A family with Xq22.3q25 interstitial deletion and normal ovarian function

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1 CASE REPORT A family with Xq22.3q25 interstitial deletion and normal ovarian function Long-Ching Kuan, M.D., a Mei-Tsz Su, M.D., b Chin-Ming Wu, M.D., c Ming Chen, M.D., Ph.D., d Pao-Lin Kuo, M.D., b and Tsung-Cheng Kuo, M.D., Ph.D. a a Department of Obstetrics and Gynecology, Kuo General Hospital Tainan, and b Department of Obstetrics and Gynecology, National Cheng Kung University Hospital and College of Medicine, Tainan; c Department of Obstetrics and Gynecology, Yuan s General Hospital, Kaoshiung; and d Department of Genomic Medicine, Changhua Christian Hospital, Changhua City, Taiwan Objective: To investigate genomic changes in a family with deletion of X chromosome q22.3-q25 associated with normal constitutional and reproductive phenotypes. Design: Case report. Setting: Academic district hospital genetic laboratory. Patient(s): A family incidentally found to have deletion of X chromosome q22.3-q25. Intervention(s): Cytogenetic analysis and array-based comparative genomic hybridization for amniotic fluid and peripheral blood lymphocyte of family members. Main Outcome Measure(s): Ovarian function and menstrual cycles. Result(s): The proband and two daughters showed deletion of Xq22.3q25. This region spans 17.4 Mb and contains 121 genes. Conclusion(s): Female subjects with deletion of Xq22.3q25 may present with normal constitutional and reproductive phenotypes. (Fertil Steril Ò 2011;96:e Ó2011 by American Society for Reproductive Medicine.) Key Words: X chromosome deletion, premature ovarian failure Premature ovarian failure (POF) is a disorder characterized by amenorrhea and elevated serum gonadotropin level before 40 years of age. Premature ovarian failure accounts for about 10% of infertile women (1). The cause of POF is not known for the majority of cases. Some best known causes include autoantibodies, chemotherapy, radiotherapy, and surgical intervention. A genetic basis is also well established and demonstrated by the report of familial cases (2). Mutations in genes acting as risk factors for POF also have been suggested by pedigree analysis (3 5). These genetic studies showed POF as an extremely heterogeneous group of diseases that can be inherited as a mendelian disorder, but more often as a multifactorial disorder (2, 6, 7). In the search for genes responsible for POF, a role for the X chromosomal genes was suggested by the frequent observation of X chromosome anomalies in patients. The cytogenetic definition of a critical region for normal ovarian function on the long arm of the X chromosome, corresponding to two regions on Xq: Xq13- Xq21 and Xq23-Xq27 (2, 6, 8 10). In the present report, we Received December 15, 2010; revised April 13, 2011; accepted April 18, 2011; published online May 31, L-C.K. has nothing to disclose. M-T.S. has nothing to disclose. C-M.W. has nothing to disclose. M.C. has nothing to disclose. P-L.K. has nothing to disclose. T-C.K. has nothing to disclose. Supported by the Department of medical education, Kuo General Hospital, Tainan, Taiwan. Reprint requests: Tsung-Cheng Kuo, M.D., Ph.D., No. 22, Sec.2, Minsheng Road, Tainan City, Taiwan ( tckuo@kgh.com.tw). present a family with deletion of Xq22.3q25 in the mother, daughter, and fetus. The mother was fertile with regular menstrual cycles after weaning of breast feeding. Our finding suggests that deletion of Xq22.3q25 may not interfere with ovarian function. CASE REPORT The proband was a 38-year-old woman, gravida 4 para 2, spontaneous abortion 1, who underwent amniotic fluid sampling at 16 weeks gestation due to advanced maternal age. Her body height was 165 cm and body weight was 62 kg. Her menarche occurred at the age of 13 years and her menstrual cycle has been regular since puberty. The karyotype of the fetus was 46,X,del (X) (q22q25) (Fig. 1A). Karyotyping for family members showed that her husband had a normal male karyotype (46,XY). However, the first (9-year-old) and the third (2-year-old) daughters had inherited the deleted X chromosome from the proband (Fig. 1B). The pedigree of this family is shown in Figure 1C. At 40 weeks gestation, a healthy female baby was delivered through vaginal route. The body weight of the newborn was 3,200 g and the body length was 46 cm. No gross anomaly was identified in the newborn, including the external genital organs. She did not breastfeed the third daughter due to mastitis occurring 2 weeks after