Role of luteinizing hormone β-subunit gene variants among South Indian women with polycystic ovary syndrome

Gene 494 (2012) 51–56

Contents lists available at SciVerse ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Role of luteinizing hormone β-subunit gene variants among South Indian women with polycystic ovary syndrome Shilpi Dasgupta a, P.V.S. Sirisha a, K. Neelaveni b, K. Anuradha c, G. Sudhakar d, B. Mohan Reddy a,⁎ a

Molecular Anthropology Group, Biological Anthropology Unit, Indian Statistical Institute, Hyderabad, Andhra Pradesh, India Department of Endocrinology, Osmania General Hospital, Hyderabad, India Anu Test Tube Baby Centre, Somajiguda, Hyderabad, India d Department of Human Genetics, Andhra University, Visakhapatanam, Andhra Pradesh, India b c

a r t i c l e

i n f o

Article history: Accepted 21 November 2011 Available online 20 December 2011 Received by A.J. van Wijnen Keywords: Luteinizing hormone β-subunit PCOS Mutation India

a b s t r a c t Abnormal luteinizing hormone (LH) secretion and action are known to affect ovarian steroidogenesis and thus playing a crucial role in manifestation of polycystic ovary syndrome (PCOS). This study is first of its kind to study association of LH β-subunit gene variants with PCOS among South-Indian women. 250 PCOS cases and 299 controls were recruited for the study. All the exons of LH β gene were screened. Allele and genotype frequencies of the SNPs were compared between the cases and controls. We identified seven SNPs in the LH β gene; one SNP in exon 3 (rs#1056917) exhibited significant difference in the allele frequency between the PCOS cases and controls (p = 0.015). Although, the LH β variants that are found to be more frequent among PCOS cases are silent in nature and not of any functional significance, they might influence other significant functional polymorphisms in the hypothalamic–pituitary–gonadal axis which needs to be explored. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Polycystic ovary syndrome (PCOS) is a common clinical disorder, occurring in 5–8% of premenopausal women. It is associated with anovulation, hirsutism, obesity, and multiple cysts in the ovaries. The disorder may reflect different etiologies and the roles of insulin resistance and hyperinsulinemia, functional ovarian hyperandrogenism, and abnormalities of gonadotropin secretion have been reviewed (Marshall et al., 2002). The actions of hyperinsulinemia and abnormal gonadotropin secretion in stimulating ovarian steroidogenesis, which in turn leads to hyperandrogenism, have been supported by various studies (cited in Marshall et al., 2002). PCOS is associated with alterations in the function of the hypothalamic–pituitary–gonadal (HPG) axis wherein the increased frequency and amplitude of the hypothalamic GnRH pulse generator leads to consistently elevated plasma luteinizing hormone (LH) levels (Valkenburg et al., 2009). This

Abbreviations: PCOS, Polycystic Ovary Syndrome; LH, Luteinizing hormone; TSH, Thyroid stimulating hormone; hCG, Human chorionic gonadotropin; vLH/VLH, Variant luteinizing hormone; DNA, Deoxyribose nucleic acid; PCR, Polymerase chain reaction; LD, Linkage disequilibrium; SNP, Single nucleotide polymorphism; HWP, Hardy-Weinberg proportion; BMI, Body mass index; SE, Standard error; FSH, Follicle stimulating hormone; RBS, Random blood sugar; HDL, High density lipoprotein; LDL, Low density lipoprotein; CI, Confidence interval. ⁎ Corresponding author at: Molecular Anthropology Group, Biological Anthropology Unit, Indian Statistical Institute, Street#8, Habsiguda, Hyderabad 500007, Andhra Pradesh, India. Tel.: + 91 40 27171906; fax: +91 40 27173602. E-mail addresses: [email protected], [email protected] (B.M. Reddy). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.11.054

