- Email: [email protected]
Complex X chromosome rearrangement delineated by array comparative genome hybridization in a woman with premature ovarian insufficiency
CASE REPORT Complex X chromosome rearrangement delineated by array comparative genome hybridization in a woman with premature ovarian insufficiency Melanie E. Ochalski, M.D.,a,c Natalie Engle, B.S.,c Anthony Wakim, M.D.,a Britt J. Ravnan, Ph.D.,d Lori Hoffner, M.S.,c Aleksandar Rajkovic, M.D., Ph.D.,a,c and Urvashi Surti, Ph.D.b,c a
Department of Obstetrics, Gynecology, and Reproductive Sciences, and b Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; c Magee-Womens Research Institute, Pittsburgh, Pennsylvania; and d Signature Genomics Laboratories, Spokane, Washington
Objective: To investigate candidate genes affected by a complex X chromosome rearrangement that may play a role in the diagnosis of spontaneous premature ovarian insufficiency (POI). Design: Prospective cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis and oligonucleotide array comparative genome hybridization (CGH). Setting: University medical center. Patient(s): A 36-year-old woman with POI found to have a highly rearrangement X chromosome. Intervention(s): FISH analysis and oligonucleotide array CGH. Main Outcome Measure(s): Oligonucleotide microarray analysis to detect duplicated, deleted, or translocated regions of the X chromosome. Result(s): Complex rearrangement of the X chromosome involving R12 breakpoints resulting in two deletions, four duplications, and several intrachromosomal translocations. At least 13 genes with possible relevance to POI may be affected by the rearrangement. Conclusion(s): Array CGH can reveal candidate genes that may have essential roles in fertility and POI. (Fertil Steril 2011;95:2433.e9–e15. 2011 by American Society for Reproductive Medicine.) Key Words: Premature ovarian insufficiency, complex chromosomal rearrangements, X chromosome aberration, amenorrhea
Premature ovarian insufficiency (POI) is defined by amenorrhea associated with elevated gonadotropin levels (FSH >40 IU/L) before the age of 40 years. POI affects 1% of the population, but accounts for 10% of all female sterility (1). Affected women face infertility and increased risk of osteoporosis and cardiovascular disease and require long-term hormone therapy. POI is a heterogeneous disorder with chromosomal, genetic, autoimmune, metabolic, infectious, and iatrogenic etiologies (2). However, the majority of spontaneous POI cases remain unexplained (2). Complex chromosomal rearrangements (CCRs) are constitutional structural rearrangements having three or more breakpoints (3). CCRs have been associated with infertility, repeated miscarriages, and chromosomal imbalances in offspring (4). The majority of cases show three-way exchanges, in which three segments from three chromosomes break off, translocate, and join. Few exceptional CCRs involve more than three breakReceived February 7, 2011; revised March 21, 2011; accepted March 23, 2011; published online April 29, 2011. M.E.O. has nothing to disclose. N.E. has nothing to disclose. A.W. has nothing to disclose. B.J.R. has nothing to disclose. L.H. has nothing to disclose. A.R. has nothing to disclose. U.S. has nothing to disclose. Reprint requests: Urvashi Surti, Ph.D., Department of Cytogenetics, Magee Women’s Hospital, Room 1233, Pittsburgh, PA 15213 (E-mail: [email protected]).
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points. Even rarer are rearrangements occurring within a single chromosome. CCRs involving the X chromosome, albeit rare, can shed insight into the genetic mechanisms responsible for X-linked POI. We report a novel CCR in a woman with sporadic POI and a highly rearranged derivative X chromosome further delineated using 135K oligonucelotide array comparative genome hybridization (CGH), and discuss the genes that may be affected (Table 1).
