Article CGG repeat sizing in the FMR1 gene in Indian women with premature ovarian failure

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1 RBMOnline - Vol 19 No Reproductive BioMedicine Online; on web 25 June 29 Article CGG repeat sizing in the FMR1 gene in Indian women with premature ovarian failure Suparna Chatterjee obtained her MSc in Microbiology from the University of Mumbai, India, in 22. She is currently a senior research scholar at the National Institute for Research in Reproductive Health, Mumbai. She works in the area of female infertility, the main focus of her work being on the genetics of premature ovarian failure. The work presented in this manuscript forms part of her PhD thesis. She has been instrumental in carrying out DNA extraction, PCR, repeat sizing, and data analysis. Ms Suparna Chatterjee Suparna Chatterjee 1,4, Anurupa Maitra 2,4, Seema Kadam 1,4, Zarine Patel 3, Jyotsna Gokral l,4, Pervin Meherji 1,4,5 1 Department of Reproductive Endocrinology and Infertility; 2 Department of Molecular Endocrinology; 3 Genetic Research Centre; 4 National Institute for Research in Reproductive Health (ICMR), Parel, Mumbai 4 12, India 5 Correspondence: meherji_per@rediffmail.com, meherjipkm@rediffmail.com Abstract The CGG repeat stretch in the FMR1 gene is polymorphic, ranging from 5 to 5 repeats in the normal population. Expansion of the repeats to the premutation range (5 2) has been associated with premature ovarian failure (POF). This case control study was conducted to enumerate CGG repeats in the FMR1 gene in 8 Indian women with non-familial, non-syndromic POF, and 7 controls from the same ethnicity. A possible association between CGG repeats and endocrine profile of these cases was investigated. All patients and controls had CGG repeats in the normal polymorphic range. Serum FSH concentrations were significantly raised in both and controls having CGG repeats in the 31 4 repeats range (P <.1). and controls had FSH concentrations of versus 84.2 miu/ml and 16. versus 6.2 miu/ml for >3 repeats versus <3 repeats respectively. Inhibin B concentrations were not associated with CGG repeats. The results of this study indicate that FMR1 premutations are rare in sporadic cases of POF with no family history of fragile X syndrome. However, although in the normal polymorphic range, expansion of the CGG repeat tract to beyond 3 repeats was associated with serum FSH concentrations in both and controls. Keywords: CGG repeats, FMR1 gene, India, POF, premutations Introduction The fragile X syndrome is the most common cause of X-linked mental retardation. The syndrome is caused by the expansion of a CGG repeat in the 5 untranslated region of the FMR1 gene, located at Xq27.3. In the normal population, the numbers of CGG repeats vary from 5 to 5, with an average of 3 repeats, and these do not change when transmitted through generations. Repeat tracts with 5 2 are unstable during transmission as a result of a mutational mechanism of trinucleotide repeat instability and expansion. They are referred to as premutation alleles in relation to their general lack of phenotype and their propensity to expand to disease-causing lengths in offspring. Expansion of the repeat beyond 2 is called full mutation; it leads to methylation, deacetylation and gene silencing. Subjects with full mutation present with mental retardation and a whole spectrum of clinical characteristics of the fragile X syndrome. Unlike full mutation alleles, the premutation alleles have no effect on FMR1 transcription. However, some females bearing the permutated allele have been shown to manifest with premature ovarian failure (POF) (Conway, 1995; Conway et al., 1998; Allingham- Hawkings et al., 1999; Bione and Toniolo, 2; Gersak et al., 23; Goswamy and Conway 25; Sullivan et al., 25). Premature ovarian failure is the cessation of ovarian function in a woman before the age of 4 years. Women with POF suffer from anovulation and hypo-oestrogenism and present with secondary amenorrhoea, infertility, sex steroid deficiency and elevated gonadotrophins (Kalantaridou et al., 1998). The condition affects approximately 1% of women in the general population and 4 18% of women with secondary amenorrhoea (Coulam et al., 1986; Anasti, 1998). Early loss of ovarian function has significant psychosocial sequelae and major health implications (Taylor, 21) A possible association between fragile X syndrome and POF was first investigated by Cronister et al. in 1991, following which several studies have detected women with POF among FRAXA Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 282 carriers (Schwartz