- Email: [email protected]
A de novo complex chromosome rearrangement involving three chromosomes (2, 13, and 18) in an oligospermic male
CASE REPORT A de novo complex chromosome rearrangement involving three chromosomes (2, 13, and 18) in an oligospermic male Emel Ergul, Ph.D.,a Thomas Liehr, Ph.D.,b Kristin Mrasek, Ph.D.,b and Ali Sazci, Ph.D.a a b
Department of Medical Biology and Genetics, Faculty of Medicine, University of Kocaeli, Kocaeli, Turkey; and Institute of Human Genetics and Anthropology, Jena, Germany
Objective: To determine an unusual complex chromosome rearrangement found in a man with oligospermia with a normal phenotype. Design: Case report with a review of the literature. Setting: Academic research environment. Patient(s): A man with oligospermia but otherwise apparently healthy. Intervention(s): Peripheral blood lymphocytes were used for karyotyping, and metaphases were analyzed by the fluorescence in situ hybridization (FISH) procedure. Further characterization of the karyotype was done by using multicolor banding (MCB) probes. Main Outcome Measure(s): Physical examination, semen analysis, GTG banding, FISH, MCB. Result(s): The semen analysis revealed oligospermia. The lymphocytic karyotype detected an unusual complex chromosome rearrangement involving chromosomes 2, 13, and 18 determined by banding cytogenetics. Karyotype was established as 46,XY,t(2;13;18)ins(2;13)(2qter/2p25.1::13q13/13q22::18q12.3/18qter;13pter/ 13q13::2p25/2pter;18pter/18q12.3::13q22/13qter) after MCB analysis. Conclusion(s): The association of an unusual complex chromosome rearrangement with three recurrent spontaneous abortions was reported. (Fertil Steril 2009;92:391.e9–e12. 2009 by American Society for Reproductive Medicine.) Key Words: Complex chromosome rearrangement (CCR), oligospermia, recurrent spontaneous abortion, multicolor banding (MCB)
Complex chromosome rearrangements (CCRs) are structural aberrations that involve exchange of genetic material between two or more chromosomes, and they are classified depending on the origin, number of breakpoints, or number of chromosomes involved (1, 2). CCRs are rare events; to our knowledge, 156 cases of CCRs have been reported up to the time of writing (3–21). Despite the fact that there are familial cases reported, most CCRs are de novo. The transmission of the familial cases occurs mainly via the maternal line. However, the origin of de novo cases can also be of paternal origin (3). According to Kouseff et al. (22), CCRs can be categorized as group I, including those with %4 breaks, and group II, including >4 breaks. CCRs are usually associated with congenital anomalies and mental retardation. The
Received January 2, 2009; revised and accepted February 12, 2009; published online April 14, 2009. E.E. has nothing to disclose. T.L. has nothing to disclose. K.M. has nothing to disclose. A.S. has nothing to disclose. Supported in part by the German Academic Exchange Service (DAAD) (316-A/08/94286). €l, Ph.D., Department of Medical Biology, FacReprint requests: Emel Ergu €rkiye ulty of Medicine, Kocaeli University, 41380, Umuttepe, Kocaeli, Tu (FAX: þ90 262 303 70 03; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.02.042
resulting abnormal phenotype may be due to the disrupting of genes at the breakpoints or the presence of submicroscopic deletions or duplications. Chromosomal rearrangements resulting in gene inactivation at the breakpoints have been reported in a number of cases (2, 19, 23, 24). On the other hand, CCRs can also occur in phenotypically normal persons, and in such cases they are referred to as truly balanced. The regulatory sequences can also be affected at the balanced chromosomal rearrangements, which are usually associated with severe reproductive impairment through meiotic disturbance or chromosomal imbalance in gametes in men and recurrent miscarriages in women (2, 7, 25). It is difficult or even impossible to identify precise breakpoints of CCRs using only conventional cytogenetic techniques. Recently developed multicolor banding (MCB), based on overlapping region-specific microdissection libraries, is a powerful molecular cytogenetic method which allows the characterization of chromosomal breakpoints at the GTG sub-band level (26). In the present study, we report a patient with oligospermia who was found to have a de novo CCR involving the three chromosomes 2, 13, and 18.
