Fragile X syndrome prenatal diagnosis: parental attitudes and reproductive responses

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1 Reproductive BioMedicine Online (2010) 21, ARTICLE Fragile X syndrome prenatal diagnosis: parental attitudes and reproductive responses M Xunclà a,b, C Badenas a,c,d, M Domínguez e, L Rodríguez-Revenga a,c, I Madrigal a,c, L Jiménez a, A Soler a,c,d, A Borrell d,f,asánchez a,c,d, M Milà a,c,d, * a Servei de Bioquímica i Genètica Molecular, Hospital Clinic, Barcelona, Spain; b Fundació Clinic per a la Recerca Biomèdica, Barcelona, Spain; c CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain; d IDIBAPS (Institut d Investigacions Biomèdiques August Pi i Sunyer), Barcelona, Spain; e Màster en Assessorament Genètic, IDEC- Universitat Pompeu Fabra, Barcelona, Spain; f Unitat de Diagnòstic Prenatal, Institut de Ginecologia i Obstetricia i Neonatologia, Hospital Clinic, Barcelona, Spain * Corresponding author. address: mmila@clinic.ub.es (M Milà). Mar Xuncla was born in 1976 in Spain. She studied Biology at the University of Girona, and Biochemistry at the University of Barcelona, Spain. She performed a master degree in the Universitat Autonoma of Barcelona in Human Biology. She is now working in the field of human genetics involved in Fragile X syndrome in the Hospital Clinic of Barcelona. Abstract Fragile X syndrome (FXS) is the most common inherited form of mental retardation. It is caused by a CGG repeat expansion, which results in hypermethylation and silencing of the FMR1 gene. The results from 213 FXS prenatal diagnoses performed in the study centre were reviewed. Family history of FXS or undiagnosed mental retardation (MR) were the reasons for referral and 64% of mothers were not aware of their status so prenatal and mother tests were performed at the same time. Among those women referred for family history of unknown MR, 17.6% were found to be FXS carriers. The attitudes and perceptions of the syndrome of 52 FXS carriers were also evaluated. Most of them had been diagnosed as carriers when the child was already born and the most common feeling was sadness, followed by impotence and guilt. The majority of them had received genetic counselling and they considered it useful. Regarding reproductive options, prenatal diagnosis was chosen by 40.5% of women. Prenatal diagnosis for FXS is a good reproductive option and it should be carried out whenever family history of MR is present. A high percentage of FXS carriers are detected following this approach. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: Fragile X syndrome, genetic counselling, molecular diagnosis, prenatal /$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 Fragile X syndrome prenatal diagnosis 561 Introduction Fragile X syndrome (FXS) is the most common inherited form of mental retardation with an estimated birth prevalence of one in 4000 males and one in 8000 females (Rifé et al., 2003). In 98% of cases the syndrome is caused by an expansion in the CGG trinucleotide repeat in the untranslated region of the first exon of the FMR1 gene (Fu et al., 1991; Oberlé et al., 1991; Verkerk et al., 1991). For normal individuals, repeats range between six and 55. Premutation carriers have between 55 and 200 repeats and are at risk of having affected children. Individuals with more than 200 repeats have the full mutation, which usually results in promotor hypermethylation leading to gene silencing and no FXS mental retardation protein production (Pieretti et al., 1991). Affected male individuals present mild to severe mental retardation, behavioural and language problems and exhibit characteristic physical features (Hagerman et al., 1991). Fully mutated females are usually less severely affected. FMR1 premutated individuals present different phenotypes and at least two associated disorders have been described: (i) Fragile X tremor/ataxia syndrome (FXTAS), affecting both male and female individuals older than 50 years; and (ii) Fragile X primary ovarian insufficiency (FXPOI), with 19% penetrance in the Spanish population (Rodriguez-Revenga et al., 2009). Molecular testing allows the detection of premutated females at risk of having affected children, making prenatal diagnosis feasible. FXS prenatal diagnosis has been offered to pregnant women since 1991, when the FMR1 gene was cloned (Fu et al., 1991; Oberlé et al., 1991; Verkerk et al., 1991). One aim of the current study was to evaluate 213 prenatal FXS diagnostics performed in the centre during a 15-year period, analysing the reasons for referral and the results obtained. Also, the attitudes and perceptions of FXS carriers were evaluated regarding their status, their risk perception, the feelings as anxiety or worry, reproductive options and the understanding of the syndrome after the genetic counselling. Materials and methods Prenatal diagnosis This study includes a total of 213 FXS prenatal tests performed between January 1993 and July 2009 in the Biochemistry and Molecular Genetics Department of the Hospital Clinic of Barcelona. The tests were performed for 178 women, 18 of whom had more than one pregnancy studied. All couples