et al., 1994; Turner et al., 1994) and FRAXA premutations among women with familial POF (Conway et al., 1995; Vianna-Morgante et al., 1996; Murray et al., 1998). An extensive international collaborative study conducted in 1999 reported POF phenotype in 16% of premutation carriers and.4% of controls. These data were further confirmed by another large study of 18 POF patients, of whom 6.5% carried the premutation allele. Murray et al. (2a,b) found no significant effect of either FMR1 repeat size or X chromosome inactivation pattern on the risk of POF. To further investigate the association of CGG repeats with ovarian functioning, some studies have explored the occurrence of endocrine signs of premature ovarian dysfunction in young fragile X premutation carriers not manifesting POF (Braat et al., 1999; Murray et al., 1999; Hundscheidet et al., 21), and have shown higher serum concentrations of FSH as compared with controls. Similarly, inhibin B, a negative regulator of FSH produced by (pre)antral follicles (Roberts et al., 1993) in the early follicular phase, was also investigated as the earliest marker of reproductive ageing (Welt et al., 1999), but no association was found. The estimated rate of POF among carriers is 21%. The frequency of premutation carriers among women with the sporadic form of POF has been found to range between 1.6 and 3.3% (Kenneson et al., 1997; Conway et al., 1998; Murray et al., 1998; Uzielli et al., 1999; Marozzi et al., 2) in a Caucasian population, with one study reporting 4.8% in a group of Slovenian patients screened (Gersak et al., 23). The frequency of premutation carriers among women with idiopathic familial POF is 6 13%, which is higher than that for the general population (1 in 25). There are differences in repeat distribution between different populations, even in controls. The Caucasian population reports a trimodal distribution, with modes at 2 and 3 and a small mode at 4 repeats (Murray, 2). Unfortunately, no such reference data are available from the Indian population, to enable a comparison between different ethnicities. Although there is evidence of an association between ovarian dysfunction and premutation alleles (Bione S and Toniolo D, 2; Gersak et al., 23; Sullivan et al., 25), the association is far from complete. Many fragile X families do not have members with POF, and some population-based studies have failed to detect the association (Kenneson et al., 1997; Marozzi et al., 1999). Various mechanisms have been invoked to explain the discrepancy in results, including linkage disequilibrium (Kenneson et al., 1997), population differences and imprinting mechanisms (Hundscheid et al., 2). The premutation allele has been found to co-segregate with POF in families (Conway et al., 1995). But how far this is true in sporadic cases of POF, with no history of fragile X syndrome or mental retardation, still needs to be investigated. This study was designed to test the above hypothesis in nonsyndromic, non-familial cases of POF in an Indian setting. Additionally, it aimed to determine an association between CGG repeats and endocrine profile in these cases. So far as is known, this is the first study to investigate CGG repeats in in the Indian population and also the first to investigate whether an association exists between FSH and inhibin B concentrations with CGG repeat numbers. Materials and methods Study subjects A total of 8 women with clinically confirmed POF visiting the Reproductive, Endocrine and Infertility Clinic of the Institute were recruited for the study (Table 1). Out of these, only 78 were enrolled, as two were found to harbour a mutation in the FOXL2 gene. POF status was defined as the cessation of ovarian function for a period of >6 months before or at the age of 4 years, with a high concentration of FSH >4 miu/ml, detected on two different occasions. All subjects had normal pubarche and menarche and were phenotypically normal with no family history of POF or fragile X syndrome. All patients gave their informed consent to check their medical history and FRAXA status and for collection of a peripheral blood sample suitable for further molecular analysis. The recruited cases, aged 2 38 years had secondary amenorrhoea for 6 months or more (Chatterjee et al., 27). There was no history of mumps, surgery, chemotherapy or radiation to pelvic region. Out of the 8 women recruited, only nine had conceived spontaneously prior to the onset of POF. Women with abnormal karyotype and autoimmune disorders were excluded from the study. Seventy normally cycling women served as age-matched controls (Table 