delivery, and her menstruation came at 8 weeks postpartum. We had checked her gynecologic organs by real-time ultrasound examination. The uterine and bilateral ovarian sizes were in normal range and a functional cyst was noted in unilateral ovary, which indicated that she had ovulation cycles soon after the last delivery. Since postpartum 8 weeks to present, her menstrual /$36.00 Fertility and Sterility â Vol. 96, No. 1, July 2011 e29 doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 FIGURE 1 (A) Karyotype of the fetus 46,X,del(X)(q22q25). (B) Three pairs of X chromosomes in proband (a), first daughter (b), and third daughter (c) 46,X,del(X)(q22q25). (C) The pedigree of this family. The proband (I-2), her first daughter (II-1), the third daughter (fetus) (II-3) had the same chromosomal abnormality. e30 Kuan et al. Xq partial deletion with normal menses Vol. 96, No. 1, July 2011

3 TABLE 1 Hormone profiles of proband and her first daughter. FSH LH PRL Proband 27 wk gestation 0.1 miu/ml 0.1 miu/ml ng/ml 5 mo postpartum 5.3 miu/ml First daughter 3.1 miu/ml 0.1 miu/ml 15.2 ng/ml cycles were in regular monthly pattern, and the FSH level in her 5- month postpartum follow-up was 5.3 miu/ml. The hormone profiles of the proband (at 27 weeks gestation and 5 months postpartum) and her first daughter were all within normal ranges (Table 1). This study had been approved by the Institutional Review Board of Kuo General Hospital (Tainan, Taiwan, Republic of China), and informed consent had been obtained from the proband, her husband, and her first daughter with the legal representative. Genetic Studies Array-based comparative genomic hybridization analysis The genomic DNA samples of the peripheral blood lymphocytes were extracted from the couple, their first daughter, and third daughter (fetus). An oligonucleotide-based array CGH (acgh; CytoScan v3 gene chip, Agilent Technologies DNA Microarray Scanner) was used for genomic analysis. This CytoScan 105 K chip included about 86,000 probes spanning the 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes, with an emphasis on more than 300 loci and regions associated with known genetic diseases. In this chip, the mean space between two probes was about 39 kb and the median of the probe space was 13 kb. The acgh analysis revealed one copy deletion in the chromosome region of Xq22.3q25, spanning about 17.4 Mb and containing 121 genes (Fig. 2). No genomic imbalance was detected in the father. X-chromosome inactivation assay The genomic DNA of the proband was subjected to the X-chromosome inactivation assay according to the method described previously (11, 12). In brief, the human androgen receptor (AR) locus was used. The AR gene is known to be methylated on the inactive X chromosome, thus this allele will be amplified by polymerase chain reaction (PCR) after the genomic DNAwas digested by the methylation-sensitive HpaII restriction enzyme. First, genomic DNA was digested with HpaII þ RsaI. Then the digested and undigested samples were amplified by PCR, and the peak area for methylated and unmethylated allele was analyzed using an ABI 3100 genetic analyzer and Genotyper 3.7 program (Applied Biosystems). The degree of skewing was calculated as FIGURE 2 (A) One copy deletion in chromosome Xq22.3q25 in the proband. (B) One copy deletion in chromosome Xq22.3q25 in the first daughter. Fertility and Sterility â e31

4 FIGURE 2 Continued (d1/u1)/(d1/u1 þ d2/u2), where d1 and d2 represent the peak area of the more intense allele and the less intense allele, respectively, from the digested sample, whereas u1 and u2 are the corresponding bands from the undigested sample. The degree of skewing was 0.54 without significant skewing from random X inactivation. DISCUSSION In the present study, we have identified the X chromosome long arm interstitial deletion involving Xq22.3-q25 in a family in which all female members were affected. Most cases of POF are idiopathic, and the underlying mechanisms are largely unknown. Although Xq deletions have long been known to be associated with POF in some cases, the mechanisms involved remain obscure (2, 6, 9). From patients with X chromosomal rearrangements and POF, two regions (critical region I, Xq13.3-q21 and critical region II, Xq23-q27) are generally believed to be critical for ovarian function (13 18). Candidate POF genes located in critical region I include choriodermia gene (CHM), premature ovarian failure 1B (POF1B), diaphanous homolog 2 (DIAPH2), and dachshund homolog 2 (DACH2). These genes are