were previously informed about risks, reproductive options and limitations of prenatal diagnosis by a genetic counsellor and they consented to undergo the invasive procedure. They were personally informed about the results. Samples Most of the tests (180/213) were performed on chorionic villi samples, 31 tests were performed on amniotic fluid and two on fetal blood. The mean ± SD gestational age at the time of sampling was 12.8 ± 1.8 weeks for chorionic villi, 15.5 ± 3.4 weeks for amniotic fluid and 20.5 ± 2.1 weeks for fetal blood. In all tests, peripheral blood from mothers was requested and, when possible, paternal samples were also studied. Cytogenetic studies Fetal karyotype was performed on all prenatal samples as previously described (Soler et al., 2008). Molecular studies Molecular analysis of the FMR1 CGG repeat region was performed from genomic DNA by PCR and Southern blotting. Primers c and f (Fu et al., 1991) were used for PCR analysis (radiolabelled or fluorescent). Radiolabelled PCR products were separated on a 5% denaturing acrylamide/urea gel (Milà et al., 1994). For fluorescent PCR, the c primer was fluorescently labelled and the PCR products were analysed with an ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and results were analysed with the GeneMapper v3.5. Premutated alleles of <100 repeats were accurately sized using both PCR methodologies. For alleles of >100 repeats, Southern blotting was performed after double digestion with EcoRI and EagI using Stb12.3 probe and allele sizes were estimated using the Seqaid II version 3.81 program as previously described (Milà et al., 1994). In some samples, indirect analysis was needed. For this purpose, two intragenic markers (FRAXAC1 and FRAXAC2) and two extragenic markers (DXS998 and DXS548) were used (Reches et al., 2009). All forward primers were fluorescently labelled with fluorochromes FAM or tetrachlorofluorescein. PCR products were analysed with an ABI3100 Genetic Analyzer. When maternal status was unknown, maternal CGG repeat number was determined at the same time as the fetus. Surveys A total of 75 women belonging to different FXS families from the FXS Catalan Association received a self-report survey by regular mail. The survey, consisting of 21 items (Table 1), was sent out and returned to the Biochemistry and Molecular Genetics Department of the Hospital Clinic. All subjects were informed about the scientific purpose of this study and they fulfilled it anonymously. Data from 52 unrelated women were obtained. Results Prenatal diagnosis FMR1 carrier status for pregnant women in which prenatal study was performed is summarized in Table 2. The main reason for prenatal diagnosis was a positive FXS family history (62%) or undiagnosed MR (38%) family history. Remarkably, 64% (114/178) of pregnant women were not aware of their status before prenatal diagnosis. Among these, 28.9% (33/114) were pre- or full mutation carriers, and 18.2% of them would have had a full mutation carrier child if prenatal diagnosis had not been carried out. Sixty-eight women were

3 562 M Xunclà et al. Table 1 Fragile X female carrier questionnaire consisting of 21 items about reproductive options, genetic counselling and the emotional state of FXS carriers. Personal information Age FXS carrier status: premutation, full mutation or unknown Number of children, gender and FXS molecular status: Number of spontaneous abortions and number of termination of pregnancies (TOP): Information about pregnancy/offspring 1. When did you realize you were a FXS carrier: before, during or after the pregnancy? If after, indicate the child s age of diagnosis 2. Which reproductive options were you offered and which one did you choose? Preimplantation genetic diagnosis (PGD) Oocyte donation Adoption Natural conception and prenatal diagnosis Natural conception and no prenatal testing I decided not to have offspring 3. Do you understand the meaning of the previous options? 4. In case of more than one pregnancy, did you choose the same reproductive option? 5. If you got to more than one pregnancy, did you choose the same reproductive option? 6. After your reproductive decision: Which was the most difficult issue? Are you satisfied with the outcome? 7. In case of a full mutation carrier fetus, did you consider termination of pregnancy? 8. If you underwent termination of pregnancy, which was the most negative issue? 9. If you underwent PGD or oocyte donation, which was the most negative issue? 10. Did you consider not having offspring after knowing your carrier status? 11. To whom did you talk to about being a FXS carrier? 12. Which would be your option/s in the case of a healthy carrier daughter? Normal education Overprotection Not make her feel different Advise her not to have children Advise visiting a genetic counsellor Information about genetic counselling 1. Did you receive genetic counselling? 2. If so: When: before, during or after the test? Who provided it? 3. Are you aware of your risk for primary ovarian insufficiency (FXPOI) or ataxia (FXTAS)? 4. Did the relationship with your partner change after knowing your carrier status? 5. Which of these feelings did you experience when you realized your status? Anxiety Sadness Impotence Indifference Stress Relief Guilt Others 6. Who gave you support after being aware of your status? 7. Did you need psychological support during that time? 8. Would you like to receive psychological support? 9. Score the genetic counselling received (from 0 to 10)