1). All women had experienced normal pubarche and menarche and had never undergone any kind of gynaecological procedures, including assisted reproduction. Out of the 7 control women, 43 were of proven fertility, having conceived spontaneously. The others were either of single marital status or yet to plan their families (Table 1). All women were phenotypically normal, displaying no features of fragile X syndrome, nor did they have a family history of mental retardation, fragile X syndrome or POF. Blood was drawn from all control women during the follicular phase of the cycle (days 3 5). Serum FSH and inhibin B concentrations were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) respectively, using commercial kits from Diagnostic Systems Laboratories (Webster, USA); karyotyping was performed by routine G-banding (Seabright, 1971). DNA extraction and polymerase chain reaction (PCR) Genomic DNA was extracted from 5 ml of blood by conventional salting out method (Lahiri, 1991). The region on exon 1 of the FMR1 gene harbouring the CGG repeats was amplified by PCR, using forward primer A and reverse primer, 571R (Chong et al., 1994). The PCR protocol used was a modified version of that used previously (Chong et al., 1994). Tris chloride 2 mmol/l, 1 mmol/l KCl, 1 nmol/l (NH4) 2 SO 4, 2 mmol/l MgCl 2,.1% Triton X1, 1 μg/ml BSA, 15% DMSO and 5 pmole of each primer were used in the reaction. Aliquots of.2 mmol/l of each dntp were used except for dgtp, which was used in the ratio of 1:1 with 7-deaza dgtp. Pfu polymerase 1.25 IU was used for amplification. PCR was performed in an MJ Research thermal cycler (GMI Inc., Ramsay, MN, USA) with global denaturation at 98ºC for 5 min. Thirtyfive cycles of amplification were carried out, with denaturation

3 at 98º for 1 min, annealing at 65ºC for 1 min and extension at 75ºC for 2 min. A final extension of 2 min was given at 75ºC. This modification helped in elimination of smears and obtaining cleaner PCR products when run on polyacrylamide gels. Inclusion of 15% DMSO facilitated enhanced amplification of GC rich fragments and incorporation of 7-deaza dgtp reduced DNA secondary structure formation in GC rich regions, hence enhancing PCR efficiency. A rapid, simple, yet accurate PCR gel-based method of determining allele sizes was used in this study. Aliquots of 1 μl of the PCR products were subjected to electrophoretic separation on 3.5% agarose gels and observed by ethidium bromide staining. Products were run for sufficient time to allow resolution. Further, 5 μl of the PCR products were separated on 1% polyacrylamide gel at 15 V for 4 h. Bands were visualized by silver staining by the conventional method (Pooart et al., 1999). Tris/borate/EDTA buffer 1 was used as the buffer system for both runs. Calculation of CGG repeats The primers used would be expected to generate a 4-base pair (bp) PCR product with a 2 CGG repeat allele. They were divided into four groups depending on the repeat size, i.e. 5 1 repeats, 11 2 repeats, 21 3 repeats and 31 4 repeats. Statistical analysis Unpaired t-test was performed to test the significance of the difference in serum FSH and inhibin B concentrations among the two groups, based on CGG repeat size. Calculations were done on Statistics Package for Social Sciences (SPSS) software (SPSS Inc., USA). Results Seventy-eight POF patients, the clinical characteristics of whom have been elaborated earlier (Chatterjee et al., 27), and 7 controls were screened to enumerate the CGG repeats in the FMR1 gene on the X chromosome. CGG repeat sizing Electrophoretic separation of the PCR products by polyacrylamide gel electrophoresis provided more accurate information as compared with agarose gels (Figure 1). All patients and controls screened were found to have CGG repeats in the normal polymorphic range (5 5), with 6% (47/78) of and 57% (4/7) of controls falling in the range of 21 3 CGG repeats (Figure 2). Some cases showed PCR products of two different sizes, indicating heterozygous alleles (Figure 1, lanes P1, P6, P7 and P8). Neither nor controls were found to have CGG repeats falling in the premutation or full mutation range. Southern blot analysis, as is commonly used for confirmation of premutation alleles, was hence not required to be performed in this study. Endocrine profiling FSH concentrations were found to be significantly raised in as compared with controls, as opposed to inhibin B concentrations, which were found to be significantly reduced in (both P <.1; Figure 3a,b). CGG repeats and endocrine profile Serum FSH concentrations were found to be significantly raised (P <.1) in samples