CASE REPORT A 36-year-old nulligravida woman of Sri Lankan descent was referred for investigation of 10-year infertility. The patient had menarche at age 11 years and regular monthly menstrual bleeding up until age 25 years, when she began oral contraceptive pills (OCPs) for contraception. At age 28, she presented with amenorrhea since discontinuing OCPs. Her laboratory tests showed an FSH >40 mIU/mL, PRL 15 ng/mL, TSH 2.3 mIU/mL, free T4 1.1 mUI/mL, and E2 14 pg/mL. Ultrasound scan revealed an endometrial thickness of 3 mm and quiescent ovaries with volumes of 1.47 cm3 and 1.11 cm3 on the right and left, respectively. She was diagnosed with POI. Her gynecologic history revealed normal sexual development, with breast and pubic hair compatible with Tanner stage V. Her
Fertility and Sterility Vol. 95, No. 7, June 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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TABLE 1 Genes with possible relevance to premature ovarian insufficiency. Gene
Location
Alteration
Function
BCOR
Xp11.4
Copy loss
Xp11.4 Xp11.2-q11.2
Copy loss Copy loss
Xp11.3
Copy loss
PCTK1 ARAF AR ARHGEF9 FOXO4
Xp11.3-p11.23 Xp11.3-p11.23 Xq11- q12 Xq22.1 Xq13.1
Copy loss Copy loss Copy Gain Copy Gain Copy Gain
OTUD6A
Xq13.1
Copy Gain
XIST POF 1B BMP 15
Xq13.2 Xq21 Xp11.2
Copy Gain Proximal to breakpoint Proximal to breakpoint
Key transcriptional regulator during early embryogenesis Encodes renin receptor Contributes to age-associated changes in tissue susceptibility to harmful stimuli Unknown, escapes X inactivation; no Y homolog Governs progress through cell cycle Critical role in cell growth Encodes androgen receptor Cell signaling Cell cycle regulation, Over expression causes growth suppression Ovarian tumor domain containing protein 6A X inactivation Susceptibility gene for POI Regulates mammalian folliculogenesis
ATP6AP2 RGN
INE1
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
medical history was positive for autoimmune hypothyroidism diagnosed at age 30 years. The family history was significant for one sister, who naturally conceived two pregnancies in her 30s that resulted in healthy children. The family history was otherwise unremarkable for infertility and POI. Her physical examination showed a stature of 62 inches, normal nipple span with normal secondary sexual development. Her gynecologic examination was within normal limits. Ovaries were not palpable on bimanual exam. Karyotyping of the patient showed a highly rearranged derivative X chromosome in all cells analyzed (46,X,der(X).ish der(X)(Xwcpþ, pcpXpwcpþþ, pcpXqwcpþ, DXZ1þ, Xistþþ, Xptelþ, Xqtelþ). Fluorescence in situ hybridization (FISH) and microarray analysis were performed to obtain a detailed picture of the genetic abnormality. The subject later underwent IVF with directed donor oocytes from her sister, which resulted in an ectopic pregnancy. A subsequent frozen embryo transfer resulted in spontaneous miscarriage at 5 weeks. She was not interested in pursuing further assisted reproductive techniques.
METHODS Routine G-banded karyotyping was performed on phytohemagglutinin-stimulated peripheral lymphocytes following routine protocols. FISH analyses were performed on metaphase spreads from cultured lymphocytes by using chromosome paint probes specific for the short and long arms (Qbiogene) as well as whole X chromosome (Vysis). DNA probes specific for Xq13.2 region (XIST); X chromosome centromere (DXZ1), Xptel, and Xqtel (Vysis) were also used. SignatureChipOS, Roche Nimblegen 135K oligonucleotide array was performed as described elsewhere (5). Additional FISH probes were used to confirm and locate the abnormalities detected by the array (Table 2).The Online Mendelian Inheritance in Man
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(OMIM) database (www.ncbi.nlm.nih.gov/omim) was used to investigate the functions of the genes involved.
RESULTS Karyotyping revealed a derivative X chromosome with an increased length, altered p/q arm ratio, and altered centromere position with a highly rearranged banding pattern in all the 55 cells examined (Fig. 1). There was no evidence of mosaicism. X chromosome inactivation studies performed at another laboratory showed a completely skewed pattern suggesting that the abnormal X was inactivated in all cells. Molecular cytogenetics demonstrated that the derivative X chromosome was fully painted with the X-specific paint probe. In addition, the XIST probe was duplicated (Fig. 2A) and there was a single copy of the centromeric marker (DXZ1), which was located closer to the p-terminal end. Chromosome Xp and Xq specific paint probes (Fig. 2B) showed that the derivative X chromosome was composed of a shorter p-arm and a relatively longer segment (Xq11.21–21.2) of the q-arm, with a small segment (Xp11.3–11.23) of p-arm material inserted in the distal q-arm, ending with a terminal segment of Xq (Fig. 2B). FISH confirmed the presence of the Xptel at the end of the shortened Xp arm and the Xqtel present at the end of the highly rearranged Xq arm of the derivative X. The karyotype was described as 46,X,der(X).ish der(X) (Xwcpþ, pcpXpwcpþ, pcpXqwcpþ, DXZ1þ, Xistþþ, Xptelþ, Xqtelþ). The microarray analysis and subsequent FISH confirmation using multiple DNA probes (Table 2; Fig. 2C–2F) revealed a complex X chromosome rearrangement with multiple segmental interstitial deletions and duplications involving both arms. In summary, microarray results revealed two deletions and four duplications, in addition to multiple insertions (Fig. 3; Table 2). All of the deletions and duplications were confirmed, as were some of the insertions (Fig. 3).
