CHAPTER V. Luteinizing hormone beta (LHβ) gene SNPs and its. association with PCOS

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1 CHAPTER V Luteinizing hormone beta (LHβ) gene SNPs and its association with PCOS

2 5.0. Introduction Luteinizing hormone (LH), a heterodimeric glycoprotein hormone secreted from anterior pituitary gonadotropes and acts primarily at ovary to regulate ovarian folliculogeneisis and luteinization. It is made up of two peptides (α and β) and have structural similarities with FSH and TSH; however its bioactivity is conferred by the LH beta (LHβ) subunit gene. LHβ originates from an ancestral cluster genes sharing >98% sequence similarity with human chorianic gonadotropin genes (hcgb) and other pseudogenes located on human chromosome 19. The LH performs its biological functions by binding with its receptor expressed on the granulosa and luteal cells of the ovary. Although LH-receptors are expressed on human follicles already from the start of the cycle, the synergizing effect of LH and FSH appears to be most prominent from the mid-follicular phase and onwards. In addition to follicular growth, LH stimulates the androgen secretion by ovarian theca cells [1, 2]. Abnormal endocrine profile in specific altered LH:FSH ratio (>1.0) is consistently seen in PCOS women [3,4,5]. High serum levels of LH are found to be associated with increased ovarian androgen production, hyperinsulinemia, and polycystic ovaries, as a result of arrested follicular development in PCOS women [6, 7, 8]. Even though LH levels are correlated with reproductive and metabolic phenotypes, role of LHβ variants is unclear in the pathogenesis of reproductive disorders. However LHβ variations are found to be associated with infertility and premature ovarian failure in Japanese women [9, 10, 11] and slow down the progression of puberty in boys [12]. In light of the clinical significance of LH in ovarian steroid synthesis and ovarian folliculogenesis, reproductive physiology, the role of LHβ SNPs rs (T/C) with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 78

3 (Exon 2; Trp28Arg), rs (T/C) (Exon 2; Ile35Thr) and SNP rs (G/A) (exon 3; Gly122Ser) were examined in the study subjects Methodology The study population consists of PCOS women (n=97) and healthy woman (n=101) with normal reproductive physiology were recruited in the study by adopting the criteria as explained in the chapter 2.1. & 2.2. About 3mL of intravenous blood sample was collected from the study subjects and DNA was extracted as phenol chloroform method as mentioned in the methodology section The PCR reactions were performed for SNPs rs , rs and rs as explained in methodology section SNP genotyping was performed by RFLP using the restriction endonucleases NcoI, BseGI and EcoOI09I respectively as described in the methodology section Confirmation of PCR-RFLP results were carried out by DNA sequencing of PCR products (section 2.5.9). Suitable statistical tools were applied to draw inference as explained in the methodology section Results Genotyping of the LHβ SNPs rs , rs and rs was done by PCR-RFLP. PCR-RFLP showed the presence of only homozygous wild genotypes in LHβ SNP [Figure 2.3. A, B and C]. The present study heterozygous and homozygous variants are completely absent in both PCOS and control subjects Table 5.1; (Figure 5.1). All the reactions were repeated to confirm the genotypes obtained. with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 79

4 Table 5.1. The allele and genotype frequencies of the LHβ SNP in the study subjects SNP Subjects Genotypes (n) Minor allele Major allele C/C C/T T/T C T rs PCOS (97) 0 (0) 0 (0) 97 (100) Controls (101) 0 (0) 0 (0) 97 (100) rs PCOS (97) 0 (0) 0 (0) 97 (100) Controls (101) 0 (0) 0 (0) 97 (100) rs PCOS (97) 0 (0) 0 (0) 97 (100) Controls (101) 0 (0) 0 (0) 97 (100) Values within the parentheses represent the percentage of genotype. Figure 5.1 Genotype distribution of LHβ SNPs in PCOS and controls The homozygous wild allele only present in both the cases and controls. Concordant results obtained by PCR-RFLP were further confirmed by DNA sequencing (Figure 2.8, 2.9 and 2.10) Discussion with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 80