Fertility and Sterility Vol. 92, No. 1, July 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1
FIGURE 1 Continued (A) GTG banded karyotype of the patient showing three derivative chromosomes. (B) The pedigree of the patient (arrow). (C) Multicolor banding (MCB) pattern of the molecular cytogenetic result. MCB with probe sets for chromosomes 2, 13, 18 are shown. The breakpoints were characterized as 2p25.1, 13q13, 13q22, and 18q12.1. The MCB pseudocolors and the underlying fluorescence profiles are shown (for detail of MCB evaluation see ref. no. 37). Probe sets used for chromosome 2, 13, and 18 are shown in the first column. The normal chromosomes are shown in the second column, and derivative chromosomes in the third, fourth, and fifth columns. MCB2 stains parts of der(2) and der(13), MCB13 stains part of der(2), der(13), and der(18), and MCB18 stains parts of der(2) and der(18). The parts that are not stained by the corresponding MCB probe sets are pseudocolored in gray. The complex chromosomal rearrangement was evaluated as balanced according to molecular cytogenetic analysis. Ergul. Complex chromosome rearrangements in men. Fertil Steril 2009.
found that the left one measured 40 21 25 mm, 11 mL in volume, and the right one measured 44 25 30 mm, 18 mL in volume. He had bilateral varicocele with grade 2. The FSH, LH, and T values were within the normal ranges. Semen analysis was performed three times according to the World Health Organization (27) guidelines for semen analysis within an interval of three months. Semen analyses revealed oligospermia with only 1.6 mL in volume, sperm concentration 20.2 106/mL, forward motility 35% at 1 hour, and after 4 hours, 80% immotile and 92% normal morphology. Total ejaculate contained only 32.3 106/1.6 mL on the average. Ethical Considerations Informed consent was obtained from the family before the study. The Institutional Review Board approved the study. Ergul. Complex chromosome rearrangements in men. Fertil Steril 2009.
MATERIALS AND METHODS Clinical Case Report The patient was a 28-year-old man working in a restaurant, from nonconsanguineous parents, who was married for 4 years and whose wife had three consecutive spontaneous abortions each within the first trimester. The first abortion was within 3 months, the second within 2 months, and the third within 1 month. His physical examination revealed a normally androgenized male with an unexpected volume difference between testes. His body mass index was 24.49 kg/m2, height 175 cm, and weight 92 kg. When his testes were investigated by using color Doppler ultrasound, it was 391.e10
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Banding Cytogenetics Cytogenetic analysis was performed by standard methods using cultured peripheral blood lymphocytes from the patient, his parents, one of his unmarried brothers, and his wife. Although the patient had two brothers and one sister, for this study only his unmarried brother was available. Twenty metaphases were analyzed from each subject by GTG banding technique. His sister and other brother were married with children. Molecular Cytogenetics The MCB probes for chromosomes 2, 13, and 18 and subtelomeric probe for 2pter (Abbott) and whole chromosome probe were performed to define the exact breakpoints of rearranged chromosomes (26). Metaphase spreads were analyzed by using a fluorescence microscope (Axioplan 2 mot, Zeiss)
Complex chromosome rearrangements in men
Vol. 92, No. 1, July 2009
equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain diaminophenylindol. For each chromosome, ten metaphases were evaluated. Image capturing and processing were carried out using the Isis mFISH imaging system (MetaSystems, Altlussheim, Germany) for the evaluation of MCB. RESULTS GTG banding revealed a reciprocal balanced triple translocation among chromosomes 2, 13, and 18 in the patient (Fig. 1A). The pedigree of the family is shown in Fig. 1B. Further characterization of the karyotype was done by using MCB probes described in Weise et al. (26). After MCB analysis, the karyotype was established as 46,XY,t(2;13;18)ins(2;13) (2qter/2p25.1::13q13/13q22::18q12.3/18qter;13pter/ 13q13::2p25/2pter;18pter/18q12.3::13q22/13qter) (Fig. 1C). The karyotypes of the proband’s parents, his unmarried brother, and his wife were normal (data not shown). DISCUSSION In the present case, three chromosomes were involved in CCRs: 2, 13, and 18. As a result, three derivative chromosomes were formed. The four breakpoints were located in 2p25.1,13q13 and 13q22, and 18q12.3. To the best of our knowledge, this is the first time a patient with such an apparently balanced translocation associated with three recurrent consecutive spontaneous abortions has been described. Compared with rearrangements up to 15 breakpoints (9), this case with CCR is considered to be at the low level of complexity.