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TABLE 2 Details of fluorescence in situ hybridization (FISH) and microarray experiments. FISH
Probe
Location
Result
WCP X PCP Xp
Whole X chromosome X p-arm
PCP Xq
X q-arm
XIST DXZ1 Xptel
Xq13.2 X centromere X p telomere
Xqtel
X q telomere
Derivative X fully painted X p-arm painted (appeared shortened); also, small insertion of p material on distal q-arm X q-arm painted (except for small distal segment) Duplicated on derivative X Single copy on derivative X Present at the end of the shortened p-arm Present at the end of highly rearranged X q-arm
RP11–204C16 DXZ1 RP11–513O15 RP11–1005N24 RP13–216E22
Xp11.4 X centromere (Xq22.3–Xq23) Xp11.3 Xq13.2
RP11–151G3
Xp11.3
RP11–107A19
Xp11.23
RP11–943J20
Xq11.1
RP13–216E22
Xq13.2
RP11–187H16
Xq21.2
RP11–187H16
Xq21.2
RP11–513O15
Xq22.3–Xq23
Diagnostic FISH
Confirmatory FISH Experiment 1 Experiment 2 Experiment 3
Experiment 4
Experiment 5
Experiment 6
Deleted Single copy at constriction Duplication visible on metaphase Deleted Duplication visible on long metaphases Present in correct position and orientation relative to RP13–216E22 (short arm visibly shorter) Normal copy number, moved to Xqter right next to RP11–943J20 Duplicated, second copy on Xqter right next to RP11–107A19 Duplication visible on long metaphases Normal copy number, moved to Xq, near end of arm Normal copy number, moved to Xq, near end of arm, distal to RP11–513O15 Duplication visible on metaphase, both copies proximal to moved RP11–187H16
Microarray Abnormality Single copy loss deletion 1.4 Mb normal intervening sequence Single copy loss deletion 8.4 Mb normal intervening sequence Single copy gain duplication 1.4 Mb normal intervening sequence Single copy gain duplication 6.2 Mb normal intervening sequence Single copy gain duplication 2.2 Mb normal intervening sequence Single copy gain duplication
No. of probes affected
Location
Size
203 oligonucleotide probes
Xp11.4p11.3
4.2 Mb
175 oligonucleotide probes
Xp11.3p11.23
2.3 Mb
750 oligonucleotide probes
Xp11.21q12
11.3 Mb
513 oligonucleotide probes
Xq13.1q21.1
15.4 Mb
17 oligonucleotide probes
Xq21.31
1.2 Mb
Xq21.33q25
30.0 Mb
1,114 oligonucleotide probes
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
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FIGURE 1 Karyotype 46,X,der(X).
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
Interestingly, although the microarray data showed a duplication for the pericentromeric region Xp11.21q12, the FISH analysis showed only one copy for the X centromere. This suggests that there must be additional breaks near the centromere and that the chromosomal rearrangements are even more complex. A total of 540 genes are located in the rearranged genomic regions, with 40 (31 OMIM) and 283 genes (183 OMIM) predicted to be deleted and duplicated, respectively. Table 1 lists a few genes relevant to POI, and among these is the Regucalcin gene, previously shown to function in age-associated cell changes and hypothesized to increase tissue susceptibility to harmful stimuli (6). Deletions in this gene may impose a susceptibility to environmental toxins, leading to early oocyte loss. BCL6 corepressor is a key transcriptional regulator that is preferentially expressed in the ovary during early embryogenesis, and it was also deleted. Several genes may have altered expression due to their proximity to the breakpoints or altered position within the X chromosome (Table 1). Both POF1B and bone morphogenetic protein 15 were present in normal copy number, but adjacent to breakpoints. These genes, which play a critical role in ovarian maintenance, may have contributed to the phenotype in the present case (7).
DISCUSSION This report of a woman with spontaneous POI and an extremely complex intrachromosomal rearrangement of the X chromosome is among the most complex rearrangements of the X chromosome reported thus far. It is the first intrachromosomal rearrangement involving solely the X chromosome associated with a POI phenotype.