falling in the 31 4 repeats range as compared with those having fewer than 3 repeats in both POF cases and controls (Figure 4). having fewer than 3 repeats had a mean FSH concentration of 84.2 miu/ml as compared with those having more than 3 repeats (133.7 miu/ ml). Although well within the normal range (3 2 miu/ml, day 3 FSH), a similar trend was observed in the controls, among whom women with fewer than 3 repeats had a mean FSH of 6.2 miu/ ml as compared with 16. miu/ml in women with more than 3 repeats (Figure 4). However, when inhibin B concentrations were compared, there was no significant difference in samples falling in the 31 4 repeats range as compared with those having fewer than 3 repeats in both and controls (Figure 5). Table 1. Characteristics of patients with premature ovarian failure (POF) and controls. Parameter Patients (n = 78) (n = 7) Age (years) 31. ± 2.7 (2 38) 32.2 ± 3.9 (22 4) Age at menarche (years) ± 3.8 (11 22) ± 1.9 (11 17) Normal cycle (%) a Menstrual cycle length (days) a 26.3 ± 1.9 (23 3) 27.1 ± 1.8 (23 3) Irregular cycle (%) a Amenorrhoeic since (age, years) 23.2 ± 15. NA Normal ovaries (%) Streak ovaries (%) LH (miu/ml) b ± b ± 1. Proven fertile (n) 9 43 Spontaneous abortions (n) 3 Failed to conceive spontaneously (n) 64 Of single marital status 2 25 Married but yet to plan a family 2 a In POF patients, prior to onset of amenorrhoea. b Values with the same superscript letter are significantly different (P <.1, Student s t-test). NA = not applicable. 283

4 Figure 1. CGG repeat sizing by polymerase chain reaction polyacrylamide gel electrophoresis (PCR PAGE). Lanes P1, P4, P5, P6, P7, P8: premature ovarian failure cases, lanes C1, C2, C3: controls, marker for PAGE. Arrows depicting the PCR amplified products of interest POF 5. % Frequency CGG repeat number Figure 2. Frequency distribution of CGG repeats in premature ovarian failure (POF) cases and controls. a FSH (miu/ml) * 284 b Inhibin B (pg/ml) * Figure 3. (a) Serum FSH concentrations in premature ovarian failure (POF) patients and controls. *Significantly different (P <.1) as compared with FSH concentrations in controls, by Student s t-test. (b) Serum inhibin B concentrations in POF patients and controls. *Significantly different (P <.1) as compared with inhibin B concentrations in POF patients, by Student s t-test. FSH (miu/ml) <3 repeats >3 repeats * <3 repeats >3 repeats Figure 4. Comparison of serum FSH concentrations and CGG repeats in premature ovarian failure (POF) cases and controls, based on repeat numbers. *Significantly different as compared with controls having <3 repeats by Student s t-test, P <.1. **Significantly different as compared with having <3 repeats by Student s t-test, P <.1. **

5 Inhibin B (pg/ml) Discussion <3 repeats >3 repeats <3 repeats >3 repeats Figure 5. Comparison of serum inhibin B concentrations in premature ovarian failure (POF) cases and controls based on repeat numbers. Seventy-eight women with clinically confirmed POF and 7 agematched controls were screened to enumerate the CGG repeats in exon 1 of the FMR1 gene. It was ensured that the recruited had no family history of POF, mental retardation or fragile X syndrome. Women from both groups were found to have CGG repeats in the normal range of 5 5, with a majority of women falling in the 21 3 repeats range. Studies from the Caucasian population report premutation frequencies of about 12 21% from familial, and a bare 1.6 3% of sporadic, though some groups have also suggested that FMR1 premutations are not a significant cause of ovarian failure (Patsalis et al., 197; Kenneson et al., 1997; Marozzi et al., 1999). The discrepancy in results maybe attributed to the choice of study population, the exclusion as well as inclusion criteria set for the study, and the ethnicity of the population. In this study, although no patient or control was found to have CGG repeats in the premutation range, it was interesting to note increased concentrations of FSH in both and controls having CGG repeats in the 31 4 repeats range. When both patients and controls were further segregated into two groups, one with fewer than 3 repeats and the other with more than 3 repeats, a significant difference was observed. FSH concentrations were found to be significantly higher in those patients and controls with more than 3 CGG repeats as compared with their counterparts with fewer than 3 CGG repeats, implying that although in the normal polymorphic range, FSH secretion is somehow affected by an expansion of the CGG repeat tract to beyond 3 repeats. This is of special importance in relation to the control