4 Fragile X syndrome prenatal diagnosis 563 Table 2 Carrier status FMR1 carrier status of pregnant women referred for prenatal FXS diagnosis. Previously known Previously unknown FXS families FXS families Undiagnosed MR families Normal Premutation Full mutation Pre/full mosaic Total Values are n. FXS = Fragile X syndrome; MR = mental retardation. Total unknown Table 3 Results of the 213 prenatal Fragile X syndrome diagnostic tests. Fetal diagnosis Male Female Total Normal Premutation Full mutation Mosaicism Failure Total Values are n. referred due to undiagnosed MR family history and in 12 of them (17.6%) molecular test yielded a FXS carrier. In all but two samples, a diagnosis regarding FXS was given. Both of them were females in which the Southern blotting analysis failed. In the first one, no linkage study was possible due to the familiar structure. In the other case, parents decided to carry on with the pregnancy irrespective of the FXS status as they already had a full mutation daughter. Indirect studies using microsatellites were performed in 10 samples (4.7%) due to insufficient material for Southern blotting analysis. The results of the 213 prenatal FXS tests are summarized in Table 3. Mosaicism was detected in four samples (1.9%): one normal/full mutation male, one pre/full mutation male and two pre/full mutation female fetuses. There were no expansions to pre- or full mutation in fetuses coming from a mother with a normal or grey zone (45 55 CGG) allele. All but three premutated mothers expanded the CGG repeat number and no contractions were observed. The smallest allele expanding to full mutation was 64 CGG repeats. In two cases, the premutated or grey zone allele was inherited from the father. One fetus, whose mother carried a 112 CGG repeat expansion, inherited the normal allele from the mother but an allele of 52 CGG repeats from the father. In another pregnancy the mother had both FMR1 alleles in the normal range (unknown before pregnancy), but the female fetus inherited one allele of 57 CGG repeats from the father. Termination of pregnancy was carried out in 65% of full mutation fetuses: all of full mutation carrier males but only 13.6% of females. Termination of pregnancy was also chosen in the two mosaic male fetuses and in a twin gestation of two male fetuses with 180 CGG repeats. None of the premutated females was terminated except one in which the co-twin was a full mutation carrier male. There were also three unaffected fetuses that were terminated due to other reasons: one female with intrauterine growth retardation, one male positive for Duchenne muscular dystrophy and one male showing a 46,XY,18q- karyotype (Rodríguez-Revenga et al., 2005). Surveys A total of 75 FXS premutated women belonging to FXS families were contacted by regular mail and 52 returned the survey, resulting in a 69.3% response rate. The women s mean age was 43.2 years old, ranging from 30 to 67 years. Most of them (96.2%) had at least one affected child, diagnosed during the childhood period and they were informed about their carrier status after receiving the child s diagnosis. FXS carrier status was revealed during pregnancy in the remaining 3.8% women. It is also important to highlight that in 11.5% of cases the FXS diagnosis was received when the couples already had another affected child. Parental attitudes towards reproductive options Most women (59.6%) reported that the child s FXS diagnosis affected their reproductive decisions and they considered the idea of not having additional children. However, some of them finally decided to have more. When they were asked about which reproductive option they would choose now they were aware of their carrier status, 40.5% of women would choose prenatal diagnosis, 9.5% oocyte donation and 7.1% preimplantation genetic diagnosis (PGD). Among those women who had already undergone a reproductive technique, 88.9% would choose the same reproductive option in a new pregnancy. None of the women chose adoption and 42.9% of them renounced to have additional offspring. In this latter group, most of them were either too old to be pregnant or they already had other children. Genetic counselling The majority of women (78.4%) reported they had received genetic counselling with an average qualification of 7.1 (from 0 to 10). In 83.1% of cases the genetic counselling