all located in Xq21-q22. Prominent POF candidates located in critical region II include FSH primary response homolog 1 (FSHPRH1) (Xq22), angiotensin type II (AT2) receptor gene (Xq22-23), P receptor membrane component-1 (PGRMC1) (Xq24), XPNPEP2 (Xq25), SOX3 (Xq26-27), FMR-1 (Xq27.3), and FMR-2 (Xq28) (2, 9). Recently, array-based comparative genomic hybridization (acgh) has been applied to detect submicroscopic chromosomal aberrations with high resolution. Genomewide microarray has emerged as the first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies (19). Gerard Tachdjian et al. (20) was the first to use acgh in POF patients. They detected Xpter duplication in one of three POF cases with terminal deletions of Xq. Aboura et al. (21) searched for e32 Kuan et al. Xq partial deletion with normal menses Vol. 96, No. 1, July 2011

5 copy number variation in POF patients by acgh. They found eight statistically significant copy number variations and five possible candidate genes for POF. Recently, Quilter et al. (22) identified some copy number variations in both Xp and Xq that might be implicated in POF. In the present report, acgh has proved to be useful in detecting genomic imbalance of Xq. The acgh also excluded involvement of other genomic regions in this family. The deletion removed two potential POF genes. The first is AT2 receptor gene (also named as AGTR2). Katsuya et al. (23) had examined this receptor from two different families of sisters with POF but revealed no change in nucleotide sequences. The other POF candidates located on the deleted region is PGRMC1 gene, mutations of which have been found to cause POF (24). The present report provides evidence that haploinsufficiency of both AT2 and PGRMC1 (and the entire 17.4-Mb region spanning from Xq22.3 to Xq25) may not be sufficient to cause ovarian dysfunction and other constitutional phenotypes. Meanwhile, the deletion also did not cause significant skewing of X chromosome inactivation. The proband (mother) had delivered three girls. She usually resumed regular menstrual cycles 2 months after delivery. She also had normal endocrine profiles, indicating normal ovarian function without any signs of incipient POF at the time of work-up. We will follow-up the endocrine profiles and menstrual status of all family members until the age of 50 years. Breakpoint mapping in POF patients with balanced X chromosome rearrangements showed that the majority of X chromosome breakpoints are located in the gene-poor region and do not disrupt X-linked genes (25, 26). Rizzolio et al. (27) proposed that balanced translocations interrupting critical region (CR) I could be responsible for POF through down-regulation of ovary-expressed autosomal genes translocated to the X chromosome. They showed that chromatin organization of CR I is likely to be responsible for epigenetic modifications in POF patients (28). In contrast to CR I, deletion mapping analysis seems to favor haploinsufficiency of genes located in CR II as causative mechanism of POF. A few isolated POF cases have been found to have interstitial deletion spanning Xq22 to q26 (e.g., case III5 and GM09332) (15, 28, 29). The deletion of this family spans the proximal part of CR II. In contiguous gene syndromes, not only the aneuploid genes but also the flanking genes several megabases away from the genomic rearrangements, should contribute to the phenotypes. In Williams syndrome, not only hemizygous genes but also normal copy neighboring genes show decreased relative levels of expression (30). This case not only provides evidence that haploinsufficiency of Xq22.3-q25 may not be sufficient to interfere with ovarian function, it also suggests that the deletion may not influence expression of neighboring POF candidates (e.g., FSHPRH1, XPNPEP, and SOX3). In the present report, we presented a family in which all female members had deletion of Xq22.3-q25. The deletion does not seem to cause ovarian dysfunction at the time of the report, nor does the deletion cause any constitutional phenotypes. This family provides a unique opportunity for us to explore the link between X chromosome and human disease. The phenotype of women who carries X chromosome deletion is highly variable. For example, some women who carried Xq27-q28 deletion in distal CR II had several children and early menopause (between 40 and 45 years of age), some had irregular menstruation and secondary POF, whereas only one case had primary POF (13, 14, 17). 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