INSR with
PCOS only in lean women was observed. This prompted
us to investigate the relationship of His1058 genotypes
with PCOS associated traits. Univariate analysis
revealed the association of polymorphic genotype with
BMI, waist circumference, fasting insulin levels, HOMA-
IR, and QUICKI, only in lean PCOS group. It is
interesting that lean PCOS women with polymorphic
genotype had significantly higher waist circumference,
which is a measure of central obesity compared with
wildtype genotype. It has now been reported that the
central obesity is more in Asian Indians, causing higher
insulin resistance despite having lean BMI (34). Indeed,
the same subgroup of PCOS women also had higher
indices of insulin resistance. Hyperinsulinemia is known
to exacerbate PCOS phenotypes by inducing hyperan-
drogenemia, through excess ovarian androgen pro-
duction and suppressing hepatic SHBG synthesis (2,
40). In keeping with this evidence, the lean PCOS
subgroup with polymorphic genotype also showed
increased free bioavailable testosterone and FAI. Even
with adjustments for age and BMI in multivariate
analysis, this association persisted with respect to
indices of insulin resistance and hyperandrogenemia
in lean PCOS. These associations suggest that the
pathogenesis of insulin resistance in PCOS may differ
among lean and obese women. Dunaif et al. also
observed that insulin resistance in 50% of PCOS
women is associated with decreased tyrosine autopho-
sphorylation and increased serine phosphophorylation
of INSR (2). Indeed, this emphasizes that molecular
mechanism of insulin resistance may differ in different
PCOS group. Improvement of hyperinsulinemia and
hyperandrogenemia in PCOS women by treatment with
insulin sensitizer metformin occurs through different
mechanisms in obese and lean women (41), which
again supports the notion that the pathogenic
mechanism is different in these two groups. A recent
study reported the presence of three subpopulations
with distinct levels of insulin resistance in PCOS women
with significantly different BMI (39). With our present
data, it is not possible to identify such subpopulations.
However, the association of His1058 SNP with lean
PCOS emphasizes that the genetic determinants of these
subpopulations might be different.
Beyond His1058, few other polymorphisms also have
been reported in exon 17 of INSR in women with PCOS
(21, 23, 28). A novel T/C polymorphism at Cys1008
has been reported to be associated with PCOS and
decreased insulin sensitivity in Chinese population (28).
Studies reported another silent C/T variation in Tyr984
in PCOS women as well as in diabetic subjects (21, 23,
42). However, none of these polymorphisms or other
novel variation in exon 17 of INSR was found in this
study cohort.
PCOS is now considered as a complex multigenic
trait, where different gene variants interact with each
other and also with environmental factors in the
manifestation of different phenotypic expressions of
the syndrome. It is possible that different subgroups of
PCOS with specific phenotypic expression are associated
with specific gene variants. Since the C/T SNP at
His1058 is a silent one, it cannot exert a major effect on
the development of insulin resistance; rather it might be
in linkage disequilibrium with some other genetic
variants, which may play a direct role in the
development of PCOS by affecting insulin sensitivity in
lean women. This polymorphism may predispose lean
women to develop insulin resistance and compensatory
hyperinsulinemia, which then may induce hyperan-
drogenemia progressing towards PCOS. The lower
prevalence of the T allele in obese compared with lean
women could also suggest that this allele either has a
protective role against obesity in lean women or may
reduce the risk of developing PCOS in obese women.
To summarize, this study in Indian women shows an
association of the C/T polymorphism at His1058 of
INSR with PCOS in lean rather than obese women. The
polymorphism is also seen to be associated with the
indices of insulin resistance and hyperandrogenemia in
the lean PCOS group. The observations made herein
strongly suggest that the genetic pathogenic
mechanism of this heterogeneous disorder may differ
between lean and obese PCOS, which needs to be
studied further.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by a grant from Department of Science and
Technology, India (SR/SO/HS-60/2005).
Acknowledgements
We thank Dr Mousumi Banerjee, Department of Biostatistics,
University of Michigan, and Ann Arbor, Michigan, USA for her help
in statistical analysis. We acknowledge Dr Padma Menon and the
860 S Mukherjee and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2009) 160
www.eje-online.org

Endocrine Department of Seth GS Medical College and KEM Hospital,
Mumbai, India for their contributions. We also thank Mr Chinnaraj
Saravanan (DNA sequencing core facility, NIRRH) for his help in DNA
sequencing.
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Received 20 January 2009
Accepted 7 February 2009
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