5 564 M Xunclà et al. Table 4 Emotional impact reported by women after knowing their Fragile X syndrome carrier status. was provided by a genetic counsellor, while in the remaining it was given by other specialists such as gynaecologists, neurologists or paediatricians. Information regarding the increased risk for developing FXPOI and FXTAS was given to 90.1% of cases. Finally, the emotional impact experienced by women when knowing their FMR1 premutation carrier status is summarized in Table 4. The most usual emotions were sadness, impotence, guilt and anxiety. Discussion Emotional impact n (total 48) Sadness Impotence Guilt Anxiety Stress Relief Indifference Others Percentage Molecular FXS prenatal diagnosis is a reliable, safe and useful procedure offered as routine diagnostic technique for FXS in most clinical laboratories. Based on previous results, chorionic villi is the most suitable tissue for FXS diagnosis, as a greater quantity of DNA is obtained and it allows a faster diagnosis compared with amniotic fluid. One of the drawbacks of chorionic villi is that sometimes the methylation pattern is not yet well established and therefore a new study in amniotic fluid is recommended in premutation carrier males in order to eliminate methylation mosaicisms (Castellví-Bel et al., 1995). FXS molecular diagnosis has also been used in PGD diagnosis and the reliability of this method has been assessed and it is feasible in the majority of carriers (Reches et al., 2009). In most centres, prenatal diagnosis for FXS requires only molecular testing for the mother, but two female fetuses were detected in which alleles of 52 and 57 repeats were inherited from the father. This fact highlights the need for paternal testing, even in an X-linked disease like FXS in order to avoid possible misdiagnoses. The present study detected a full mutation carrier fetus (including mosaics) in 28% of the 213 pregnancies. It is important to mention that only 36% of the women were aware of their carrier status before getting pregnant. Moreover, in 18.2% of cases in which the maternal carrier status was unknown, the molecular study yielded a full mutation carrier fetus. It is also important to highlight that 17.6% (12/68) of women in which there was family history of undiagnosed MR were FXS carriers. Nevertheless, the number of FXS prenatal diagnoses in which the maternal status is unknown is decreasing. There may be several reasons for this decrease: molecular FXS diagnosis has been available for the last 15 years, information about the syndrome has been given to families and professionals, and, as the FXS test is fast and accurate, it is performed whenever a family history of undiagnosed MR is detected. The present study reinforces molecular testing for FXS in such families. The present also aimed to assess the emotional impact of knowing their carrier status and their reproductive problems. Most women became aware of their carrier status at the same time that their child was diagnosed. So it is difficult to analyse both facts separately. The most common feeling when knowing both results was sadness, followed by impotence and guilt. Another part of the study was to assess how FXS carriers felt about prenatal diagnosis after this 15-year period. Carriers were asked about their preferred reproductive option, prenatal diagnosis being the most common (40.5%). In 42.9% of cases, the option of not having children was chosen, but most of the participants were too old to be pregnant or they already had other children, either affected or not. Other technologies such as oocyte donation (9.5%) and PGD (7.1%) were also selected. The main drawbacks of the latter technologies are the technical difficulties, poor ovarian response due to FXPOI, as well as the price and waiting time. These options should be directed to a selected group of FXS carrier women: if there is a poor ovarian response, oocyte donation is advisable while PGD is feasible in the remaining cases (Platteau et al., 2002). Another objective of the survey was to know the emotional impact of undergoing termination of pregnancy due to a positive FXS result, but only three of all surveyed women had undergone this procedure. They related that termination was not as difficult to overcome as to receive the positive result. The majority of carrier women received genetic counselling and considered it positively. Moreover, 92% of them would advise their carrier daughters to go to the genetic counsellor. Information about FXPOI and FXTAS risk was not given to 10% of cases, mainly because this information was only available from the year 2000, when these FXS-associated disorders were described (Allingham-Hawkins et al., 1999; Hagerman et al., 2001). In conclusion, this study shows that FXS prenatal diagnosis is a good reproductive option even though other alternatives like oocyte donation or PGD are also available nowadays. FXS molecular screening for women with undiagnosed MR family history is recommended before pregnancy because of the high risk of having affected children. A high percentage of FXS fetuses in this study were diagnosed from such mothers, who became aware of their carrier status at the same time they received a full mutation fetus report. Genetic counselling should always be carried out both before and after the genetic test for FXS. Acknowledgements The authors thank the Fragile X families, the Associació Catalana Síndrome X Fràgil, for their co-operation. This work was supported by MARATÓ TV3 (TV ). The CI- BER de Enfermedades Raras is an initiative of the ISCIII.

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