5 Genetic variants in LHβ are found to be associated with impaired reproductive functions and can lead to infertility in both sexes. In the present study, PCR-RFLP analysis of LHβ SNPs showed the presence of only homozygous wild type genotypes such as T/T for rs , T/T for rs and G/G for SNPs rs in both PCOS and control subjects. SNP rs and rs exists in complete linkage and their frequency distribution varies among different ethnic populations ranging from 0% in Kota tribe from South India to 54% in Australian aboriginals [13]. Dasgupta et al. [14], conducted a large scale study in Indian women comprising PCOS (n=250) and controls (n=299), reported a prevalence of SNP rs / rs less than 2% and complete absence of SNP rs in PCOS and controls. Similarly, the other SNP rs was reported only in Chinese woman in Singapore, however did not found in Indian and Malaysian population [15], Korean women [16], Finland, Denmark, Bengali/North-East India and Rwanda population [17]. The variant genotypes of SNP rs / rs are proposed as an ancestral allele found to have increased serum bioactivity and shorter half life, however, found to have more potent action at the receptor level compared to wild-type genotypes [18]. SNP rs /rs has high sensitivity towards GnRH stimulation in comparison to homozygotes [19]. Studies on hormonal levels in Brazilian PCOS women showed an association of variant genotypes with increased testosterone levels [20]. In controlled ovarian stimulation, women with LHβ SNP rs /rs variant genotypes are found to be hyposensitive for exogenous FSH supplementation [21]. Association of variant LHβ with female infertility has not been established with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 81

6 clearly, however, an association with reproductive disorders in Japanese women was widely reported [9, 10, 11]. On the other hand, there is evidence that LHβ SNP rs /rs protects obese women from developing symptomatic polycystic ovarian syndrome [8], however Indian women did not show any effect between obese and non-obese PCOS [14]. SNP rs /rs was found to be associated with higher testosterone levels in devoloping hyperandrogenism in PCOS in Brazilian women [20]. Functional studies on SNP rs in HEK293 cell lines did not show any affect on biological activity and receptor binding properties [17]. SNP rs found to be associated with endometriosis in Japanese [11], European [22] and Brazilian women with PCOS [23]. Biochemical studies showed an association between SNP rs A/A genotype low levels of LH and higher fasting glucose levels in PCOS women [24]. Though excess serum LH levels are involved in the androgen synthesis and recruitment of primordial follicles from the growing follicles in ovarian folliculogenesis, a key etiological factor in the pathogenesis of PCOS, the study results did not find any prevalence of LHβ variants. Concordant results obtained by RFLP and then with direct sequencing confirm the genotypes were not an artifact due to experiment and data analysis. References 1. Rahman NA and Rao CV. Recent progress in luteinizing hormone/human chorionic gonadotrophin hormone research. Molecular Human Reproduction. 2009; 15: with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 82

7 2. Jeppesen JV, Kristensen SG, Nielsen ME, Humaidan P, Dal Canto M, Fadini R et al. LH-Receptor Gene Expression in Human Granulosa and Cumulus Cells from Antral and Preovulatory Follicles. J Clin Endocrinol Metab. 2012; 97: Taylor AE, McCourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D et al. Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab. 1997; 82: Themmen APN, Huhtaniemi IT. Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function. Endocr Rev. 2000; 21: Patel K, Coffler MS, Dahan MH, Malcom PJ, Deutsch R, Chang RJ. Relationship of GnRH-stimulated LH release to episodic LH secretion and baseline endocrine-metabolic measures in women with polycystic ovary syndrome. Clin Endocrinol. 2004; 60: Rajkhowa M, Talbot JA, Jones PW, Pettersson K, Haavisto AM, Huhtaniemi I et al. Prevalence of an immunological LH b-subunit variant in a UK population of healthy women and women with polycystic ovary syndrome. Clin Endocrinol. 1995; 43: Elter K, Erel CT, Cine N, Ozbek U, Hacihanefioglu B, Ertungealp E. Role of the mutations Trp83Arg and Ile153Thr of the human luteinizing hormone b-subunit in women with polycystic ovary syndrome. Fertil Steril. 1999; 71: with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 83