suggests a central role for persistent LH stimulation of the ovary in the production of hyperandrogenism and thus playing a central role in the pathophysiology of the syndrome. Luteinizing hormone (LH) is a heterodimeric protein belonging to a family of glycoprotein hormones that also includes FSH, thyroidstimulating hormone (TSH) and hCG (Liao et al., 1998). All these hormones share a similar structure consisting of a common α-subunit and a specific β-subunit. It is this unique β-subunit which confers biological specificity for the hormone receptor in the target organ. LH plays an important role in gonadal function that includes development of follicle growth, stimulation of steroidogenesis, and maturation of the oocyte (Liao et al., 1998). Therefore, it is speculated that the abnormal LH secretion, which may in turn result from mutant form of LH gene, affects gonadal function and that the microheterogeneity is associated with anovulation, luteal insufficiency, and premature oocyte maturation, leading to menstrual disorders and infertility characteristic of PCOS. The LHβ variants differ from each other in several aspects, including their distribution among the human population, frequency of occurrence and functional properties. The most common and also the most extensively studied is the common variant, V-LH, with two amino acid replacements Trp8Arg and Ile15Thr. The allelic frequency of this variant varies extensively among different ethnic groups, and its distribution shows that the V-LHβ allele is a universally occurring polymorphism, and thus different from the other LH variants so far detected, which have a more restricted distribution. Other variants in LH β subunit gene resulting in amino-acid alterations: Glu54-

52

S. Dasgupta et al. / Gene 494 (2012) 51–56

Arg54 and Ser102-Gly102 as well as six silent polymorphisms have also been reported (Roy et al., 1996). Overall, there have been a limited number of studies on the mutational analysis of the LH β subunit gene (Furui et al., 1994; Liao et al., 1998; Pettersson et al., 1994; Ramanujam et al., 1999; Takahashi et al., 2003). Further, these investigations have addressed the role of LH β mutations among the infertility cases, in general, including among others, PCOS, premature ovarian failure and endometriosis. In this paper, we report findings of our pioneering study among the Indian women on the possible association of LH β variants with PCOS. 2. Materials and methods 2.1. Study population A total of 549 women consisting of 250 PCOS cases (aged 14–40 years) and 299 controls (aged 14–47 years) were recruited for the study. Patients were enrolled from the Gynecology clinic of the Osmania General Hospital, Hyderabad as well as from an infertility clinic (Anu's Test Tube Baby Centre, Hyderabad) as per the Rotterdam criteria, 2003 (The Rotterdam ESHRE/ASRM-sponsored PCOS Concensus Workshop Group, 2004) according to which any two of the following three conditions need to be fulfilled for the inclusion: (i) presence of clinical and/or biochemical signs of hyperandrogenism, (ii) infrequent periods with intermenstrual interval of more than 35 days, and (iii) polycystic ovaries; an ovary with the ultrasound appearance of more than 10 subcapsular follicles (b10 mm in diameter) in the presence of prominent ovarian stroma was considered polycystic. Patients with hyperprolactinemia, thyroid and adrenal diseases, 21-hydroxylase deficiency, and androgen-secreting tumors were excluded. The weight and height of the subjects were recorded. Hirsutism was defined as a Ferriman–Gallwey score of more than 5 (Mifsud et al., 2000). Hormonal assays that were recorded included serum levels of gonadotrophic hormones (LH and FSH), TSH, prolactin and testosterone (total). Normal controls with no history of treatment for fertility, and with normal menstrual cycles every 25–32 days were recruited from the family planning center of the Osmania hospital and from the general population. Intravenous blood samples (~5 ml) were collected from both the patients and controls after obtaining their informed written consent. The study protocol was approved by the Indian Statistical Institute Review Committee for Protection of Research Risks to Humans. 2.2. DNA extraction, amplification and sequencing DNA was extracted from the peripheral blood samples of the patients and control using the phenol-chloroform method (Sambrook et al., 1989). We carried out PCR amplification and sequencing to screen the LH β exonic regions using the forward and reverse primers described previously by Furui et al. (1994). Each PCR was optimized with respect to the concentration of Mg 2 + ions. The PCR-mix consisted of 10xPCRBuffer, 10 μM dNTP-mix, 1 μM of each primer, 1 U Taq-polymerase and 40 ng template DNA in a reaction volume of 10 μl. Reactions were carried out in an ABI GeneAmp9700 thermal cycler (Applied Biosystems, Foster City, CA). An initial denaturing cycle of 94 °C for 5 min was followed by 35 cycles with the conditions: 1 min at 94 °C, 2 min at 65 °C and 3 min at 72 °C. A final extension cycle at 72 °C for 10 min followed. Cycle Sequencing of PCR products were carried out with either the forward or the reverse primers using the Big-Dye Terminator ready reaction kit (Applied Biosystems, Foster City, CA). Extended products were purified by ethanol precipitation and analyzed on an ABI 3730 automated DNA Analyzer (Applied Biosystems, Foster City, CA). The polymorphisms in the LHβ gene were noted down and allele/genotype frequencies were calculated.