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To date, only four previous reports of complex intra–X chromosomal rearrangements have been described (Table 3) (4, 8–10). It is likely that the rearrangement in the present case occurred de novo, because such rearrangements tend to be more complex. Unfortunately, no family members were available for testing. However, it is not likely that the patient’s mother was a carrier for the rearrangement, because she would most certainly be infertile, and if the father had the rearranged X he would be expected to have a severely abnormal phenotype. Due to its presence in every cell examined, this mutation likely occurred very early. Because most CCRs reported are of paternal origin we propose that this event occurred during paternal meiosis (8). It appears that only the X chromosome was destabilized and shattered during a catastrophic event and repaired in a haphazard manner, resulting in this complex rearrangement. This rearrangement involves the ‘‘susceptibility’’ region of the X chromosome (Xp11.4 to Xq25). Two genes associated with POI, BMP 15 and POF1B, were both present in normal copy numbers, which underscores the possible positional effect of genes on the X chromosome (11). Alternatively, one or both of these genes may have been disrupted by their proximity to the breakpoint. The increased number of breakpoints in this CCR would have been expected to cause a number of phenotypic abnormalities; however, the patient appeared phenotypically normal, excluding her POI and early-onset hypertension, which were likely due to the completely skewed X inactivation. In general, duplications are less deleterious than deletions, and the number of breakpoints in a CCR is not necessarily predictive of the severity of the phenotype. In fact,
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FIGURE 2 FISH showing (A) duplicated XIST probe in red, and the centromeric marker in green. (B) Chromosome paint probes for the short arm (green) and long arm (red) show that a piece of p is moved to the q arm in the derivative X. (C) Xq13.2 (red) x3 RP13-216E22 Xp11.3 (green) x2 RP11-151G3. (D) Xq21.2, red RP11-187H16 – normal copy number, moved to Xq, near end of arm, distal to RP11-513O15. RP11513O15 (Xq22.3q23, green) – duplication visible on metaphase, both copies proximal to moved RP11-187H16. (E) RP13-216E22 (Xq13.2, red) – duplication visible on long metaphases; RP11-187H16 (Xq21.2, green) – normal copy number, moved to Xq, near end of arm. (F) RP11-107A19 (Xp11.23, red) – normal copy number, moved to Xqter right next to RP11-943J20; RP11-943J20 (Xq11.1, green) – duplicated, second copy on Xqter right next to RP11-107A19.
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
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FIGURE 3 Microarray results showing X chromosome alterations.
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
CCRs with no phenotypic effect have been described in the literature (4). In the present case, severe reproductive impairment may have occurred because of mispairing during meiosis activating DNA damage checkpoint mechanism or because X chromosome haploinsufficiency may be detrimental during germ cell development because biallelic expression of certain X chromosome genes may be required for proper oogenesis (12).
Array CGH is essential in the evaluation of complex chromosome rearrangements, because it describes the specific chromosomal segments involved and exact genes disturbed. There are known regions of the X chromosome that are critical to normal ovarian function; however, many genes have yet to be characterized, and new DNA technology can help identify those that may have essential roles in fertility.
TABLE 3 Published cases of complex intrachromosomal rearrangements of the X chromosome. Case no. 1 2 3 4
Origin
Phenotype
Breakpoints
References
De novo De novo De novo De novo
Primary amenorrhea Primary amenorrhea Primary amenorrhea Failure to thrive, muscular hypotonia, and minor facial anomalies
Xq22-q24 and Xp11 Xq22 and Xp11.2 Xp11.2 and Xq24 Xp11.21-p11.22
Grass et al., 1981 (9) Letterie, 1995 (8) Hernando et al., 2004 (4) Shchelochkov et al., 2008 (10)
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REFERENCES 1. Lindstrand A, Malmgren H, Sahlen S, Xin H, Schoumans J, Blennow E. Molecular cytogenetic characterization of a constitutional, highly complex intrachromosomal rearrangement of chromosome 1, with 14 breakpoints and a 0.5 Mb submicroscopic deletion. Am J Med Genet A 2008;146A:3217–22. 2. Pal L, Santoro N. Premature ovarian failure (POF): discordance between somatic and reproductive aging. Ageing Res Rev 2002;1:413–23. 3. Gardner RaSG. Chromosome abnormalities and genetic counseling. 3rd Ed. Oxford: Oxford University Press; 2004. 4. Hernando C, Plaja A, Catala V, Sarret E, Egozcue J, Fuster C. Primary amenorrhea in a woman with a cryptic complex chromosome rearrangement involving the critical regions Xp11.2 and Xq24. Fertil Steril 2004;82:1666–71.
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5. Ballif BC, Hornor SA, Sulpizio SG, Lloyd RM, Minier SL, Rorem EA, et al. Development of a high-density pericentromeric region BAC clone set for the detection and characterization of small supernumerary marker chromosomes by array CGH. Genet Med 2007;9:150–62. 6. Fujita T. Senescence marker protein-30 (SMP30): structure and biological function. Biochem Biophys Res Commun 1999;254:1–4. 7. Lacombe A, Lee H, Zahed L, Choucair M, Muller JM, Nelson SF, et al. Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure. Am J Hum Genet 2006;79:113–9. 8. Letterie GS. Unique unbalanced X;X translocation (Xq22;p11.2) in a woman with primary amenorrhea but without Ullrich-Turner syndrome. Am J Med Genet 1995;59:414–6.
9. Grass FS, Schwartz RP, Deal JO, Parke JC Jr. Gonadal dysgenesis, intra-X chromosome insertion, and possible position effect in an otherwise normal female. Clin Genet 1981;20:28–35. 10. Shchelochkov OA, Patel A, Weissenberger GM, Chinault AC, Wiszniewska J, Fernandes PH, et al. Duplication of chromosome band 12q24.11q24.23 results in apparent Noonan syndrome. Am J Med Genet A 2008;146A:1042–8. 11. Toniolo D. X-linked premature ovarian failure: a complex disease. Curr Opin Genet Dev 2006;16:293–300. 12. Lespinasse J, North MO, Paravy C, Brunel MJ, Malzac P, Blouin JL. A balanced complex chromosomal rearrangement (BCCR) in a family with reproductive failure. Hum Reprod 2003;18:2058–66.