plained by a similar mechanism (33). Although the mechanism of CCRs is unknown, molecular and cytogenetic analyses indicate that familial cases are due to the mistakes in female gametogenesis but most of de novo CCRs are of paternal origin occurring during spermatogenesis (7, 19). De Gregory et al. (17) suggested that some cells escape the mechanism for proper crossing-over during spermatogenesis in which chaotic breaks allow reunion of several chromosomes resulting in CCRs. Owing to the malsegregation of derivative chromosomes or the generation of a recombinant chromosome, the carrier of CCR has a risk for abnormal conception. Malsegregation follows the general principles as set forth for the simple translocation, but naturally the range of unbalanced combinations is greater. For the three-way CCR, the broad categories of malsegregation are 3:3 and 4:2, and theoretically 5:1 and 6:0. Recombination is rare indeed, and only eight examples have been recorded (34). There are several possibilities for a higher incidence of abnormal pregnancy outcome in our case. At the pachytene stage, the normal and derivative chromosomes 2, 13, and 18 are likely to have two possible configurations: 1) as a hexavalent with chromosomes 2, 13, and18 and the derivative der(2)t(2;13;18); or 2) tetravalent between chromosomes 13 and 18 and the derivatives der(13)t(2;13) and der(18)t(13;18) in addition to bivalent configuration. Either the tetravalent or the hexavalent configurations will give rise to many possible combinations of rearranged chromosomes in the embryos. Consequently, the unbalanced segregations are most likely to produce much greater genotypic imbalance and are expected to be nonviable, with all of them resulting in early pregnancy loss (35).
Lurie et al. (28) found that some chromosomes have more recurrent involvement in CCRs than others; among frequently involved chromosomes, 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, and 21 are usually altered, chromosomes 3 and 7 most frequently. Batanian and Eswara (3) reviewed 114 cases with CCR and proposed a hot spot on 18q21. Because there are two breakpoints on chromosome 18, they concluded that their case contradicts the general suggestion that only large chromosomes are susceptible to more than one break; this was also contradicted by a case with nine breaks in chromosome 21 (10). Battisti et al. (29) confirmed the hot spot on 18q21 and showed that chromosomes 3, 4, 8, 13, and 18 were involved in CCRs. The present study doesn’t confirm this hot spot on 18q21, but two breakpoints present on chromosome 13 confirm the suggestion by Batanian and Eswara that not only large chromosomes are prone to more than one breakpoint.
Precise definition of CCRs is not possible by classic banding cytogenetics. Identification of the exact breakpoints requires using molecular methods such as fluorescence in situ hybridization (FISH) with locus-specific probes or array CGH (36). Development of banding techniques based on FISH allows characterization of chromosomal subregions (37). The recently developed MCB technique is a powerful molecular cytogenetic method that produces changing florescence intensity ratios along the chromosome, by which it is possible to identify the exact breakpoints in the chromosomes at the band and subband level (38). We performed the MCB technique, following Weise et al. (26), as a very suitable approach to clarify the breakpoints and other chromosomal changes in CCRs to define exact breakpoints in the present case.
The fact that the present case is an oligospermic man raises the question of whether the involvement of autosomal chromosomes is the reason, because it is known that sex chromosome abnormalities are predominant in azoospermic men (12.6%), whereas autosomal abnormalities are more frequent in patients with oligospermia (3%) (30). Chromosomal rearrangements alter chromosome synapsis during meiosis, and this might cause impaired sperm production (31). In mice, asynapsed regions may trigger the meiotic checkpoint machinery to eliminate spermatocytes (32). Association of chromosomal abnormalities with deficient spermatogenesis might be ex-
The CCR presented here may be the cause of oligospermia and is, with high probability, responsible for the consecutive recurrent spontaneous abortions in the corresponding partnership. It is very important to characterize exact breakpoints in CCRs to understand the mechanism underlying the formation of CCRs and to provide correlation between phenotype and chromosomal rearrangement. It is also important to give adequate genetic counseling for the patients. The occurrence of CCRs is rare, but the number of reported cases has increased over the recent years, and therefore accumulating data may help to reveal the mechanism of CCRs.