2.3. Statistical analysis Allele frequencies were determined by the gene-counting method. All the statistical analyses were performed with the help of SPSS statistical software (version 18.0, SPSS Inc, Chicago, IL, USA). Power of the study was calculated using G*Power software (version 3.1). The Hardy–Weinberg equilibrium was estimated by the χ 2 test using Pypop software. Haploview and Pypop softwares were used to estimate LD and generate haplotype frequencies. The nucleotide positions mentioned for individual SNP is according to Takahashi et al. (2003). 3. Results The anthropometric/clinical characteristics of PCOS cases and controls are presented in Table 1. PCOS subjects had a significantly higher mean value for body mass index (BMI). The age of menarche is however significantly lower in the PCOS group compared to the controls. The proportion of obese women (BMI ≥ 25) within the PCOS group was significantly higher than in the control group (55.1% vs 15.1% respectively, p b 0.001). Comparison of the biochemical parameters between the lean and obese PCOS cohort revealed that although the mean levels of LH and FSH are not significantly different between the lean and obese PCOS cases, a higher LH:FSH ratio (characteristic feature of PCOS) is evident among the obese group. Moreover, obese PCOS cases had significantly higher mean level of cholesterol and triglycerides than the lean PCOS cases (Table 2). 3.1. Allele and genotype frequency All the exons of LHβ gene were successfully amplified by PCR in the entire cohort. We were able to identify seven polymorphisms that include two successive missense mutations T986-C (rs# 1800447), T1008-C (rs#34349826), and two silent mutations in exon 2: G1018-C (rs#6521) and C1036-A (rs#1056914) in exon 2, two silent mutations in intron 2: C1098-T (rs#2387588) and CAG/C-G at nt 1105 (rs# 4287687) and one silent mutation in exon 3 T1423-C (rs#1056917). Apart from the previously known SNPs that we found in our study, we could not identify any novel variant/s specific to the Indian population. The allele frequency of each of these SNPs among the PCOS cases and controls is given in Table 3. Out of the seven mutations, only the silent mutation in exon 3 exhibited significant difference in the allele frequency between the PCOS cases and controls (χ2 = 5.83, df= 1, p = 0.015). The mutant allele C is in significantly higher frequency among the cases (48.8%) compared to the controls (41.3%) yielding an odds ratio of 1.349 along with the statistical power (1-β error probability) of 98.8%. However, after Bonferroni correction for multiple testing, the difference in the allele frequency was no longer significant. The genotype frequencies were also compared between the cases and controls but the χ2 test did not suggest any significant heterogeneity between the two groups (Supplementary Table 1). Using PyPop software, the observed genotype counts were compared with those expected under Hardy–Weinberg proportions (HWPs), and a χ2 test was carried out to check for the significance of deviation from HWP for each SNP locus in PCOS women and controls separately. All the Table 1 Anthropometric characteristics of PCOS subjects and controls. PCOS

Age(years) BMI(kg/m2) WHR Age at menarche(years)

Controls

n

Mean

n

Mean

250 176 147 250

24.6 ± 0.34 25.5 ± 0.64 0.84 ± 0.005 11.5 ± 0.28

299 232 232 299

23.7 ± 0.33 22.5 ± 0.23 0.86 ± 0.003 13.0 ± 0.11

Data are Mean ± S.E. BMI (Body Mass Index). WHR (Waist Hip ratio).

p-value

0.06 b 0.001 b 0.001 b 0.001

S. Dasgupta et al. / Gene 494 (2012) 51–56

3.2. Internal consistency

Table 2 Hormonal profile of PCOS cases.

Testosterone(ng/ml) LH(mIU/ml)a FSH(mIU/ml)a RBS(mg/dl) Cholesterol(mg/dL) HDL(mg/dL) LDL(mg/dL) Triglyceride(mg/dL)

PCOS (lean)

PCOS (obese)

N

N

Mean

53

p-value Normal range

Mean

79 1.47±0.54 95 0.82±0.13 64 15.9±1.86 76 15.2±3.60 70 8.4±1.45 83 5.9±0.40 16 71.1±4.4 26 87.7±9.7 72 120±10.4 88 145±9.4 72 28±2.4 88 31 ± 2.0 72 71±6.3 88 84 ± 5.9 72 106±11.7 88 152 ± 14.9

0.24 0.87 0.10 0.21 0.08 0.37 0.13 0.02

0.1–0.9 ng/ml 4.6–12.4 mIU/ml 6.9–12.5 mIU/ml b70–170 mg/dl b200 mg/dl 40–60 mg/dl b100 mg/dl b150 mg/dl

Data are Mean ± S.E. a Values represent follicular phase levels.

loci, except the two missense mutations in exon 2, showed a significant deviation from HWP, in both the cases and the controls (Supplementary Table 1). These departures from HWP were observed to be due to increased proportion of the homozygotes without exception in both cases and controls. This is expected given the highly endogamous nature of the Indian populations, in general, and particularly the Southern Indian populations which practice close consanguineous marriages resulting in high levels of inbreeding. The case cohort was also analyzed in two groups, based on body mass index (BMI), i.e. lean PCOS (BMI b 25) and obese PCOS (BMI ≥ 25) cases. The allele and genotype frequencies were compared between these two groups as well as each of them with controls separately (Table 4 and Supplementary Table 2, respectively). Contingency χ 2 suggests homogeneity in the allele frequencies between lean PCOS and obese PCOS cases. Significant heterogeneity is observed in the allele frequency of variant allele A in exon 2 (rs#1056914) between the lean PCOS cases and controls. 61.4% of the lean PCOS cases harbor the variant allele A as compared to 49.5% of the controls (χ 2 = 5.157, df = 1, p = 0.023). Similar heterogeneity is also observed in the allele frequency of mutant allele C in exon 3 (rs#1056917) between obese PCOS cases (50%) and controls (39.1%) (χ 2 = 4.893, df = 1, p = 0.027). Odds ratios for these significant observations were estimated to be 1.62 (95% CI: 1.067–2.46) and 1.56 (95% CI: 1.051–2.31), respectively. However, this finding is not statistically significant after correction for multiple testing. The control group was also categorized according to BMI, and similar comparison was carried out between the BMI matched case and control groups (Lean cases vs lean controls, and obese cases vs obese controls). The analysis did not yield any significant observation (results not presented).

Despite socioeconomic hierarchy and religious heterogeneity, the populations of Andhra Pradesh were found to be genetically homogenous (Reddy et al., 2005). Nevertheless, given that our sample consisted of sizeable cohort of Muslim subjects, we repeated the above analysis for the Hindu caste and Muslim subjects separately. But this categorization does not seem to have any significant effect on the allele distribution profiles (Table 5). Since the Muslim and Hindu cases were predominantly drawn from the Osmania General Hospital and Anu Test Tube Baby Centre, respectively, which represented lower and higher socioeconomic status respectively, the results also do not suggest any effect of socioeconomic hierarchy in the pattern of manifestation of PCOS in relation to LH mutations. Further, we drew 50, 60 & 70% random subset of case and control samples and repeated the above analysis to test for internal consistency in our data. Overall, the results suggest that the distribution of LH β alleles is homogenous (results not presented) between cases and controls indicating internal consistency. 3.3. Linkage disequilibrium and haplotypes We determined the LH β haplotypes based on the seven SNPs identified in our study. Linkage disequilibrium (LD) plot for all the SNP loci was generated through Haploview software. SNP loci were designated as LHP1 (rs#1800447 T/C), LHP2 (rs#34349826 T/C), LHP3 (rs#6521 G/C), LHP4 (rs#1056914 C/A), LHP5 (rs#2387588 C/ T), LHP6 (rs#4287687 CAG/C-G) and LHP7 (rs#1056917 T/C). LHP1, LHP2, LHP3, LHP4 were found to be in strong LD with LHP5 (D′ = 1) (Fig. 1). LD estimates were obtained from 245 PCOS cases and 291 controls for all possible haplotype combinations using Pypop software. A total of 29 haplotype combinations were identified with frequency > 1% in the entire group of 536 individuals. From the frequency estimates of the haplotypes, χ 2 values and odds ratio were calculated. The haplotype that exhibited significant association with PCOS was T:T:C:A:T:C-:T with an odds of 4.7 (p b 0.001, 95% C.I: 2.05–10.04). This haplotype comprises of the mutations at the third, fourth, fifth and sixth SNP positions. 3.4. Clinical correlation The controls in this study were of similar age with that of the PCOS cases (mean ± SE, 24.6 ± 0.34, 23.7 ± 0.33, respectively). We obtained the serum luteinizing hormone levels of the PCOS cases and compared the mean level of the hormone in each genotype category of the four SNP positions (LHP3, LHP4, LHP5,LHP7) (Fig. 2). Though the

Table 3 Allele frequency of LH β gene mutations in PCOS cases and controls. SNP

rs ID

Location

LHP1

1800447

Exon 2

LHP2

34349826

Exon 2

LHP3

6521

Exon 2

LHP4

1056914

Exon 2

LHP5

2387588

Intron 2

LHP6

4287687

Intron 2

LHP7 a

1056917

Exon 3

Allele

T C T C G C C A C T CAG C-G T C

Amino acid

All subjects frequency (2 N = 1072)

PCOS cases (2 N = 490)

Controls (2 N = 582)

p-value

Tryp Arg Ile Thr Val Val Pro Pro – – – – Gly Gly

0.959 0.041 0.959 0.041 0.508 0.492 0.473 0.527 0.519 0.481 0.634 0.366 0.553 0.447

0.961 0.039 0.961 0.039 0.502 0.498 0.447 0.553 0.522 0.478 0.608 0.392 0.512 0.488

0.959 0.041 0.959 0.041 0.514 0.486 0.495 0.505 0.515 0.485 0.656 0.344 0.587 0.413

Not significant after Bonferroni correction for multiple testing.

Odds ratio

CI for odds ratio Lower

Upper

0.837

0.938

0.507

1.73

0.837

0.938

0.507

1.73

0.702

1.048

0.824

1.3

0.118

1.212

0.953

1.543

0.820

1.028

0.809

1.308

1.231

0.959

1.579

1.349

1.058

1.722

0.103 0.015

a

54

S. Dasgupta et al. / Gene 494 (2012) 51–56

Table 4 Allele frequency distribution among Lean PCOS, Obese PCOS and control subjects. SNP

rs ID

Location

LHP1

1800447

Exon 2

LHP2

34349826

Exon 2

LHP3

6521

Exon 2

LHP4

1056914

Exon 2

LHP5

2387588

Intron 2

LHP6

4287687

Intron 2

LHP7

1056917

Exon 3

Allele

T C T C G C C A C T CAG C-G T C

PCOS Lean (2 N = 158)

Obese (2 N = 194)

0.943 0.057 0.943 0.057 0.456 0.544 0.386 0.614*1 0.481 0.519 0.671 0.329 0.557 0.443

0.954 0.046 0.954 0.046 0.521 0.479 0.474 0.526 0.536 0.464 0.588 0.412 0.50 0.50#1

Controls (2 N = 214)

0.967 0.033 0.967 0.033 0.486 0.514 0.505 0.495 0.486 0.514 0.607 0.393 0.609 0.391

*significantly different from controls (p = 0.02), (1-β error prob) = 0.982. #significantly different from controls (p = 0.03), (1-β error prob) = 0.974. 1 Not significant after Bonferroni correction for multiple testing.

