Intermediate and premutation FMR1 alleles in women with occult primary ovarian insufficiency

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1 GENETICS Intermediate and premutation FMR1 alleles in women with occult primary ovarian insufficiency Isabelle Streuli, M.D., a Timothee Fraisse, M.D., a Victoria Ibecheole, M.D., a Isabelle Moix, M.Sc., b Michael A. Morris, D.Phil., b and Dominique de Ziegler, M.D. c a Division of Reproductive Endocrinology and Infertility and the b Molecular Diagnostic Laboratory, Service of Genetic Medicine, University Hospitals of Geneva, Geneva; and c Reproductive Medicine Unit, University Hospital of Lausanne, Lausanne, Switzerland Objective: To compare the prevalence of intermediate and premutation FMR1 alleles in women with occult primary ovarian insufficiency (opoi) and in controls. Design: Observational study. Setting: Division of Infertility and Service of Genetic Medicine, Geneva University Hospitals. Patient(s): The study group consisted of 27 infertile women with opoi referred by infertility specialists for FMR1 testing in because of unexplained poor response to controlled ovarian hyperstimulation or altered hormonal profiles. The control group consisted of 32 women undergoing genetic testing for conditions unrelated to mental retardation or ovarian function. The DNA samples were anonymized. Intervention(s): In the study group, data were collected concerning reproductive/family history, hormonal markers, possible fertility treatment outcomes, and results of karyotype and FMR1 testing. In the control group, FMR1 gene testing was done. The only clinical data available in controls were sex and indication for genetic testing. Main Outcome Measure(s): Distribution of FMR1 alleles. Result(s): Six (22%) of 27 women with opoi had FMR1 alleles of >40 repeats (intermediate to premutation range), compared with one (3%) of 32 controls. Conclusion(s): These results suggest that women with opoi might be at risk of carrying alleles in the intermediate and premutation range. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Fragile X syndrome, FMR1 gene, occult primary ovarian insufficiency, premature ovarian failure, premutation, intermediate allele Received August 19, 2007; revised June 6, 2008; accepted July 9, 2008; published online October 29, I.S. has nothing to disclose. T.F. s spouse is an employee of Merck. V.I. has nothing to disclose. I.M. has nothing to disclose. M.A.M. has nothing to disclose. D.Z. is a consultant for Institut Biochimique SA (IBSA) and Ferring and owns stock in Ultrast. Reprint requests: Isabelle Streuli, M.D., Reproductive Endocrinology and Infertility, Geneva University Hospitals, 30 Boulevard de la Cluse, Geneva, Switzerland (FAX: ; Isabelle.Streuli@ hcuge.ch). Fragile X syndrome (FXS) is the most common inherited cause of mental impairment and autism (1). This X-linked disorder is caused by the absence of a protein coded by the fragile X mental retardation 1 gene (FMR1) (2). The genetic determinants of FMR1 are complex and are related to the length of a polymorphic expansion of CGG trinucleotides in the 5 untranslated region of the gene (2). The lengths of repeats define alleles in normal, intermediate, premutation, and full mutation ranges. Consensus has not been reached for the intermediate and premutation allele endpoints. For this article, we will use the boundaries set by the American College of Obstetricians and Gynecologists (ACOG) (3). Alleles with %40 CGG repeats are considered normal, the most common alleles being of repeats (4). Alleles ranging from 41 to 60 are defined as intermediate and can become unstable when transmitted from parent to child, potentially leading to full mutations after several generations (5, 6). Premutation alleles are defined by the presence of repeats and have a high probability of expanding to full mutations after one to three generations (7). Approximately one of 250 to one of 450 Caucasian women are carriers of FMR1 premutations (8). Full mutations have over 200 repeats and are hypermethylated and transcriptionally silent, leading to the absence of the FMR1 protein (FMRP) and the phenotypic expression of classic FXS (9, 10). The FMR1 premutation is now a well-established cause of primary ovarian insufficiency (POI) (11 16). Full mutation carriers, however, have not been shown to have altered ovarian function. Recent studies suggest a nonlinear association between the number of repeats and ovarian function. Women with premutations in the midsize range seem to be at the highest risk of ovarian insufficiency (17). Different alterations in markers of ovarian function have been described 464 Fertility and Sterility â Vol. 92, No. 2, August /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 in