women who are of reproductive age, as high FSH concentration is indicative of poorer prognosis for future pregnancy. Out of the 1 control women with >3 repeats, four were proven fertile. The other six women in their mid and late 2s were either unmarried or yet to plan their families. It is rare for women to be fertile with a follicular phase FSH >2 IU/l (O Herlihy et al., 198), and therefore the results of this study have important implications for fertility. Earlier reports have shown an association between serum FSH concentrations and CGG repeats in premenopausal premutation carriers, where increased serum FSH concentration was detected in premutation carriers as compared with the other women, irrespective of menstrual cycle pattern, indicating that they may already be suffering from ovarian ageing. However, this association was not found to hold good with other repeat ranges (Hundscheid et al., 21). Although inhibin B concentrations were found to be reduced significantly in as opposed to controls (as is true in such cases), no association was obtained within the two groups of <3 and >3 CGG repeats. Increase in CGG repeat numbers was not reflected by a corresponding decrease in inhibin B concentrations, or the converse. This is in line with two studies that found no significant association when inhibin B concentrations were compared among women with CGG repeats in the premutation range (Murray et al., 2a,b; Hundscheid et al., 21). Although inhibin B has in some instances been proposed to be the first marker of ovarian ageing (Welt et al., 1999), its clinical relevance is still elusive and somewhat controversial. Even though, in this study, CGG repeats in the premutation range were not detected, it is of importance to note the increased FSH concentrations detected in patients and controls in the 31 4 repeats range, which is precariously close to the intermediate allele range (41 58 CGG). It has been previously reported that intermediate alleles have the potential for being unstable when transmitted, leading to a full mutation in several generations (Bodega et al., 26). This suggests that alleles shorter than the canonical FMR1 premutation range, but characterized by specific molecular features, could also play a role in POF manifestation. Hence it would be of interest to carry out sequence analysis of these alleles to see if molecular features similar to those reported earlier, also exist in this group of patients. Since the risk for CGG expansion to a full mutation is directly related to the number of CGG repeats in the carrier mother, mutation and premutation carriers with >7 repeats or those already with an affected offspring, preimplantation genetic diagnosis (PGD) has been suggested. Earlier studies have shown that PGD performed by single cell PCR are reliable and sensitive (Malcov et al., 27). Results of this study, from an Indian setting, indicate that FMR1 premutations are rare in sporadic cases of POF who have no family history of fragile X syndrome or mental retardation. To date, this is the first study to have explored the association of FSH profile with CGG repeats in and controls with repeats in the normal polymorphic range. Women with POF may only hope to conceive, as is seen to naturally occur in.1% of cases. However, those in the control group may be informed of a high possibility of early ovarian ageing, as from a practical point of view, women found to have raised FSH concentrations respond poorly to all modes of fertility treatment (Scott and Hofman, 1995). However, if detected at an early stage (FSH <15 IU/l), conception may still be possible, perhaps with the assistance of ovulation induction techniques. Acknowledgements The authors would like to acknowledge Dr Chander P Puri, former Director, NIRRH, for his constant encouragement and support during the course of the study. Sincere thanks are also 285

6 286 due to the Lady Tata Memorial Trust for providing Research Scholarship to Miss Suparna Chatterjee. This manuscript NIRRH/MS/23/28 has been supported by the Indian Council of Medical Research (ICMR), New Delhi, India under the Genomics and Molecular Medicine Programme. References Allingham-Hawkins DJ, Babul-Hirji R, Chitayat D et al Fragile X premutation is a significant risk factor for premature ovarian failure: the International collaborative POF in fragile X study preliminary data. American Journal of Medical Genetics 83, Anasti JN 1998 Premature ovarian failure: an update. Fertility and Sterility 7, Bione S, Toniolo D 2 X chromosome genes and premature ovarian failure. 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