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Department of Obstetrics, Gynecology, and Reproductive Sciences, and b Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; c Magee-Womens Research Institute, Pittsburgh, Pennsylvania; and d Signature Genomics Laboratories, Spokane, Washington
Objective: To investigate candidate genes affected by a complex X chromosome rearrangement that may play a role in the diagnosis of spontaneous premature ovarian insufficiency (POI). Design: Prospective cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis and oligonucleotide array comparative genome hybridization (CGH). Setting: University medical center. Patient(s): A 36-year-old woman with POI found to have a highly rearrangement X chromosome. Intervention(s): FISH analysis and oligonucleotide array CGH. Main Outcome Measure(s): Oligonucleotide microarray analysis to detect duplicated, deleted, or translocated regions of the X chromosome. Result(s): Complex rearrangement of the X chromosome involving R12 breakpoints resulting in two deletions, four duplications, and several intrachromosomal translocations. At least 13 genes with possible relevance to POI may be affected by the rearrangement. Conclusion(s): Array CGH can reveal candidate genes that may have essential roles in fertility and POI. (Fertil Steril 2011;95:2433.e9–e15. 2011 by American Society for Reproductive Medicine.) Key Words: Premature ovarian insufficiency, complex chromosomal rearrangements, X chromosome aberration, amenorrhea
Premature ovarian insufficiency (POI) is defined by amenorrhea associated with elevated gonadotropin levels (FSH >40 IU/L) before the age of 40 years. POI affects 1% of the population, but accounts for 10% of all female sterility (1). Affected women face infertility and increased risk of osteoporosis and cardiovascular disease and require long-term hormone therapy. POI is a heterogeneous disorder with chromosomal, genetic, autoimmune, metabolic, infectious, and iatrogenic etiologies (2). However, the majority of spontaneous POI cases remain unexplained (2). Complex chromosomal rearrangements (CCRs) are constitutional structural rearrangements having three or more breakpoints (3). CCRs have been associated with infertility, repeated miscarriages, and chromosomal imbalances in offspring (4). The majority of cases show three-way exchanges, in which three segments from three chromosomes break off, translocate, and join. Few exceptional CCRs involve more than three breakReceived February 7, 2011; revised March 21, 2011; accepted March 23, 2011; published online April 29, 2011. M.E.O. has nothing to disclose. N.E. has nothing to disclose. A.W. has nothing to disclose. B.J.R. has nothing to disclose. L.H. has nothing to disclose. A.R. has nothing to disclose. U.S. has nothing to disclose. Reprint requests: Urvashi Surti, Ph.D., Department of Cytogenetics, Magee Women’s Hospital, Room 1233, Pittsburgh, PA 15213 (E-mail: [email protected]).
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points. Even rarer are rearrangements occurring within a single chromosome. CCRs involving the X chromosome, albeit rare, can shed insight into the genetic mechanisms responsible for X-linked POI. We report a novel CCR in a woman with sporadic POI and a highly rearranged derivative X chromosome further delineated using 135K oligonucelotide array comparative genome hybridization (CGH), and discuss the genes that may be affected (Table 1).
CASE REPORT A 36-year-old nulligravida woman of Sri Lankan descent was referred for investigation of 10-year infertility. The patient had menarche at age 11 years and regular monthly menstrual bleeding up until age 25 years, when she began oral contraceptive pills (OCPs) for contraception. At age 28, she presented with amenorrhea since discontinuing OCPs. Her laboratory tests showed an FSH >40 mIU/mL, PRL 15 ng/mL, TSH 2.3 mIU/mL, free T4 1.1 mUI/mL, and E2 14 pg/mL. Ultrasound scan revealed an endometrial thickness of 3 mm and quiescent ovaries with volumes of 1.47 cm3 and 1.11 cm3 on the right and left, respectively. She was diagnosed with POI. Her gynecologic history revealed normal sexual development, with breast and pubic hair compatible with Tanner stage V. Her
Fertility and Sterility Vol. 95, No. 7, June 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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TABLE 1 Genes with possible relevance to premature ovarian insufficiency. Gene
Location
Alteration
Function
BCOR
Xp11.4
Copy loss
Xp11.4 Xp11.2-q11.2
Copy loss Copy loss
Xp11.3