premutation carriers, such as shortened follicular phases, raised FSH levels, and low anti-m ullerian hormone (AMH) levels. Menopause seems to be shifted 5 years earlier in that population compared to controls (18 24). However, precise data on the influence of the FMR1 gene on fertility and the prevalence of the premutation in the infertile population are still missing. Different terms have been used in medical literature to describe the spectrum of clinical signs associated with ovarian function decline. Certain authors recommend that the term premature ovarian failure (POF) should be abandoned in favor of the use of the scientifically more accurate term primary ovarian insufficiency (POI) (25 27). Descriptors to identify the severity of ovarian dysfunction may precede the term POI. Throughout our article, we will use occult and overt to qualify the severity of ovarian insufficiency. Occult POI (opoi) describes partial ovarian insufficiency in women under the age of 40 including infertility, slightly raised FSH levels, low AMH levels, and/or a resistance to ovarian stimulation in women with either regular or irregular cycles (28 33). Overt POI is defined as the association of amenorrhea for at least 4 consecutive months and elevated gonadotropin levels in women under the age of 40 years, a condition previously called POF (34). However, the lack of evidence of natural progression from early signs of ovarian insufficiency to a state of frank hypergonadotropic amenorrhea must be borne in mind. Overt POI differs from menopause in that affected women might experience periods of remission of their ovarian function with follicular development and ovulation (35). Spontaneous pregnancies have been reported in women diagnosed with overt POI (36, 37). Overt POI affects approximately 1% of women in the general population (35), but the prevalence can be as high as 13% 26% (11) in premutation carriers. Conversely, 0.8% 7.5% of women affected by overt POI are carriers of the premutation (11, 13 16); this percentage rises to 13% if there is a family history of POF. The lower boundary of FMR1 repeat sizes that alter ovarian function has not been defined. Some recent articles have suggested that intermediate alleles, and even high normal range alleles, are more frequent (4.7%) in POI patients than in the general population (38, 39). Full mutation carriers, however, appear to have no alterations of their ovarian function (11, 20), which suggests that the ovarian dysfunction in premutation carriers is not caused by the absence of FMRP. In contrast, the increased expression of mrna (40, 41) that is observed as repeat numbers increase is believed to be the cause of the two pathologies seen in the premutation carriers, POI and FXTAS (FXS-associated ataxia/tremor syndrome), as RNA protein complexes accumulate and interfere with normal cell function, finally causing cell death (42, 43). As the link between ovarian insufficiency and the FMR1 gene became apparent, recommendations for testing were actualized. The 2006 ACOG Committee Opinion states that testing for a premutation should be considered if a woman has ovarian failure or an elevated follicle-stimulating hormone level before the age 40 years without a known cause (3). The American College of Medical Genetics (ACMG) also recommends considering testing in women who are experiencing reproductive or fertility problems associated with elevated FSH levels, especially if they have family history of POF, family history of FXS, or male or female relatives with undiagnosed mental retardation (44). The increased awareness of links between FMR1 premutations and POI led infertility specialists in Geneva to request FMR1 testing more frequently from early 2005; 30 tests were requested by infertility specialists in 2005, compared with a previous average of half a dozen per year. Forty-four women were referred by infertility specialists for FMR1 testing between January 2005 and September Most women referred had signs of occult ovarian insufficiency but did not meet the criteria for overt POI. The link between premutations and overt ovarian insufficiency has been well established, but whether women with opoi might also be at risk for expansions in the FMR1 gene is not clear. The primary aim of our study is to establish whether intermediate and premutation alleles (alleles of >40 repeats) are more frequent in the population of infertile women with opoi than in a control population. MATERIALS AND METHODS The study protocol was reviewed and accepted by our Institutional Review Board. Clinical Data A case report form was completed retrospectively for each woman by her infertility specialist. The form included questions related to the reproductive status of the patient (motive of consultation, duration