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REFERENCES 1. Pai GS, Thomas GH, Mahoney W, Migeon BR. Complex chromosome rearrangements. Report of a new case and literature review. Clin Genet 1980;18:436–44. 2. Borg K, Stankiewicz P, Bocian E, Kruczek A, Obersztyn E, Lupski JR, Mazurczak T. Molecular analysis of a constitutional complex genome rearrangement with 11 breakpoints involving chromosomes 3, 11, 12, and 21 and an approximately 0.5-Mb submicroscopic deletion in a patient with mild mental retardation. Hum Genet 2005;118:267–75. 3. Batanian JR, Eswara MS. De novo apparently balanced complex chromosome rearrangement (CCR) involving chromosomes 4, 18, and 21 in a girl with mental retardation: report and review. Am J Med Genet A 1998;78:44–51. 4. Zahed L, Der Kaloustian V, Batanian JR. Familial complex chromosome rearrangement giving rise to balanced and unbalanced recombination products. Am J Med Genet 1998;79:30–4. 5. R€ othlisberger B, Kotzot D, Brecevic L, Koehler M, Balmer D, Binkert F, Schinzel A. Recombinant balanced and unbalanced translocations as a consequence of a balanced complex chromosomal rearrangement involving eight breakpoints in four chromosomes. Eur J Hum Genet 1999;8:873–83. 6. Berend SA, Bodamer OA, Shapira SK, Shaffer LG, Bacino CA. Familial complex chromosomal rearrangement resulting in a recombinant chromosome. Am J Med Genet 2002;109:311–7. 7. 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. 8. Lespinasse J, Rethore MO, North MO, Bovier-Lapierre M, Lundsteen C, Fert-Ferrer S, et al. Balanced complex chromosomal rearrangements (BCCR) with at least three chromosomes and three or more breakpoints: report of three new cases. Ann Genet 2004;47:315–24. 9. Houge G, Liehr T, Schouman J, Ness GO, Solland K, Starke H, et al. Ten years follow up of a boy with a complex chromosomal rearrangement: going from a >5 to 15-breakpoint CCR. Am J Med Genet A 2003;118:235–40. 10. Weise A, Rittinger O, Starke H, Ziegler M, Claussen U, Liehr T. De novo 9-break-event in one chromosome 21 combined with a microdeletion in 21q22.11 in a mentally retarded boy with short stature. Cytogenet Genome Res 2003;103:14–6. 11. Grasshoff U, Singer S, Liehr T, Starke H, Fode B, Sch€oning M, Dufke A. A complex chromosomal rearrangement with a translocation 4;10;14 in a fertile male carrier: ascertainment through an offspring with partial trisomy 14q13/q24.1 and partial monosomy 4q27/q28. Cytogenet Genome Res 2003;103:17–23. 12. Patsalis PC, Evangelidou P, Charalambous S, Sismani C. Fluorescence in situ hybridization characterization of apparently balanced translocation reveals cryptic complex chromosomal rearrangements with unexpected level of complexity. Eur J Hum Genet 2004;12:647–53. 13. Kuechler A, Ziegler M, Blank C, Rommel B, Bullerdiek J, Ahrens J, et al. A highly complex chromosomal rearrangement between five chromosomes in a healthy female diagnosed in preparation for intracytoplasmatic sperm injection. J Histochem Cytochem 2005;53:355–7. 14. Chen CP, Chern SR, Lee CC, Lin CC, Li YC, Hsieh LJ, et al. Prenatal diagnosis of de novo t(2;18;14)(q33.1;q12.2;q31.2), dup(5)(q34q34), del(7)(p21.1p21.1), and del(10)(q25.3q25.3) and a review of the prenatally ascertained de novo apparently balanced complex and multiple chromosomal rearrangements. Prenat Diagn 2006;26:138–46. 15. Karmous-Benailly H, Giuliano F, Massol C, Bloch C, De Ricaud D, Lambert JC, Perelman S. Unbalanced inherited complex chromosome rearrangement involving chromosome 8, 10, 11 and 16 in a patient with congenital malformations and delayed development. Eur J Med Genet 2006;49:431–8. 16. Giardino D, Corti C, Ballarati L, Finelli P, Valtorta C, Botta G, et al. Prenatal diagnosis of a de novo complex chromosome rearrangement (CCR) mediated by six breakpoints, and a review of 20 prenatally ascertained CCRs. Prenat Diagn 2006;26:565–70. 17. De Gregori M, Ciccone R, Magini P, Pramparo T, Gimelli S, Messa J, et al. Cryptic deletions are a common finding in ‘‘balanced’’ reciprocal and complex chromosome rearrangements: a study of 59 patients. J Med Genet 2007;44:750–62.