comparison did not reach statistical significance, the pattern observed depicted a higher mean value of the luteinizing hormone among the cases who were homozygous for the mutant alleles. The comparison was not made for the variant LH genotype since only one PCOS case was found to be homozygous for the mutant allele. 4. Discussion Of the LH α and β subunits, the role of LHβ subunit in reproductive physiology is widely studied. Although mutations in the LHβ-subunit gene are very rare, the study of their biological actions is important in elucidating normal and abnormal functions of LH. The previous studies have primarily focused on the variant luteinizing hormone (v-LH) and the two point mutations that cause amino acid replacements from Trp 8 to Arg 8 and Ile 15 to Thr 15 leading to this immunologically anomalous form and its association with infertility in general (Elter et al., 1999; Furui et al., 1994; Rajkhowa et al., 1995; Ramanujam et .al., 1999). Some of the earlier studies have also estimated the prevalence of v-LH in different populations (Haavisto et al., 1995; Nilsson et al., 1997; Rajkhowa et al., 1995). Only two earlier studies have focused on the PCOS cases in particular but they have reported only the prevalence of v-LH in the Caucasian populations (Rajkhowa et al., 1995; Tapanainen et al., 1999). However, the relation between a mutant LH gene and female infertility has not yet been established clearly. While some investigators have reported the clinical significance of the variant LH in Japanese patients with reproductive disorders

Fig. 1. Linkage Disequilibrium plot of the seven LHβ mutations.

including infertility and/or menstrual disorders (Furui et al., 1994; Huhtaniemi et al., 1999; Takahashi et al., 2003), Finnish women carrying the variant LH (detected in 28% of the population) were reported to be fertile (Haavisto et al., 1995; Nilsson et al., 1997). Thus, evidence concerning the clinical significance of the variant LH with respect to reproductive disorders has been contradictory. In an attempt to validate the results of earlier genetic studies and given the biological specificity of the LH β-subunit for hormone action, we investigated the possible association of LH β gene polymorphisms and PCOS in the largest cohort of women with PCOS so far examined for the LH β gene mutations. In our Indian cohort, we genotyped the variant LH and identified few heterozygotes. However, the genotype frequency distribution was not significantly different between the PCOS cases and controls. Therefore, the functional role of v-LH in PCOS susceptibility could not be ascertained. Nevertheless, our comparison of the obese and lean PCOS cohort for v-LH frequency distribution yielded similar inference to that of a previous study of PCOS women from Finland, Netherlands and the United States where the prevalence of v-LH gene in obese PCOS cases was observed to be lower than in lean PCOS women (Tapanainen et al., 1999). Inspite of the ethnic differences between the populations studied, such a similar v-LH frequency adds to the evidence that the obese women with v-LH are somehow protected from developing symptomatic PCOS, and those with wildtype-LH are more likely to develop

Table 5 Allele frequency distribution among Hindu PCOS, Muslim PCOS and control subjects. SNP

rs ID

Location

LHP1

1800447

Exon 2

LHP2

34349826

Exon 2

LHP3

6521

Exon 2

LHP4

1056914

Exon 2

LHP5

2387588

Intron 2

LHP6

4287687

Intron 2

LHP7

1056917

Exon 3

Allele

T C T C G C C A C T CAG C-G T C

(i) Hindus

(ii) Muslims

(iii) Hindus Vs Muslims

Case (2 N = 314)

Control (2 N = 440)

Case (2 N = 160)

Control (2 N = 122)

Hindu PCOS (2 N = 314)

Muslim PCOS (2 N = 160)

0.968 0.032 0.968 0.032 0.484 0.516 0.430 0.570 0.487 0.513 0.611 0.389 0.542 0.458

0.955 0.045 0.955 0.045 0.493 0.507 0.482 0.518 0.505 0.495 0.659 0.341 0.616 0.384

0.950 0.050 0.950 0.050 0.537 0.463 0.481 0.519 0.587 0.413 0.575 0.425 0.449 0.551

0.967 0.033 0.967 0.033 0.566 0.434 0.525 0.475 0.541 0.459 0.672 0.328 0.508 0.492

0.968 0.032 0.968 0.032 0.484 0.516 0.430 0.570 0.487 0.513 0.611 0.389 0.542 0.458

0.950 0.050 0.950 0.050 0.537 0.463 0.481 0.519 0.587 0.413 0.575 0.425 0.449 0.551