and etiology of infertility, length and regularity of menstrual cycles, and response to controlled ovarian hyperstimulation [COH]), hormonal profile (day 3 FSH, E 2, and AMH levels), and family history of POF, mental retardation, and neurodegenerative disease. For women who had undergone cycles of COH, data regarding the antral follicular count (AFC) and the number of follicles retrieved were not available in most cases. If more than one hormonal sample was available per patient, the highest FSH and E 2 levels and the lowest AMH levels were considered. Classification of Ovarian Function The 44 women tested for FMR1 premutations were classified into three categories according to the results of the retrospective assessment of their ovarian function. Women with regular or irregular menstrual cycles, normal hormonal levels, and a normal response to COH were classified as having a normal ovarian function. Women with regular or irregular cycles and elevated FSH levels >10 IU/L and/or Fertility and Sterility â 465

3 decreased AMH levels <7 pmol/l and/or a poor response to COH were included in the opoi group. Women with amenorrhea for at least 4 consecutive months and elevated FSH levels (>10 UI/L) were considered to have overt POI. According to this classification, nine women had a normal ovarian function, 28 had opoi, and seven met the diagnostic criteria for overt POI. An FSH cutoff level of 10 IU/L measured by enzyme-linked immunoassay was chosen, since FSH levels above that level are associated with diminished pregnancy and birth rates after assisted reproductive techniques (45). Study and Control Populations In the study group, we included 28 women with opoi referred by infertility specialists for FMR1 testing at the Geneva University Hospitals between January 2005 and September One woman was excluded after she was found to have a 45,X/ 46,XX mosaicism, with X chromosome monosomy in 8% of cells but no phenotypical signs of Turner syndrome. Women with overt POI and those with a normal ovarian function referred in the context of a positive family history were not included. In the control group, the FMR1 gene was tested in a population of 32 women referred for genetic testing for a condition unrelated to fertility or to mental retardation. After selection based on ethnic origin, sex, and the indication for referral, the DNA samples were irreversibly anonymized. No data concerning fertility were available in these women. The FMR1 testing in the control group was approved by the Institutional Review Board in an amendment. FMR1 Repeat Length DNA was isolated from peripheral blood lymphocytes by conventional techniques and stored at 4 C. The FMR1 CGG repeat length size was determined in both groups by our accredited diagnostic procedure. The repeat region was polymerase chain reaction amplified with fluorescently labeled primers and sized by capillary electrophoresis (precision 1 repeat). When only one allele size was identified, Southern blotting was performed to exclude the presence of a large (unamplifiable) full mutation. We used the endpoints defined by ACOG (3) and considered alleles of %40 repeats as normal, of repeats as intermediate, of repeats as premutation, and of >200 repeats as full mutation alleles. Statistical Analysis Power analysis was performed using PS Power and Sample Size Calculations, version (WD Dupont and WD Plummer, Nashville, TN). Power was 80% to show an odds ratio of 8.6 between alleles and 9.9 between individuals in the two groups with a type I error of 5%. Statistical analysis was performed using SPSS software, version (SPSS, Inc., Chicago) and Acluster software, version 2.0. (World Health Organization, Geneva, Switzerland). Fisher s exact test was used to compare the proportion of women with alleles of >40 CGG repeats in the study group and controls. The same test was also used to compare biallelic means. To take into account a possible correlation between alleles, we also used Acluster software to compute P-values and odd ratios adjusted for this correlation when comparing alleles. P<.05 was considered statistically significant. Results are expressed as odd ratio and 95% confidence interval (CI). RESULTS The mean age of the study population is 34.5 years (SD 4.3). The characteristics of the study population are described in Table 1. TABLE 1 FMR1 expansions in the study group. Proportion (%) Motive of consultation: Infertility 26/27 (96%) Family history of POF 1/27 (4%) Family history: POF 4/27 (15%) Mental retardation 3/27 (11%) Neurodegenerative 2/27 (7%) disease Fragile X syndrome 0/27 (0%) Menstrual cycles: Regular 17/27 (63%) Irregular 10/27 (37%) Amenorrhea 0/27 (0%) Controlled ovarian hyperstimulation (COH): No COH 8/27 (30%) COH 19/27 (70%) Response to COH : Normal response 0/19 (0%) Poor response 19/19 (100%) FSH levels : a. with E 2 levels <40 pg/ml: < 10 IU/L 3/27 (11%) IU/L 9/27 (33%) > 15 IU/L 11/27 (41%) b. with E 2 levels >40 pg/ml 4/27 (15%) AMH levels : > 7 pmol/l 5/26 (19%) < 7 pmol/l 21/26 (81%) Streuli. FMR1 expansions in ovarian insufficiency. Fertil Steril Streuli et al. FMR1 expansions in ovarian insufficiency Vol. 92, No. 2, August 2009