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18. Park JK, Lee JI, Jo HC, Shin JK, Choi WJ, Lee SA, et al. Molecular cytogenetic investigation of a balanced complex chromosomal rearrangement carrier ascertained through a neonate with partial trisomies of 13 and 22. Am J Med Genet A 2007;143A:1502–9. 19. Borg K, Nowakowska B, Obersztyn E, Cheung SW, Brycz-Witkowska J, Korniszewski L, et al. Complex balanced translocation t(1;5;7) (p32.1;14,3;p21.3) and two microdeletions del(1)(p31.1p31.1) and del(7)(p14.1p14.1) in a patient with features of Greig cephalopolysyndactyly and mental retardation. Am J Med Genet A 2007;143:2738–43. 20. Surace C, Digilio MC, Lombardo A, Sirleto P, Tomaiuolo AC, Roberti MC, et al. Masked complex chromosome rearrangement in a child thought to have del(8qter) as the sole cytogenetic abnormality. Cytogenet Genome Res 2008;121:215–21. 21. Karadeniz N, Mrasek K, Weise A. Further delineation of complex chromosomal rearrangements in fertile male using multicolor banding. Mol Cytogenet 2008;1:17. 22. Kousseff BG, Papenhausen P, Essig YP, Torres MP. Complex chromosome rearrangement with ankyloblepharon filiforme adnatum. J Med Genet 1993;30:167–70. 23. Bodrug SE, Roberson JR, Weiss L, Ray PN, Worton RG, Van Dyke DL. Prenatal identification of a girl with a t(X;4)(p21;q35) translocation: molecular characterization, paternal origin, and association with muscular dystrophy. J Med Genet 1990;27:426–32. 24. Pichon B, Vankerckhove S, Bourrouillou G, Duprez L, Abramowicz MJ. A translocation breakpoint disrupts the ASPM gene in a patient with primary microcephaly. Eur J Hum Genet 2004;12:419–21. 25. Kleinjan DA, van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease. Am J Hum Genet 2005;76:8–32. 26. Weise A, Mrasek K, Fickelscher I, Claussen U, Cheung SW, Cai WW, et al. Molecular definition of high-resolution multicolor banding probes: first within the human DNA sequence anchored FISH banding probe set. J Histochem Cytochem 2008;56:487–93. 27. World Health Organisation. WHO laboratory manual for the examination of human semen and sperm–cervical mucus interaction. 4th ed. Cambridge (U.K.): Cambridge University Press, 1999. 28. Lurie IW, Wulfsberg EA, Prabhakar G, Rosenblum-Vos LS, Supovitz KR, Cohen MM. Complex chromosomal rearrangements: some breakpoints may have cellular adaptive significance. Clin Genet 1994;46:244–7. 29. Battisti C, Bonaglia MC, Giglio S, Anichini C, Pucci L, Dotti MT, et al. De novo double translocation 3;13 and 4;8;18 in a patient with mental retardation and skeletal abnormalities. Am J Med Genet A 2003;117:207–11. 30. Lissitsina J, Mikelsaar R, Punab M. Cytogenetic analyses in infertile men. Arch Androl 2006;52:91–5. 31. Burgoyne PS, Baker TG. Meiotic pairing and gametogenic failure. Symp Soc Exp Biol 1984;38:349–62. 32. Odorisio T, Rodriguez TA, Evans EP, Clarke AR, Burgoyne PS. The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53independent apoptosis. Nat Genet 1998;18:257–61. 33. Bache I, Assche EV, Cingoz S, Bugge M, T€umer Z, Hjorth M, et al. An excess of chromosome 1 breakpoints in male infertility. Eur J Hum Genet 2004;12:993–1000. 34. Berend SA, Spikes AS, Kashork CD, Wu JM, Daw SC, Scambler PJ, et al. Dual-probe fluorescence in situ hybridization assay for detecting deletions associated with VCFS/DiGeorge syndrome I and DiGeorge syndrome II loci. Am J Med Genet 2000;91:313–7. 35. De Braekeleer M, Dao TN. Cytogenetic studies in male infertility: a review. Hum Reprod 1991;6:245–50. 36. Gribble SM, Prigmore E, Burford DC, Porter KM, Ng BL, Douglas EJ, et al. The complex nature of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes. J Med Genet 2005;42:8–16. 37. Liehr T, Starke H, Heller A, Kosyakova N, Mrasek K, Gross M, et al. Multicolor fluorescence in situ hybridization (FISH) applied to FISHbanding. Cytogenet Genome Res 2006;114:240–4. 38. Liehr T, Heller A, Starke H, Rubtsov N, Trifonov V, Mrasek K, et al. Microdissection based high resolution multicolor banding for all 24 human chromosomes. Int J Mol Med 2002;9:335–9.