S. Dasgupta et al. / Gene 494 (2012) 51–56

55

for this haplotype makes its impact somewhat imperceptible. We had obtained serum LH levels from the PCOS cases and could thus compare the mean hormone levels in different genotype categories. The results essentially demonstrate that the cases harboring the variant alleles had higher mean levels of LH indicating plausible genetic basis for the altered LH secretion pattern in PCOS. Though all the polymorphisms reported in our study did not lead to any amino acid alterations, they may influence other missense mutations leading to an abnormal LH secretion and/or action as mentioned earlier. Unfortunately, it was not feasible for us to obtain the hormonal parameters for the control group, which would have given us much better insights. 4.1. Conclusion

Fig. 2. Serum Luteinizing hormone levels (mean) among PCOS cases according to the genotype.(WT-wild type, HZ-heterozygote, MT- mutant).

the disease. It is therefore imperative that similar evidence is supplemented from populations of Asian ethnic background before establishing the significance of v-LH in PCOS. Apart from the two point mutations leading to v-LH that are located in exon 2, other mutations were also reported in LHβ exon 2 and 3 (Roy et al., 1996). While the remaining mutations in exon 2 and 3 are silent in nature, one of the mutations in exon 3 was reported to cause amino acid replacement from Glycine 102 to Serine 102. In the present study, we have identified seven polymorphisms which were reported earlier spanning from exon 2 to exon 3, but the missense polymorphism in exon 3 (Gly 102 to Ser 102) was absent in our cohort. We have also been able to identify a single nucleotide deletion polymorphism (CAG/C-G) in intron 2 which has so far not been reported among the studies involving PCOS cases. Though it does not show any significant difference in the frequency between the cases and controls, the relative frequency of the deletion allele is found to be more among the cases. This difference was more pronounced between the obese PCOS cases and controls than between lean PCOS cases and controls. The overall pattern of allele distribution between the PCOS cases and controls was quite homogenous. However, only the silent mutation in exon 3 exhibited a significantly higher frequency among the cases than controls, although this statistical significance was not retained after Bonferroni correction. The silent mutations of the LHβ subunit may be considered to have influenced the missense mutation at two sites and/or other unknown missense mutations, which might lead to ovulatory disorders (Takahashi et al., 2003). The significant heterogeneity for the silent mutation in exon 3 was retained when the obese PCOS cases were compared with the controls. The lean cases, on the other hand, had a significantly higher frequency of the variant allele A in exon 2 (rs#1056914).This pattern of association of different alleles among the PCOS cases, when categorized according to BMI, supports the notion that different patterns of physiological alterations determine the metabolic complications involved with PCOS, paralleling the observation in our previous study with respect to the androgen receptor gene (Dasgupta et al., 2010). The linkage disequilibrium results depicted that majority of the alleles were in strong LD confirming with the observations of Roy et al. (1996). Since haplotypes provide a more comprehensive information about the pattern of association, we expected to obtain some specific haplotypes in association with PCOS. However, the haplotype that was found to be most frequent among the PCOS cases and controls comprised of the wild type alleles. Even though the haplotype that exhibited significant association with PCOS cases contained the variant alleles in exon 2 and intron 2, the small number of cases observed