4 FIGURE 1 Distribution of FMR1 alleles in women with opoi and in controls. Streuli. FMR1 expansions in ovarian insufficiency. Fertil Steril FMR1 Allele Size We compared the distribution of the 54 FMR1 alleles of women with opoi with the distribution of the 64 alleles of controls (Fig. 1). The most common allele in both groups is 30 repeats. In the study group, the mean allele size is 33 repeats, with a range of repeats. In the control group, the mean allele size is 28 repeats, with a narrower range of alleles from 16 to 41 repeats. We also compared biallelic means between the two groups. In the study group, the mean biallelic mean is 32 repeats (SD, 8) compared with 28 repeats (SD, 4) in the control group. This difference is significant, with P ¼.04. In the control group, one (1.5%) allele of 62 is in the intermediate range, and none is in the premutation range. In the study group, seven (13%) of 54 alleles have 41 or more repeats; four (7.5%) alleles are in the intermediate range, and three (5.5%) are in the premutation range. In the study group, there are four (7.5%) of 54 upper normal alleles compared with one (1.5%) of 64 in controls. Separate comparisons of upper normal, intermediate, and premutation alleles between groups are not statistically significant, with P>.1. The difference is statistically significant, with P ¼.02, when comparing alleles of >40 repeats in both groups. The odds ratio of having an allele of >40 repeats in alleles of Fertility and Sterility â 467

5 women with opoi compared with alleles of controls is 9.4 (95% CI ¼ ). A separate odds ratio for combined high-normal and intermediate alleles (35 60 repeats) was calculated. In the study group, four alleles are in the high-normal and four in the intermediate ranges (8/54, 15%). In the control group there are one high-normal and one intermediate allele (2/62, 3%). This represents an odds ratio of 5.4, with a 95% CI of (P¼.015). One woman was found to have both alleles equal to or longer than 41 repeats, so that the seven expanded alleles were identified in six women. Six (22%) of 27 women with ovarian insufficiency have alleles higher than 40, compared with one (3%) of 32 control women. This difference is significant, with P ¼.04. The odds ratio of having an allele of 40 or more repeats in women with opoi compared with controls is 8.8, with a 95% CI of One woman was found to have both alleles equal to 40 repeats, so that seven (26%) of 27 women have alleles in the upper to intermediate range (35 60 repeats) in the study group compared with two (6%) of 32 in the control group. This difference is nonsignificant, with P ¼.065. Clinical Profile of Women with Premutations Patient 1 is a 37-year-old nulligravida consulting for infertility and reporting irregular cycles of days for 7 months. She has a family history of premature cessation of menstruation in her mother and her sister, before age 40. Investigations show an AFC of 2 follicles, FSH levels of 9.8 and later of 87 IU/L, undetectable AMH levels, and a 46,XX karyotype. She is found to be a carrier of a premutation of 76 repeats and an intermediate allele of 42 repeats. Patient 2 is a 39-year-old gravida 2, para 1 consulting for infertility and reporting irregular cycles of days since she stopped using contraception 1 year earlier. Her sister had experienced a premature cessation of menstruation at the age of 27 years. Investigations show FSH levels of 29 IU/L, undetectable AMH, and a 46,XX karyotype. She is diagnosed with a premutation allele of 77 repeats and a normal allele of 33 repeats. Patient 3 is a 31-year-old gravida 3, para 0 who consulted for infertility with a history of repeated early pregnancy loss. She has regular 28-day cycles. Investigations show FSH levels of 4.9 and 8.3 IU/L, AMH at 1.9 pmol/l, an AFC of 5 follicles, and a normal 46,XX karyotype. She responded poorly to ovarian hyperstimulation. She is diagnosed as a premutation carrier with an allele of 78 repeats and a normal allele of 30 repeats. DISCUSSION The results of our study suggest that FMR1 alleles of >40 repeats are more frequent in the study population of infertile women with occult ovarian insufficiency than in controls. Alleles ranging from 35 to 60 repeats (upper normal to intermediate) were more frequent among alleles of women with opoi compared with controls. This difference could not be