Complex chromosome rearrangements in men
Vol. 92, No. 1, July 2009
Department of Medical Biology and Genetics, Faculty of Medicine, University of Kocaeli, Kocaeli, Turkey; and Institute of Human Genetics and Anthropology, Jena, Germany
Objective: To determine an unusual complex chromosome rearrangement found in a man with oligospermia with a normal phenotype. Design: Case report with a review of the literature. Setting: Academic research environment. Patient(s): A man with oligospermia but otherwise apparently healthy. Intervention(s): Peripheral blood lymphocytes were used for karyotyping, and metaphases were analyzed by the fluorescence in situ hybridization (FISH) procedure. Further characterization of the karyotype was done by using multicolor banding (MCB) probes. Main Outcome Measure(s): Physical examination, semen analysis, GTG banding, FISH, MCB. Result(s): The semen analysis revealed oligospermia. The lymphocytic karyotype detected an unusual complex chromosome rearrangement involving chromosomes 2, 13, and 18 determined by banding cytogenetics. Karyotype was established as 46,XY,t(2;13;18)ins(2;13)(2qter/2p25.1::13q13/13q22::18q12.3/18qter;13pter/ 13q13::2p25/2pter;18pter/18q12.3::13q22/13qter) after MCB analysis. Conclusion(s): The association of an unusual complex chromosome rearrangement with three recurrent spontaneous abortions was reported. (Fertil Steril 2009;92:391.e9–e12. 2009 by American Society for Reproductive Medicine.) Key Words: Complex chromosome rearrangement (CCR), oligospermia, recurrent spontaneous abortion, multicolor banding (MCB)
Complex chromosome rearrangements (CCRs) are structural aberrations that involve exchange of genetic material between two or more chromosomes, and they are classified depending on the origin, number of breakpoints, or number of chromosomes involved (1, 2). CCRs are rare events; to our knowledge, 156 cases of CCRs have been reported up to the time of writing (3–21). Despite the fact that there are familial cases reported, most CCRs are de novo. The transmission of the familial cases occurs mainly via the maternal line. However, the origin of de novo cases can also be of paternal origin (3). According to Kouseff et al. (22), CCRs can be categorized as group I, including those with %4 breaks, and group II, including >4 breaks. CCRs are usually associated with congenital anomalies and mental retardation. The
Received January 2, 2009; revised and accepted February 12, 2009; published online April 14, 2009. E.E. has nothing to disclose. T.L. has nothing to disclose. K.M. has nothing to disclose. A.S. has nothing to disclose. Supported in part by the German Academic Exchange Service (DAAD) (316-A/08/94286). €l, Ph.D., Department of Medical Biology, FacReprint requests: Emel Ergu €rkiye ulty of Medicine, Kocaeli University, 41380, Umuttepe, Kocaeli, Tu (FAX: þ90 262 303 70 03; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.02.042
resulting abnormal phenotype may be due to the disrupting of genes at the breakpoints or the presence of submicroscopic deletions or duplications. Chromosomal rearrangements resulting in gene inactivation at the breakpoints have been reported in a number of cases (2, 19, 23, 24). On the other hand, CCRs can also occur in phenotypically normal persons, and in such cases they are referred to as truly balanced. The regulatory sequences can also be affected at the balanced chromosomal rearrangements, which are usually associated with severe reproductive impairment through meiotic disturbance or chromosomal imbalance in gametes in men and recurrent miscarriages in women (2, 7, 25). It is difficult or even impossible to identify precise breakpoints of CCRs using only conventional cytogenetic techniques. Recently developed multicolor banding (MCB), based on overlapping region-specific microdissection libraries, is a powerful molecular cytogenetic method which allows the characterization of chromosomal breakpoints at the GTG sub-band level (26). In the present study, we report a patient with oligospermia who was found to have a de novo CCR involving the three chromosomes 2, 13, and 18.