Copy loss
PCTK1 ARAF AR ARHGEF9 FOXO4
Xp11.3-p11.23 Xp11.3-p11.23 Xq11- q12 Xq22.1 Xq13.1
Copy loss Copy loss Copy Gain Copy Gain Copy Gain
OTUD6A
Xq13.1
Copy Gain
XIST POF 1B BMP 15
Xq13.2 Xq21 Xp11.2
Copy Gain Proximal to breakpoint Proximal to breakpoint
Key transcriptional regulator during early embryogenesis Encodes renin receptor Contributes to age-associated changes in tissue susceptibility to harmful stimuli Unknown, escapes X inactivation; no Y homolog Governs progress through cell cycle Critical role in cell growth Encodes androgen receptor Cell signaling Cell cycle regulation, Over expression causes growth suppression Ovarian tumor domain containing protein 6A X inactivation Susceptibility gene for POI Regulates mammalian folliculogenesis
ATP6AP2 RGN
INE1
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
medical history was positive for autoimmune hypothyroidism diagnosed at age 30 years. The family history was significant for one sister, who naturally conceived two pregnancies in her 30s that resulted in healthy children. The family history was otherwise unremarkable for infertility and POI. Her physical examination showed a stature of 62 inches, normal nipple span with normal secondary sexual development. Her gynecologic examination was within normal limits. Ovaries were not palpable on bimanual exam. Karyotyping of the patient showed a highly rearranged derivative X chromosome in all cells analyzed (46,X,der(X).ish der(X)(Xwcpþ, pcpXpwcpþþ, pcpXqwcpþ, DXZ1þ, Xistþþ, Xptelþ, Xqtelþ). Fluorescence in situ hybridization (FISH) and microarray analysis were performed to obtain a detailed picture of the genetic abnormality. The subject later underwent IVF with directed donor oocytes from her sister, which resulted in an ectopic pregnancy. A subsequent frozen embryo transfer resulted in spontaneous miscarriage at 5 weeks. She was not interested in pursuing further assisted reproductive techniques.
METHODS Routine G-banded karyotyping was performed on phytohemagglutinin-stimulated peripheral lymphocytes following routine protocols. FISH analyses were performed on metaphase spreads from cultured lymphocytes by using chromosome paint probes specific for the short and long arms (Qbiogene) as well as whole X chromosome (Vysis). DNA probes specific for Xq13.2 region (XIST); X chromosome centromere (DXZ1), Xptel, and Xqtel (Vysis) were also used. SignatureChipOS, Roche Nimblegen 135K oligonucleotide array was performed as described elsewhere (5). Additional FISH probes were used to confirm and locate the abnormalities detected by the array (Table 2).The Online Mendelian Inheritance in Man
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Ochalski et al.
(OMIM) database (www.ncbi.nlm.nih.gov/omim) was used to investigate the functions of the genes involved.
RESULTS Karyotyping revealed a derivative X chromosome with an increased length, altered p/q arm ratio, and altered centromere position with a highly rearranged banding pattern in all the 55 cells examined (Fig. 1). There was no evidence of mosaicism. X chromosome inactivation studies performed at another laboratory showed a completely skewed pattern suggesting that the abnormal X was inactivated in all cells. Molecular cytogenetics demonstrated that the derivative X chromosome was fully painted with the X-specific paint probe. In addition, the XIST probe was duplicated (Fig. 2A) and there was a single copy of the centromeric marker (DXZ1), which was located closer to the p-terminal end. Chromosome Xp and Xq specific paint probes (Fig. 2B) showed that the derivative X chromosome was composed of a shorter p-arm and a relatively longer segment (Xq11.21–21.2) of the q-arm, with a small segment (Xp11.3–11.23) of p-arm material inserted in the distal q-arm, ending with a terminal segment of Xq (Fig. 2B). FISH confirmed the presence of the Xptel at the end of the shortened Xp arm and the Xqtel present at the end of the highly rearranged Xq arm of the derivative X. The karyotype was described as 46,X,der(X).ish der(X) (Xwcpþ, pcpXpwcpþ, pcpXqwcpþ, DXZ1þ, Xistþþ, Xptelþ, Xqtelþ). The microarray analysis and subsequent FISH confirmation using multiple DNA probes (Table 2; Fig. 2C–2F) revealed a complex X chromosome rearrangement with multiple segmental interstitial deletions and duplications involving both arms. In summary, microarray results revealed two deletions and four duplications, in addition to multiple insertions (Fig. 3; Table 2). All of the deletions and duplications were confirmed, as were some of the insertions (Fig. 3).