In conclusion, we find heterogeneity in the LHβ polymorphic pattern between PCOS cases and controls suggesting that despite the lack of evidence for any direct functional role, these genetic variants may contribute to the susceptibility of PCOS by way of influencing polymorphisms in other loci leading to physiological alterations at the hypothalamic–pituitary–gonadal level. However, these results need to be interpreted with caution given the consistent departures from the Hardy–Weinberg proportions. The present study indicating association of LHβ polymorphisms and PCOS warrants further validation in other ethnicities of Indian population. Additionally, the exploration of promoter region mutations in this gene might help in understanding the transcription pattern and its relation to the normal and pathological pituitary-gonadal function. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2011.11.054. Funding This work is funded by the Indian Statistical Institute. Acknowledgments The authors thank Director, Indian Statistical Institute for logistic support and the Director, Centre for Cellular and Molecular biology, Hyderabad, for providing us access to the DNA sequencing facility to run the plates processed at ISI. References Dasgupta, S., et al., 2010. Androgen receptor CAG repeat polymorphism and epigenetic influence among the South Indian women with polycystic ovary syndrome. PLoS One 5, e12401. Elter, K., Erel, C.T., Cine, N., Ozbek, U., Hacihanefioglu, B., Ertungealp, E., 1999. Role of the mutations Trp8 => Arg and Ile15 => Thr of the human luteinizing hormone beta-subunit in women with polycystic ovary syndrome. Fertil. Steril. 71, 425–430. Furui, K., et al., 1994. Identification of two point mutation in the gene coding luteinizing hormone (LH) β subunit associated with immunologically anomalous LH variant. J. Clin. Endocrinol. Metab. 78, 107–113. Haavisto, A., Pettersson, K., Bergendahl, M., Virkah'lkki, A., Huhtaniemi, I., 1995. Occurrence and biological properties of a common genetic variant of luteinizing hormone. J. Clin. Endocrinol. Metab. 80, 1257–1263. Huhtaniemi, I., Jiang, M., Nilsson, C., Pettersson, K., 1999. Mutations and polymorphisms in gonadotropin genes. Mol. Cell. Endocrinol. 51, 89–94. Liao, W.X., Roy, A.C., Chan, C., Arulkumaran, S., Ratnam, S.S., 1998. A new molecular variant of luteinizing hormone associated with female infertility. Fertil. Steril. 69, 102–106. Marshall, J.C., Eagleson, C.A., McCartney, C.R., 2002. Neuroendocrine dysfunction in polycystic ovary syndrome. In: Chang, R.J., Heindel, J.J., Dunaif, A. (Eds.), Polycystic Ovary Syndrome. Marcel Dekker Inc., New York, pp. 89–103. Mifsud, A., Ramirez, S., Yong, E.L., 2000. Androgen receptor gene CAG trinucleotide repeats in anovulatory infertility and polycystic ovaries. J. Clin. Endocrinol. Metab. 85, 3484–3488. Nilsson, C., Pettersson, K., Millar, R.P., Coerver, K.A., Matzuk, M.M., Huhtaniemi, I.T., 1997. Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. Fertil. Steril. 67, 998–1004. Pettersson, K., Makela, M.M., Dahlen, P., Lamminen, T., Huoponen, K., Huhtaniemi, I., 1994. Gene polymorphism found in the LH beta gene of an immunologically

56

S. Dasgupta et al. / Gene 494 (2012) 51–56

anomalous variant of human luteinizing hormone. Eur. J. Endocrinol. 130, S17.03 (abstract). Rajkhowa, M., et al., 1995. Prevalence of an immunological LH β-subunit variant in a UK population of healthy women and women with polycystic ovary syndrome. Clin. Endocrinol. 43, 297–303. Ramanujam, L.N., Liao, W.X., Roy, A.C., Loganath, A., Goh, H.H., Ng, S.C., 1999. Association of molecular variants of luteinizing hormone with menstrual disorders. Clin. Endocrinol. 51, 243–246. Reddy, B.M., Naidu, V.M., Madhavi, V.K., Thangaraj, K., Kumar, V., 2005. Microsatellite diversity in Andhra Pradesh, India: genetic stratification versus social stratification. Hum. Biol. 77, 803–823. Roy, A.C., Liao, W.X., Chen, Y., Arulkumaran, S., Ratnam, S.S., 1996. Identification of seven novel mutations in LH β -subunit genes by SSCP. Mol. Cell. Biochem. 165, 151–153.

Sambrook, J., Fritschi, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Takahashi, K., et al., 2003. Influence of missense mutation and silent mutation of LHb-subunit gene in Japanese patients with ovulatory disorders. Eur. J. Hum. Gen. 11, 402–408. Tapanainen, J.S., et al., 1999. A new contributing factor to polycystic ovary syndrome: the genetic variant of luteinizing hormone. J. Clin. Endocrinol. Metab. 84, 1711–1715. The Rotterdam ESHRE/ASRM-sponsored PCOS Concensus Workshop Group, 2004. Revised 2003 Concensus on diagnostic criteria and long term health risk related to polycystic ovary syndrome. Fertil. Steril. 81, 19–25. Valkenburg, O., et al., 2009. Genetic polymorphisms of GnRH and gonadotrophic hormone receptors affect the phenotype of polycystic ovary syndrome. Hum. Reprod. 24, 2014–2022.