confirmed when comparing individuals. These findings support the results of two studies that showed that intermediate, premutation, and even highnormal alleles are more frequent in patients (38, 39) with POI. However, these earlier studies included only women with overt POI and did not include milder forms of ovarian insufficiency. Different mechanisms have been hypothesized to explain the reduced ovarian reserve in premutation carriers. Either a decreased antral follicular pool or an accelerated follicular atresia may account for this depletion. Conway et al. proposed that alleles in the premutation range might interfere with the transcription of the FMR1 gene in the fetal ovary and reduce the follicular pool in utero (46). Premutation carriers have been shown to have a reduced FMRP expression with increased mrna levels (41, 42), leading to the hypothesis of the possible toxic effect of FMR1 premutation transcripts that could induce follicular atresia and apoptosis. These transcripts could also be the cause for an early ovarian dysfunction leading to a reduction in the initial follicular reserve (11). Our study s primary limitation stems from its design and from the selection of the study population. The study was designed to evaluate the current practice in Geneva, which is to refer certain women with signs of ovarian insufficiency for FMR1 premutation testing. This selection bias possibly leads to a study population with a greater clinical suspicion of FMR1-related ovarian insufficiency. A proportion of women was probably referred because of a combination of family history suggestive of fragile X related disorders and signs of POI. Our population might therefore not be representative of all women with ovarian insufficiency. Other limitations stem from the retrospective data collection and from its small sample size. The data were collected by a questionnaire sent to each subject s infertility specialist and might therefore be subjected to variability in the interpretation of the questions. Certain questions concerning menstrual cycles or the response to COH had categorical answers and might therefore lack precision. Our results suggest that alleles of >40 repeats (intermediate to premutation range) are more frequent in women with opoi. However, the study did not have sufficient power to show a difference when analyzing upper normal, intermediate, and premutation alleles separately. We were able to show a difference in alleles ranging from 35 to 60 repeats when comparing alleles. When comparing individuals the difference was not significant. Our results emphasize the need to initiate further prospective testing with a larger sample to confirm our findings and to determine the odds ratio of FMR1 expansions in women with occult ovarian insufficiency more precisely. 468 Streuli et al. FMR1 expansions in ovarian insufficiency Vol. 92, No. 2, August 2009

6 FMR1 testing has a dual role in patients with ovarian insufficiency: determining the probable cause of ovarian failure and identifying women at risk of transmitting full mutations to their offspring. Premutation carriers are clearly at risk of transmitting a full mutation, since the repeats of that size are unstable and prone to expansions. Women with intermediate mutations are more difficult to counsel since their number of CGG repeats might not be unstable but could still be the cause of their ovarian insufficiency. If expansion carriers are identified, genetic counseling is required to discuss the consequences for the patient herself and for other members of her family, who may also carry an expanded allele. Prenatal diagnosis is available on request in specialized medical genetic laboratories for women at risk of transmitting a full mutation. In conclusion, our study suggests that women with signs of ovarian insufficiency are at increased risk of having FMR1 alleles in the intermediate and premutation ranges. In light of our findings, further efforts should be put into studying the incidence of FMR1 expansions in all forms of ovarian insufficiency including otherwise unexplained infertility. We suggest that FMR1 testing has a role in the comprehension of the etiology of ovarian insufficiency and in the prevention of transmission of FXS and should be considered before infertility treatments are initiated. Acknowledgments: The authors thank the infertility specialists of Geneva for their participation in the data collection: Dr. B. Bourrit, Dr. G. de Candolle, Dr. D. Chardonnens, Dr. N. Fournet, Dr. R. Martin du Pan, Dr. P. Mock, Dr. C. Reuse, and Dr. A. Stalberg. We also thank the reviewers for their help and suggestions and Dr. M. Boulvain for his help in the statistical analysis. REFERENCES 1. 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