Fertility and Sterility Vol. 92, No. 1, July 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
391.e9
FIGURE 1
FIGURE 1 Continued (A) GTG banded karyotype of the patient showing three derivative chromosomes. (B) The pedigree of the patient (arrow). (C) Multicolor banding (MCB) pattern of the molecular cytogenetic result. MCB with probe sets for chromosomes 2, 13, 18 are shown. The breakpoints were characterized as 2p25.1, 13q13, 13q22, and 18q12.1. The MCB pseudocolors and the underlying fluorescence profiles are shown (for detail of MCB evaluation see ref. no. 37). Probe sets used for chromosome 2, 13, and 18 are shown in the first column. The normal chromosomes are shown in the second column, and derivative chromosomes in the third, fourth, and fifth columns. MCB2 stains parts of der(2) and der(13), MCB13 stains part of der(2), der(13), and der(18), and MCB18 stains parts of der(2) and der(18). The parts that are not stained by the corresponding MCB probe sets are pseudocolored in gray. The complex chromosomal rearrangement was evaluated as balanced according to molecular cytogenetic analysis. Ergul. Complex chromosome rearrangements in men. Fertil Steril 2009.
found that the left one measured 40 21 25 mm, 11 mL in volume, and the right one measured 44 25 30 mm, 18 mL in volume. He had bilateral varicocele with grade 2. The FSH, LH, and T values were within the normal ranges. Semen analysis was performed three times according to the World Health Organization (27) guidelines for semen analysis within an interval of three months. Semen analyses revealed oligospermia with only 1.6 mL in volume, sperm concentration 20.2 106/mL, forward motility 35% at 1 hour, and after 4 hours, 80% immotile and 92% normal morphology. Total ejaculate contained only 32.3 106/1.6 mL on the average. Ethical Considerations Informed consent was obtained from the family before the study. The Institutional Review Board approved the study. Ergul. Complex chromosome rearrangements in men. Fertil Steril 2009.
MATERIALS AND METHODS Clinical Case Report The patient was a 28-year-old man working in a restaurant, from nonconsanguineous parents, who was married for 4 years and whose wife had three consecutive spontaneous abortions each within the first trimester. The first abortion was within 3 months, the second within 2 months, and the third within 1 month. His physical examination revealed a normally androgenized male with an unexpected volume difference between testes. His body mass index was 24.49 kg/m2, height 175 cm, and weight 92 kg. When his testes were investigated by using color Doppler ultrasound, it was 391.e10
Ergul et al.
Banding Cytogenetics Cytogenetic analysis was performed by standard methods using cultured peripheral blood lymphocytes from the patient, his parents, one of his unmarried brothers, and his wife. Although the patient had two brothers and one sister, for this study only his unmarried brother was available. Twenty metaphases were analyzed from each subject by GTG banding technique. His sister and other brother were married with children. Molecular Cytogenetics The MCB probes for chromosomes 2, 13, and 18 and subtelomeric probe for 2pter (Abbott) and whole chromosome probe were performed to define the exact breakpoints of rearranged chromosomes (26). Metaphase spreads were analyzed by using a fluorescence microscope (Axioplan 2 mot, Zeiss)
Complex chromosome rearrangements in men
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equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain diaminophenylindol. For each chromosome, ten metaphases were evaluated. Image capturing and processing were carried out using the Isis mFISH imaging system (MetaSystems, Altlussheim, Germany) for the evaluation of MCB. RESULTS GTG banding revealed a reciprocal balanced triple translocation among chromosomes 2, 13, and 18 in the patient (Fig. 1A). The pedigree of the family is shown in Fig. 1B. Further characterization of the karyotype was done by using MCB probes described in Weise et al. (26). After MCB analysis, the karyotype was established as 46,XY,t(2;13;18)ins(2;13) (2qter/2p25.1::13q13/13q22::18q12.3/18qter;13pter/ 13q13::2p25/2pter;18pter/18q12.3::13q22/13qter) (Fig. 1C). The karyotypes of the proband’s parents, his unmarried brother, and his wife were normal (data not shown). DISCUSSION In the present case, three chromosomes were involved in CCRs: 2, 13, and 18. As a result, three derivative chromosomes were formed. The four breakpoints were located in 2p25.1,13q13 and 13q22, and 18q12.3. To the best of our knowledge, this is the first time a patient with such an apparently balanced translocation associated with three recurrent consecutive spontaneous abortions has been described. Compared with rearrangements up to 15 breakpoints (9), this case with CCR is considered to be at the low level of complexity.
plained by a similar mechanism (33). Although the mechanism of CCRs is unknown, molecular and cytogenetic analyses indicate that familial cases are due to the mistakes in female gametogenesis but most of de novo CCRs are of paternal origin occurring during spermatogenesis (7, 19). De Gregory et al. (17) suggested that some cells escape the mechanism for proper crossing-over during spermatogenesis in which chaotic breaks allow reunion of several chromosomes resulting in CCRs. Owing to the malsegregation of derivative chromosomes or the generation of a recombinant chromosome, the carrier of CCR has a risk for abnormal conception. Malsegregation follows the general principles as set forth for the simple translocation, but naturally the range of unbalanced combinations is greater. For the three-way CCR, the broad categories of malsegregation are 3:3 and 4:2, and theoretically 5:1 and 6:0. Recombination is rare indeed, and only eight examples have been recorded (34). There are several possibilities for a higher incidence of abnormal pregnancy outcome in our case. At the pachytene stage, the normal and derivative chromosomes 2, 13, and 18 are likely to have two possible configurations: 1) as a hexavalent with chromosomes 2, 13, and18 and the derivative der(2)t(2;13;18); or 2) tetravalent between chromosomes 13 and 18 and the derivatives der(13)t(2;13) and der(18)t(13;18) in addition to bivalent configuration. Either the tetravalent or the hexavalent configurations will give rise to many possible combinations of rearranged chromosomes in the embryos. Consequently, the unbalanced segregations are most likely to produce much greater genotypic imbalance and are expected to be nonviable, with all of them resulting in early pregnancy loss (35).
Lurie et al. (28) found that some chromosomes have more recurrent involvement in CCRs than others; among frequently involved chromosomes, 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, and 21 are usually altered, chromosomes 3 and 7 most frequently. Batanian and Eswara (3) reviewed 114 cases with CCR and proposed a hot spot on 18q21. Because there are two breakpoints on chromosome 18, they concluded that their case contradicts the general suggestion that only large chromosomes are susceptible to more than one break; this was also contradicted by a case with nine breaks in chromosome 21 (10). Battisti et al. (29) confirmed the hot spot on 18q21 and showed that chromosomes 3, 4, 8, 13, and 18 were involved in CCRs. The present study doesn’t confirm this hot spot on 18q21, but two breakpoints present on chromosome 13 confirm the suggestion by Batanian and Eswara that not only large chromosomes are prone to more than one breakpoint.
Precise definition of CCRs is not possible by classic banding cytogenetics. Identification of the exact breakpoints requires using molecular methods such as fluorescence in situ hybridization (FISH) with locus-specific probes or array CGH (36). Development of banding techniques based on FISH allows characterization of chromosomal subregions (37). The recently developed MCB technique is a powerful molecular cytogenetic method that produces changing florescence intensity ratios along the chromosome, by which it is possible to identify the exact breakpoints in the chromosomes at the band and subband level (38). We performed the MCB technique, following Weise et al. (26), as a very suitable approach to clarify the breakpoints and other chromosomal changes in CCRs to define exact breakpoints in the present case.
The fact that the present case is an oligospermic man raises the question of whether the involvement of autosomal chromosomes is the reason, because it is known that sex chromosome abnormalities are predominant in azoospermic men (12.6%), whereas autosomal abnormalities are more frequent in patients with oligospermia (3%) (30). Chromosomal rearrangements alter chromosome synapsis during meiosis, and this might cause impaired sperm production (31). In mice, asynapsed regions may trigger the meiotic checkpoint machinery to eliminate spermatocytes (32). Association of chromosomal abnormalities with deficient spermatogenesis might be ex-
The CCR presented here may be the cause of oligospermia and is, with high probability, responsible for the consecutive recurrent spontaneous abortions in the corresponding partnership. It is very important to characterize exact breakpoints in CCRs to understand the mechanism underlying the formation of CCRs and to provide correlation between phenotype and chromosomal rearrangement. It is also important to give adequate genetic counseling for the patients. The occurrence of CCRs is rare, but the number of reported cases has increased over the recent years, and therefore accumulating data may help to reveal the mechanism of CCRs.
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Vol. 92, No. 1, July 2009