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TABLE 2 Details of fluorescence in situ hybridization (FISH) and microarray experiments. FISH
Probe
Location
Result
WCP X PCP Xp
Whole X chromosome X p-arm
PCP Xq
X q-arm
XIST DXZ1 Xptel
Xq13.2 X centromere X p telomere
Xqtel
X q telomere
Derivative X fully painted X p-arm painted (appeared shortened); also, small insertion of p material on distal q-arm X q-arm painted (except for small distal segment) Duplicated on derivative X Single copy on derivative X Present at the end of the shortened p-arm Present at the end of highly rearranged X q-arm
RP11–204C16 DXZ1 RP11–513O15 RP11–1005N24 RP13–216E22
Xp11.4 X centromere (Xq22.3–Xq23) Xp11.3 Xq13.2
RP11–151G3
Xp11.3
RP11–107A19
Xp11.23
RP11–943J20
Xq11.1
RP13–216E22
Xq13.2
RP11–187H16
Xq21.2
RP11–187H16
Xq21.2
RP11–513O15
Xq22.3–Xq23
Diagnostic FISH
Confirmatory FISH Experiment 1 Experiment 2 Experiment 3
Experiment 4
Experiment 5
Experiment 6
Deleted Single copy at constriction Duplication visible on metaphase Deleted Duplication visible on long metaphases Present in correct position and orientation relative to RP13–216E22 (short arm visibly shorter) Normal copy number, moved to Xqter right next to RP11–943J20 Duplicated, second copy on Xqter right next to RP11–107A19 Duplication visible on long metaphases Normal copy number, moved to Xq, near end of arm Normal copy number, moved to Xq, near end of arm, distal to RP11–513O15 Duplication visible on metaphase, both copies proximal to moved RP11–187H16
Microarray Abnormality Single copy loss deletion 1.4 Mb normal intervening sequence Single copy loss deletion 8.4 Mb normal intervening sequence Single copy gain duplication 1.4 Mb normal intervening sequence Single copy gain duplication 6.2 Mb normal intervening sequence Single copy gain duplication 2.2 Mb normal intervening sequence Single copy gain duplication
No. of probes affected
Location
Size
203 oligonucleotide probes
Xp11.4p11.3
4.2 Mb
175 oligonucleotide probes
Xp11.3p11.23
2.3 Mb
750 oligonucleotide probes
Xp11.21q12
11.3 Mb
513 oligonucleotide probes
Xq13.1q21.1
15.4 Mb
17 oligonucleotide probes
Xq21.31
1.2 Mb
Xq21.33q25
30.0 Mb
1,114 oligonucleotide probes
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FIGURE 1 Karyotype 46,X,der(X).
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
Interestingly, although the microarray data showed a duplication for the pericentromeric region Xp11.21q12, the FISH analysis showed only one copy for the X centromere. This suggests that there must be additional breaks near the centromere and that the chromosomal rearrangements are even more complex. A total of 540 genes are located in the rearranged genomic regions, with 40 (31 OMIM) and 283 genes (183 OMIM) predicted to be deleted and duplicated, respectively. Table 1 lists a few genes relevant to POI, and among these is the Regucalcin gene, previously shown to function in age-associated cell changes and hypothesized to increase tissue susceptibility to harmful stimuli (6). Deletions in this gene may impose a susceptibility to environmental toxins, leading to early oocyte loss. BCL6 corepressor is a key transcriptional regulator that is preferentially expressed in the ovary during early embryogenesis, and it was also deleted. Several genes may have altered expression due to their proximity to the breakpoints or altered position within the X chromosome (Table 1). Both POF1B and bone morphogenetic protein 15 were present in normal copy number, but adjacent to breakpoints. These genes, which play a critical role in ovarian maintenance, may have contributed to the phenotype in the present case (7).
DISCUSSION This report of a woman with spontaneous POI and an extremely complex intrachromosomal rearrangement of the X chromosome is among the most complex rearrangements of the X chromosome reported thus far. It is the first intrachromosomal rearrangement involving solely the X chromosome associated with a POI phenotype.
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To date, only four previous reports of complex intra–X chromosomal rearrangements have been described (Table 3) (4, 8–10). It is likely that the rearrangement in the present case occurred de novo, because such rearrangements tend to be more complex. Unfortunately, no family members were available for testing. However, it is not likely that the patient’s mother was a carrier for the rearrangement, because she would most certainly be infertile, and if the father had the rearranged X he would be expected to have a severely abnormal phenotype. Due to its presence in every cell examined, this mutation likely occurred very early. Because most CCRs reported are of paternal origin we propose that this event occurred during paternal meiosis (8). It appears that only the X chromosome was destabilized and shattered during a catastrophic event and repaired in a haphazard manner, resulting in this complex rearrangement. This rearrangement involves the ‘‘susceptibility’’ region of the X chromosome (Xp11.4 to Xq25). Two genes associated with POI, BMP 15 and POF1B, were both present in normal copy numbers, which underscores the possible positional effect of genes on the X chromosome (11). Alternatively, one or both of these genes may have been disrupted by their proximity to the breakpoint. The increased number of breakpoints in this CCR would have been expected to cause a number of phenotypic abnormalities; however, the patient appeared phenotypically normal, excluding her POI and early-onset hypertension, which were likely due to the completely skewed X inactivation. In general, duplications are less deleterious than deletions, and the number of breakpoints in a CCR is not necessarily predictive of the severity of the phenotype. In fact,
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FIGURE 2 FISH showing (A) duplicated XIST probe in red, and the centromeric marker in green. (B) Chromosome paint probes for the short arm (green) and long arm (red) show that a piece of p is moved to the q arm in the derivative X. (C) Xq13.2 (red) x3 RP13-216E22 Xp11.3 (green) x2 RP11-151G3. (D) Xq21.2, red RP11-187H16 – normal copy number, moved to Xq, near end of arm, distal to RP11-513O15. RP11513O15 (Xq22.3q23, green) – duplication visible on metaphase, both copies proximal to moved RP11-187H16. (E) RP13-216E22 (Xq13.2, red) – duplication visible on long metaphases; RP11-187H16 (Xq21.2, green) – normal copy number, moved to Xq, near end of arm. (F) RP11-107A19 (Xp11.23, red) – normal copy number, moved to Xqter right next to RP11-943J20; RP11-943J20 (Xq11.1, green) – duplicated, second copy on Xqter right next to RP11-107A19.
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FIGURE 3 Microarray results showing X chromosome alterations.
Ochalski. Complex rearranged X chromosome and POI. Fertil Steril 2011.
CCRs with no phenotypic effect have been described in the literature (4). In the present case, severe reproductive impairment may have occurred because of mispairing during meiosis activating DNA damage checkpoint mechanism or because X chromosome haploinsufficiency may be detrimental during germ cell development because biallelic expression of certain X chromosome genes may be required for proper oogenesis (12).
Array CGH is essential in the evaluation of complex chromosome rearrangements, because it describes the specific chromosomal segments involved and exact genes disturbed. There are known regions of the X chromosome that are critical to normal ovarian function; however, many genes have yet to be characterized, and new DNA technology can help identify those that may have essential roles in fertility.
TABLE 3 Published cases of complex intrachromosomal rearrangements of the X chromosome. Case no. 1 2 3 4
Origin
Phenotype
Breakpoints
References
De novo De novo De novo De novo
Primary amenorrhea Primary amenorrhea Primary amenorrhea Failure to thrive, muscular hypotonia, and minor facial anomalies
Xq22-q24 and Xp11 Xq22 and Xp11.2 Xp11.2 and Xq24 Xp11.21-p11.22
Grass et al., 1981 (9) Letterie, 1995 (8) Hernando et al., 2004 (4) Shchelochkov et al., 2008 (10)
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REFERENCES 1. Lindstrand A, Malmgren H, Sahlen S, Xin H, Schoumans J, Blennow E. Molecular cytogenetic characterization of a constitutional, highly complex intrachromosomal rearrangement of chromosome 1, with 14 breakpoints and a 0.5 Mb submicroscopic deletion. Am J Med Genet A 2008;146A:3217–22. 2. Pal L, Santoro N. Premature ovarian failure (POF): discordance between somatic and reproductive aging. Ageing Res Rev 2002;1:413–23. 3. Gardner RaSG. Chromosome abnormalities and genetic counseling. 3rd Ed. Oxford: Oxford University Press; 2004. 4. Hernando C, Plaja A, Catala V, Sarret E, Egozcue J, Fuster C. Primary amenorrhea in a woman with a cryptic complex chromosome rearrangement involving the critical regions Xp11.2 and Xq24. Fertil Steril 2004;82:1666–71.
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5. Ballif BC, Hornor SA, Sulpizio SG, Lloyd RM, Minier SL, Rorem EA, et al. Development of a high-density pericentromeric region BAC clone set for the detection and characterization of small supernumerary marker chromosomes by array CGH. Genet Med 2007;9:150–62. 6. Fujita T. Senescence marker protein-30 (SMP30): structure and biological function. Biochem Biophys Res Commun 1999;254:1–4. 7. Lacombe A, Lee H, Zahed L, Choucair M, Muller JM, Nelson SF, et al. Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure. Am J Hum Genet 2006;79:113–9. 8. Letterie GS. Unique unbalanced X;X translocation (Xq22;p11.2) in a woman with primary amenorrhea but without Ullrich-Turner syndrome. Am J Med Genet 1995;59:414–6.
9. Grass FS, Schwartz RP, Deal JO, Parke JC Jr. Gonadal dysgenesis, intra-X chromosome insertion, and possible position effect in an otherwise normal female. Clin Genet 1981;20:28–35. 10. Shchelochkov OA, Patel A, Weissenberger GM, Chinault AC, Wiszniewska J, Fernandes PH, et al. Duplication of chromosome band 12q24.11q24.23 results in apparent Noonan syndrome. Am J Med Genet A 2008;146A:1042–8. 11. Toniolo D. X-linked premature ovarian failure: a complex disease. Curr Opin Genet Dev 2006;16:293–300. 12. Lespinasse J, North MO, Paravy C, Brunel MJ, Malzac P, Blouin JL. A balanced complex chromosomal rearrangement (BCCR) in a family with reproductive failure. Hum Reprod 2003;18:2058–66.
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