8 8. Tapanainen JS, Koivunen R, Fauser BC, Taylor AE, Clayton RN, Rajkhowa M et al. A new contributing factor to polycystic ovary syndrome: the genetic variant of luteinizing hormone. J Clin Endocrinol Metab.1999; 84: Takahashi K, Ozaki T, Okada M, Kurioka H, Kanasaki H, Miyazaki K. Increased prevalence of luteinizing hormone beta-subunit variant in patients with premature ovarian failure. Fertil Steril. 1999; 71: Suganuma N, Furui K, Furuhashi M, Asada Y, Kikkawa F, Tomoda Y. Screening of the mutations in luteinizing hormone beta-subunit in patients with menstrual disorders. Fertil Steril. 1995; 63: Takahashi K, Karino K, Kanasaki H, Kurioka H, Ozaki T, Yonehara T et al. Influence of missense mutation and silent mutation of LHβeta-subunit gene in Japanese patients with ovulatory disorders. Eur J Hum Genet. 2003;11: Raivio T, Huhtaniemi I, Anttila R, Siimes MA, Hagena SL, Nilsson C et al. The role of luteinizing hormone-b gene polymorphism in the onset and progression of puberty in healthy boys. J Clin Endocrinol Metab. 1996; 81: Nilsson C, Pettersson K, Millar RP, Coerver KA, Matzuk MM, Huhtaniemi IT. Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. International Collaborative Research Group. Fertil Steril. 1997; 67: Dasgupta S, Sirisha PV, Neelaveni K, Anuradha K, Sudhakar G, Reddy BM. Role of luteinizing hormone β-subunit gene variants among South Indian women with polycystic ovary syndrome. Gene. 2012; 494: with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 84

9 15. Ramanujam L, Liao WX, Roy AC, Ng SC, Ratnam SS. Molecular variants of luteinizing hormone in three populations of Southeast Asia. Hum Hered. 1998; 48: Kim NK, Nam YS, Ko JJ, Chung HM, Chung KW, Cha KY. The luteinizing hormone beta-subunit exon 3 (Gly102Ser) gene mutation is rare in Korean women with endometriosis and polycystic ovary syndrome. Fertil Steril. 2001; 75: Lamminen T, Jiang M, Manna PR, Pakarinen P, Simonsen H, Herrera RJ. Functional study of a recombinant form of human LHβ-subunit variant carrying the Gly102Ser mutation found in Asian populations. Molecular Human Reproduction 2002; 8: Huhtaniemi I, Jiang M, Nilsson C, Pettersson K. Mutations and polymorphisms in gonadotropin genes. Mol Cell Endocrinol. 1999; 25:151: Takahashi K, Kurioka H, Ozaki T, Kanasaki H, Miyazaki K, Karino K. Pituitary response to luteinizing hormone-releasing hormone in women with variant luteinizing hormone. European Journal of Endocrinology. 2000; 143: Batista MC, Duarte Ede F, Borba MD, Zingler E, Mangussi-Gomes J, dos Santos BT et al. Trp28Arg/Ile35Thr LHΒ gene variants are associated with elevated testosterone levels in women with polycystic ovary syndrome. Gene. 2014; 550: Alviggi C, Pettersson K, Longobardi S, Andersen CY, Conforti A, De Rosa P et al. A common polymorphic allele of the LH beta-subunit gene is associated with with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 85

10 higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology. Reprod Biol Endocrinol. 2013; 11: Christofolini DM, Vilarino FL, Mafra FA, Andre GM, Bianco B, Barbosa CP. Combination of polymorphisms in luteinizing hormone β, estrogen receptor β and progesterone receptor and susceptibility to infertility and endometriosis. Eur J Obstet Gynecol Reprod Biol. 2011; 158: Mafra FA, Bianco B, Christofolini DM, Souza AM, Zulli K, Barbosa CP. Luteinizing hormone beta-subunit gene (LHβeta) polymorphism in infertility and endometriosis-associated infertility. Eur J Obstet Gynecol Reprod Biol. 2010; 151: Liu N, Ma Y, Wang S, Zhang X, Zhang Q, Zhang X et al. Association of the genetic variants of luteinizing hormone, luteinizing hormone receptor and polycystic ovary syndrome. Reprod Biol Endocrinol. 2012; 10:36. with polycystic ovary syndrome [Ph.D (RR)/146-FT/VII/08] Page 86

X/99/$03.00/0 Vol. 84, No. 5 The Journal of Clinical Endocrinology & Metabolism Copyright 1999 by The Endocrine Society

0021-972X/99/$03.00/0 Vol. 84, No. 5 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society A New Contributing Factor to Polycystic Ovary Syndrome: