http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic) Syst Biol Reprod Med, 2014; 60(6): 367–372 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2014.948580
RESEARCH ARTICLE
Different segregation patterns in five carriers due to a pericentric inversion of chromosome 1 Yuqin Luo1,2, Chenming Xu1,2, Yixi Sun1,2, Liya Wang1,2, Songchang Chen1,2, and Fan Jin2,3* 1
Department of Reproductive Genetics, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China, 2Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of Education, Hangzhou, P.R. China, and 3Centre of Reproductive Medicine, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China Abstract
Keywords
Pericentric inversion can produce recombinant gametes; however, meiotic segregation studies on the relationship between the frequency of recombinants and the inverted segment size are rare. Triple-color fluorescence in situ hybridization (FISH) was performed to analyze the meiotic behavior in five inv(1) carriers with different breakpoints. Recombination gametes were absent in Patient 1, whereas the percentages of the recombinants in Patients 2, 3, 4, and 5 were of 9.2%, 15.3%, 17.3%, and 40.9%, respectively. A significant difference was present for the frequencies of the recombinant spermatozoa among the five patients (p50.001). For each patient, the frequency of the two types of recombinant gametes (dup(1p)/del(1q) or del(1p)/ dup(1q)) did not exhibit a significant difference in comparison with the expected 1:1 ratio (p40.05). The meiotic segregation of nine inv(1) carriers (including those presented in this paper) is now available. A significant correlation was discovered between the rate of recombination and the proportion of the chromosome implicated in the inversion (R ¼ 0.9435, p50.001). The frequency of the recombinant gametes was directly related to the proportion of the chromosome that was inverted. Sperm-FISH allowed an additional comprehension of the patterns of meiotic segregation and provided accurate genetic counseling.
Chromosome 1, infertility, meiotic segregation, pericentric inversion, sperm FISH
Introduction Pericentric inversion is a structural chromosomal rearrangement, which is the result of two breaks on both sides of the centromere within a single chromosome and is followed by a 180 rotation and reintegration of the inverted segment in the same chromosome. In the general population, the frequency of pericentric inversions is estimated to be variable from 0.089 % [Ravel et al. 2006] to 0.34% [Nielsen and Wohlert 1991], or even at 1–2% [Kaiser 1984]. Most pericentric inversions affect the pericentric region of chromosome 2 and the heterochromatic region of chromosomes 1, 9, 16, or Y, which are considered as non-pathological polymorphisms. Generally, carriers of pericentric inversions do not show phenotypic alteration but they might have reproductive risks, such as the production of chromosomally imbalanced gametes and/or spermatogenetic failure caused by chromosome reorganization [Anton et al. 2005; Ferfouri et al. 2009]. During meiosis, the complete pairing of an inverted and its normal homologous chromosome requires the formation of an inversion loop, which may produce two abnormal gametes with both duplicated and deleted chromosome segments for *Address correspondence to Fan Jin, MD, Centre of Reproductive Medicine, Women’s Hospital, School of Medicine, Zhejiang University, 1 Xueshi Road, Hangzhou, Zhejiang, 310006, China. Tel: 86571-87013891. E-mail: [email protected]
History Received 25 March 2014 Revised 9 June 2014 Accepted 11 June 2014 Published online 6 August 2014
regions distal to the inversion (duplication p/deletion q (dup p/del q) or deletion p/duplication q (del p/dup q)). To date, few studies have analyzed the segregation products on the sperm nuclei using fluorescence in situ hybridization (FISH) in the case of pericentric inversion [Anton et al. 2002; Anton et al. 2006; Bernicot et al. 2010; Caer et al. 2008; Chantot-Bastaraud et al. 2007; Malan et al. 2006; Mikhaail-Philips et al. 2004; Mikhaail-Philips et al. 2005]. The frequency of the two abnormal gametes ((dup p/ del q and del p/dup q) was highly variable from 0% to 37.85%. Such variability indicated that the inversion couples had a divergent effect on the reproductive fitness. To determine the meiotic segregation behavior in spermatozoa in pericentric inversion carriers, we used sperm-FISH in five cases of a pericentric inversion of chromosome 1 with different breakpoints: inv(1)(p11;q12), inv(1) (p32q21), inv(1)(p36.3q21), inv(1)(p32q32), and inv(1) (p36.2q42). As far as we know, this is the first report which analyzes the segregation pattern in spermatozoa from five carriers of a pericentric inversion involving the same chromosome with different meiotic segregation. There has yet to be a statistical study that analyzes the correlation between the frequency of the recombinants and the inverted segment size for the same chromosome. This primarily reflects the absence of the carrier of a pericentric inversion within the same chromosome with different breakpoints.
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Results Five inv(1) patients between 32 and 41 years of age were consulted for primary infertility (n ¼ 4) or repeated firsttrimester spontaneous abortions (n ¼ 1). The same sperm sample was analyzed for sperm parameters and FISH. According to the criteria of the World Health Organization [WHO 1999], the inv (1) carriers revealed abnormal sperm parameters with oligoasthenospermia or teratozoospermia. Seminal parameters are shown in Table 1. FISH was performed on metaphase spreads obtained from peripheral blood lymphocytes revealing a normal localization of the signals (Figure 1A) except an inv(1) (p36.3q21) carrier (Figure 2). The size of the inverted segment represented 8.54%, 36.9%, 59.9%, 62.3%, and 91.5% of the whole chromosome 1. The 1p subtelomere probe (Spectrum Green) was shown near the centromere of the inv(1) (p36.3q21) chromosome (Figure 1B). In the segregation analysis, 1438 spermatozoa from Patient 1, 1630 spermatozoa from Patient 2, 654 spermatozoa from
Patient 3, 867 spermatozoa from Patient 4, and 1858 spermatozoa from Patient 5 were analyzed. The frequencies of the non-recombinant products were 98.7%, 89.2%, 82.9%, 80.7%, and 56.6% for Patients 1, 2, 3, 4, and 5, respectively. Recombination gametes were absent in Patient 1, whereas Patients 2, 3, 4, and 5 had recombination gamete percentages of 9.2%, 15.3%, 17.3%, and 40.9%, respectively. A significant difference was presented for the frequencies of the recombinant spermatozoa among the five patients (p50.001). The remaining ratio of spermatozoa was 1.3%, 1.6%, 1.8%, 2.0%, and 2.5%, which had other signal combinations that probably resulted from incomplete hybridization or were classical recombinants with incomplete hybridization. The frequency of the dup(1p)/del(1q) recombinant sperm was 0% (Patient 1), 4.4% (Patient 2), 6.8% (Patient 3), 9.4% (Patient 4), and 19.7% (Patient 5), while the frequency of the del(1p)/dup(1q) recombinants was 0%, 4.8%, 8.5%, 7.9%, and 21.2% for Patients 1, 2, 3, 4, and 5, respectively. For each patient, the frequency of the two types of recombinant
Table 1. Patients, cytogenetic analysis, and sperm parameters. Patient 1 2 3 4 5
Karyotype
Ascertainment
Age (years)
Sperm concentration (106/ml)
Motility (%)
Abnormal forms (%)
inv(1)(p11q12) inv(1)(p32q21) inv(1)(p36.3q21) inv(1)(p32q32) inv(1)(p36.2q42)
infertility infertility infertility infertility repeated abortion
34 34 35 32 41
10 1.8 101 2 103 3 103 40
25 15 1 5 51
70 52 96 99 85
Figure 1. FISH analysis of five inversion carriers. Hybridization of the green chromosome 1p subtelomeric probe, red chromosome 1q subtelomeric probe, and aqua chromosome 18 centromeric probe are shown. (A) Hybridization of lymphocyte metaphase from carrier with inv(1)(p32q21). (B) Hybridization of lymphocyte metaphase from carrier with inv(1)(p36.3q21). (C) Sperm with normal or inv(1) chromosome (aqua, green, and red). (D) One dup(1q)/del(1p) sperm (two red and aqua). (E) Two dup(1p)/del(1q) sperm (two green and aqua).
DOI: 10.3109/19396368.2014.948580
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Figure 2. Idiograms and GTG banding. The idiograms for the normal chromosome 1 and five inversion carriers are shown. Breakpoints are indicated.
gametes (dup(1p)/del(1q) or del(1p)/dup(1q)) did not exhibit a statistically significant difference in comparison with the expected 1:1 ratio (p40.05).
Discussion In this study, sperm-FISH was used to analyze the production of recombinant gametes of five carriers due to a pericentric inversion of chromosome 1. The frequencies of the recombinant products were significantly different among the five male patients. No significant difference was discovered between the frequencies of the two types of recombinant gametes and the expected 1:1 ratio, therefore, genotypic selection was not apparent. The inverted segment size corresponds to only 8.54% of chromosome 1 for the inv(1)(p11q12) patient, and no recombinant spermatozoa were discovered. The heterochromatic region of chromosome 1 was always considered a nonpathological polymorphism, and this inversion is not linked to an increased risk of abnormal progeny in comparison with the general population. This group of patients would not benefit from preimplantation genetic diagnosis or amniocentesis and chorion villus sampling. For the inv(1)(p32q21) carrier, the inverted segment is approximately one-third of the chromosome length, and both breakpoints are in the G light bands. The rate of spermatozoa exhibiting a chromosomal imbalance was 9.2%. In comparison, Jaarola et al. [1998] discovered that few recombinant spermatozoa appeared as inv(1)(p31q12) carriers, and the inversion size was approximately 30% of the total chromosomal length. The breakpoint is present at the 1q12 of the heterochromatic region where recombination is restrained, which may explain this low frequency of recombinant spermatozoa. For Patients 3 and 4, the inversion corresponded to more than half of the total chromosome length (59.9 and 62.3%,
respectively), and the imbalanced spermatozoa were 15.3% (Patient 3) and 17.3% (Patient 4). In addition, two similar cases of pericentric inversion of chromosome 1 have been reported. In one study, Yakut et al. [2003] discovered that the rate of the recombinant gametes was 16% when the inversion size was 47.97% of the total chromosomal length. In another study, Chantot-Bastaraud et al. [2007] reported that the frequency of the recombinant products was 14.8% while the size of the inverted segment represented 52.2% in an inv(1)(p22q42) carrier. A high level of recombinant product (40.9%) was discovered for the inv(1) (p36.2q42) carrier, and the inverted segment size was 91.5% of the total length of chromosome 1. A similar result was observed in a inv(1) (p36.3q43) carrier with a large chromosome inversion. The inverted segment size was 95%, and the percentage of imbalanced gametes was 30.43% [Morel et al. 2007]. Thus, the risk of recombination aneusomy in the offspring was high, consistent with two early miscarriages. The high risk of miscarriage (13/38) was observed in the same inversion inv(1)(p36.2q42) in five generational pedigrees [Honeywell et al. 2012]. A disequilibrium recombinant can lead to repeated spontaneous abortions in some conceptuses because of an increase or decrease in the size of the distal segment. In this study, the range of the recombinant spermatozoa varied from 0 to 40.9% causing the variable reproductive risks for inv (1). The rate of imbalanced recombinants seemed to be influenced by many factors, such as the chromosome involved, the region affected, the position of the breakpoints, or the size of the inverted segment [Anton et al. 2005; Morel et al. 2007]. The frequency of the recombinant gametes was plotted according to the relative length of the inverted segment of nine male carriers of a pericentric inversion of -chromosome 1. The meiotic segregation of nine inv(1) carriers (including those presented in the paper) is now available and summarized in Table 2. A significant
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Table 2. Frequencies of non-recombinant and recombinant sperm in nine men heterozygous for a pericentric inversion of chromosome 1 by spermFISH analysis.
Patient 1 2 3 4 5 [Jaarola et al. 1998] [Yakut et al. 2003] [Chantot-Bastaraud et al. 2007] [Morel et al. 2007]
Inversion inv(1) inv(1) inv(1) inv(1) inv(1) inv(1) inv(1) inv(1)
Inverted segment (%)
Total Spermatozoa
Nonrecombinants (%)
Recombinants (%)
dup(1p)/ del(1q)
del(1p)/ dup(1q)
Others
8.54 36.9 59.9 62.3 91.5 30.49 47.97 52.2
1438 1630 654 867 1858 6006 1636 2133
98.7 89.2 82.9 80.7 56.6 99.33 82.5 80.1
0 9.2 15.3 17.3 40.9 0.38 16.0 14.8
0 4.4 6.8 9.4 19.7 0.25 8.7 7.6
0 4.8 8.5 7.9 21.2 0.13 7.3 7.2
1.3 1.6 1.8 2.0 2.5 0.28 1.5 5.1
69
68.12
30.43
11.59
18.84
1.45
(p11q12) (p32q21) (p36.3q21) (p32q32) (p36.2q42) (p31q12) (p36q32) (p22q42)
inv(1) (p36.3q43)
95
Figure 3. Relative size of inversions observed. The distribution of the percentages of the recombinant gametes as a function of the relative size of the inversion is shown. Individual data points are shown as well as the regression line (R ¼ 0.9435).
correlation (R ¼ 0.9435, p50.001) was discovered between the rate of recombination and the proportion of the chromosome implicated in the inversion (Figure 3). To our knowledge, this is the first report to analyze the relation between the frequency of the recombinants and the inverted segment size in the case of pericentric inversion for the same chromosome. The frequency of the recombinants was strongly associated with the inverted segment size, but not with the affected chromosome. This study sheds new light on the role of the proportion of the inverted segment on the final outcome of recombinant gametes. The risk of unbalanced gametes must be considered with care as a function of the increase of the inverted segment proportion. If the inverted segment was nearly the total chromosome size, approximately 50% of recombinant chromosomes could be produced. Sperm-FISH provided a personalized risk assessment of unbalanced spermatozoa analyzing the patterns of meiotic segregation. The inv (1) cases appeared to be at a special risk because four of the five carriers were infertile. Chromosome
1 harbors a critical chromosomal domain, the integrity of which was essential for normal spermatogenesis [Bache et al. 2004]. Spermatogenic failure associated with rearrangements on chromosome 1 can be correlated with spermatogenic failure 2 (SPGF2;OMIM 108420). The detection of particular breakpoint sites can be correlated with a degree of spermatogenic failure in male carriers. The inv(1) carrier displayed synapsis impairment for all unsynapsed bivalents exhibiting markers of transcriptional silencing, which may cause impaired spermatogenesis [Kirkpatrick et al. 2012]. A meiotic failure could be due to a reduction in recombination events with an absence of a full loop formation and the rare occurrence of a partial loop leading [Chandley et al. 1987]. In conclusion, the frequency of the recombinant gametes was directly related to the proportion of the chromosome inverted. Sperm-FISH provided additional understanding of the patterns of meiotic segregation which should aid accurate genetic counseling. The inv(1) carriers appeared to be at high risk for male infertility.
DOI: 10.3109/19396368.2014.948580
Materials and Methods Patients Five inv(1) patients (Figure 2) between 32 and 41 years old were consulted for primary infertility (n ¼ 4) or for two firsttrimester spontaneous abortions (n ¼ 1). The size of the inverted segment represented 8.54%, 36.9%, 59.9%, 62.3%, and 91.5% of the whole chromosome 1. Each patient provided their informed consent for the genetic investigation and sperm chromosomal evaluation as approved by our institutional ethics committee. None of the five patients had any positive familial history for congenital malformations or mental retardation. No AZF microdeletions were found using six sequence targeted sites (sY254, sY86, sY127, sY84, sY134, and sY255) in four inv(1) infertility carriers [Simoni et al. 2004]. Sperm parameters The same sperm sample was analyzed for sperm parameters and FISH. According to the criteria of the WHO [1999], the inv (1) carriers revealed abnormal sperm parameters with oligoasthenospermia or teratozoospermia. Seminal parameters are shown in Table 1. Sperm FISH analysis The sperm samples of the five carriers were analyzed by triple-FISH with the 1p subtelomeric probe (Tel1p, CEB108/ T7, Spectrum Green, Vysis, Downers Grove, IL, USA), the 1q subtelomeric probe (Tel1q, VIJ2YRM2123, Spectrum Orange, Vysis), and the chromosome 18 centromeric probe (CEP18, Spectrum Aqua, Vysis). The triple-FISH was performed on the blood cell metaphases in the five inv (1) carriers in order to avoid chromosome 1 translocation. The sperm sample was washed three times with phosphatebuffered saline (PBS) and was fixed in fresh fixative (3:1 methanol: acetic acid). The spermatozoa were then spread onto a clean glass slide and air-dried. The sperm heads were decondensed by incubation in 1 M NaOH for 2 min at room temperature. Probe hybridization and detection were performed in accordance with the manufacturer’s instructions (Vysis). The slides were examined using a Zeiss Imager. An A2 microscope (Zeiss, France) equipped with a FISH Imaging System (Isis, MetaSystems, Germany) and filter sets for DAPI, FITC, RBITC, Texas Red, and Aqua were used. Chromosome 1 segregation pattern In the segregation outcome analysis, the sperm heads containing one blue, one green, and one orange signal represented normal or inverted chromosome 1, while those containing one blue signal and two green signals were considered as a recombinant dup(1p)/del(1q) chromosome, and those with one blue signal and two orange signals were characterized as recombinant del(1p)/dup(1q) chromosome. Statistical analysis Data were statistically analyzed with the chi-square test to compare the frequencies of the recombinant products of both types and the non-recombinant spermatozoa for each patient.
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Pearson’s correlation coefficient was used to analyze the relation between the inversion size and the frequency of the recombinants.
Declaration of interest This work was supported by the National Basic Research Program of China (No. 2012CB944901), the Scientific Research Foundation of Zhejiang Medical and Health System (2010KYB065), and Grant of Science and Technology Plan of Zhejiang Province (2010C33096).The authors have no conflicts of interest to declare.
Author contributions Conceived and designed the experiments: Y-QL, FJ; Performed the experiments: Y-QL, C-MX; Analyzed the data: Y-XS, L-YW; Wrote the manuscript: Y-QL; Revised the manuscript: L-YW; S-CC; All authors approved the revisions to the manuscript.
References Anton, E., Blanco, J., Egozcue, J., and Vidal, F. (2002) Risk assessment and segregation analysis in a pericentric inversion inv6p23q25 carrier using FISH on decondensed sperm nuclei. Cytogenet Genome Res 97: 149–54. Anton, E., Blanco, J., Egozcue, J., and Vidal, F. (2005) Sperm studies in heterozygote inversion carriers: a review. Cytogenet Genome Res 111: 297–304. Anton, E., Vidal, F., Egozcue, J., and Blanco, J. (2006) Genetic reproductive risk in inversion carriers. Fertil Steril 85:661–6. Bache, I., Assche, E.V., Cingoz, S., Bugge, M., Tumer, Z., Hjorth, M., et al. (2004) An excess of chromosome 1 breakpoints in male infertility. Eur J Hum Genet 12:993–1000. Bernicot, I., Dechanet, C., Mace, A., Hedon, B., Hamamah, S., Pellestor, F., et al. (2010) Predictive value of sperm-FISH analysis on the outcome of preimplantation genetic diagnosis (PGD) for a pericentric inversion inv5(p15.3q11.2) carrier. Hum Reprod 25: 1818–23. Caer, E., Perrin, A., Douet-Guilbert, N., Amice, V., De Braekeleer, M., and Morel, F. (2008) Differing mechanisms of meiotic segregation in spermatozoa from three carriers of a pericentric inversion of chromosome 8. Fertil Steril 89:1637–40. Chandley, A.C., McBeath, S., Speed, R.M., Yorston, L., and Hargreave, T.B. (1987) Pericentric inversion in human chromosome 1 and the risk for male sterility. J Med Genet 24:325–34. Chantot-Bastaraud, S., Ravel, C., Berthaut, I., McElreavey, K., Bouchard, P., Mandelbaum, J., et al. (2007) Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46, XY, inv(1)(p22q42), associated with infertility. Mol Hum Reprod 13:55–9. Ferfouri, F., Clement, P., Gomes, D.M., Minz, M., Amar, E., Selva, J., et al. (2009) Is classic pericentric inversion of chromosome 2 inv(2)(p11q13) associated with an increased risk of unbalanced chromosomes? Fertil Steril 92:1491–7. Honeywell, C., Argiropoulos, B., Douglas, S., Blumenthal, A.L., Allanson, J., McGowan-Jordan, J., et al. (2012) Apparent transmission distortion of a pericentric chromosome one inversion in a large multigeneration pedigree. Am J Med Genet A 158A:1262–8. Jaarola, M., Martin, R.H., and Ashley, T. (1998) Direct evidence for suppression of recombination within two pericentric inversions in humans: a new sperm-FISH technique. Am J Hum Genet 63: 218–24. Kaiser, P. (1984) Pericentric inversions. Problems and significance for clinical genetics. Hum Genet 68:1–47. Kirkpatrick, G., Chow, V., and Ma, S. (2012) Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier. Reprod Biomed Online 24:91–100. Malan, V., Pipiras, E., Sifer, C., Kanafani, S., Cedrin-Durnerin, I., Martin-Pont, B., et al. (2006) Chromosome segregation in an infertile
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man carrying a unique pericentric inversion, inv(21)(p12q22.3), analysed using fluorescence in situ hybridization on sperm nuclei: Significance for clinical genetics. A case report. Hum Reprod 21: 2052–6. Mikhaail-Philips, M.M., Ko, E., Chernos, J., Greene, C., Rademaker, A., and Martin, R. H. (2004) Analysis of chromosome segregation in sperm from a chromosome 2 inversion heterozygote and assessment of an interchromosomal effect. Am J Med Genet A 127A: 139–43. Mikhaail-Philips, M.M., McGillivray, B.C., Hamilton, S.J., Ko, E., Chernos, J., Rademaker, A., et al. (2005) Unusual segregation products in sperm from a pericentric inversion 17 heterozygote. Hum Genet 117:357–65. Morel, F., Laudier, B., Guerif, F., Couet, M.L., Royere, D., Roux, C., et al. (2007) Meiotic segregation analysis in spermatozoa of pericentric inversion carriers using fluorescence in-situ hybridization. Hum Reprod 22:136–41.
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Nielsen, J., and Wohlert, M. (1991) Chromosome abnormalities found among 34,910 newborn children: Results from a 13-year incidence study in Arhus, Denmark. Hum Genet 87:81–3. Ravel, C., Berthaut, I., Bresson, J.L., and Siffroi, J.P. (2006) Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: Large-scale survey of over 10,000 sperm donor karyotypes. Hum Reprod 21:1484–9. Simoni, M., Bakker, E., and Krausz, C. (2004) EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art 2004. Int J Androl 27:240–9. World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction. Cambridge University Press; Cambridge, United Kingdom. Yakut, T., Acar, H., Egeli, U., and Kimya, Y. (2003) Frequency of recombinant and nonrecombinant products of pericentric inversion of chromosome 1 in sperm nuclei of carrier: By FISH technique. Mol Reprod Dev 66:67–71.
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RESEARCH ARTICLE
Different segregation patterns in five carriers due to a pericentric inversion of chromosome 1 Yuqin Luo1,2, Chenming Xu1,2, Yixi Sun1,2, Liya Wang1,2, Songchang Chen1,2, and Fan Jin2,3* 1
Department of Reproductive Genetics, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China, 2Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of Education, Hangzhou, P.R. China, and 3Centre of Reproductive Medicine, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China Abstract
Keywords
Pericentric inversion can produce recombinant gametes; however, meiotic segregation studies on the relationship between the frequency of recombinants and the inverted segment size are rare. Triple-color fluorescence in situ hybridization (FISH) was performed to analyze the meiotic behavior in five inv(1) carriers with different breakpoints. Recombination gametes were absent in Patient 1, whereas the percentages of the recombinants in Patients 2, 3, 4, and 5 were of 9.2%, 15.3%, 17.3%, and 40.9%, respectively. A significant difference was present for the frequencies of the recombinant spermatozoa among the five patients (p50.001). For each patient, the frequency of the two types of recombinant gametes (dup(1p)/del(1q) or del(1p)/ dup(1q)) did not exhibit a significant difference in comparison with the expected 1:1 ratio (p40.05). The meiotic segregation of nine inv(1) carriers (including those presented in this paper) is now available. A significant correlation was discovered between the rate of recombination and the proportion of the chromosome implicated in the inversion (R ¼ 0.9435, p50.001). The frequency of the recombinant gametes was directly related to the proportion of the chromosome that was inverted. Sperm-FISH allowed an additional comprehension of the patterns of meiotic segregation and provided accurate genetic counseling.
Chromosome 1, infertility, meiotic segregation, pericentric inversion, sperm FISH
Introduction Pericentric inversion is a structural chromosomal rearrangement, which is the result of two breaks on both sides of the centromere within a single chromosome and is followed by a 180 rotation and reintegration of the inverted segment in the same chromosome. In the general population, the frequency of pericentric inversions is estimated to be variable from 0.089 % [Ravel et al. 2006] to 0.34% [Nielsen and Wohlert 1991], or even at 1–2% [Kaiser 1984]. Most pericentric inversions affect the pericentric region of chromosome 2 and the heterochromatic region of chromosomes 1, 9, 16, or Y, which are considered as non-pathological polymorphisms. Generally, carriers of pericentric inversions do not show phenotypic alteration but they might have reproductive risks, such as the production of chromosomally imbalanced gametes and/or spermatogenetic failure caused by chromosome reorganization [Anton et al. 2005; Ferfouri et al. 2009]. During meiosis, the complete pairing of an inverted and its normal homologous chromosome requires the formation of an inversion loop, which may produce two abnormal gametes with both duplicated and deleted chromosome segments for *Address correspondence to Fan Jin, MD, Centre of Reproductive Medicine, Women’s Hospital, School of Medicine, Zhejiang University, 1 Xueshi Road, Hangzhou, Zhejiang, 310006, China. Tel: 86571-87013891. E-mail: [email protected]
History Received 25 March 2014 Revised 9 June 2014 Accepted 11 June 2014 Published online 6 August 2014
regions distal to the inversion (duplication p/deletion q (dup p/del q) or deletion p/duplication q (del p/dup q)). To date, few studies have analyzed the segregation products on the sperm nuclei using fluorescence in situ hybridization (FISH) in the case of pericentric inversion [Anton et al. 2002; Anton et al. 2006; Bernicot et al. 2010; Caer et al. 2008; Chantot-Bastaraud et al. 2007; Malan et al. 2006; Mikhaail-Philips et al. 2004; Mikhaail-Philips et al. 2005]. The frequency of the two abnormal gametes ((dup p/ del q and del p/dup q) was highly variable from 0% to 37.85%. Such variability indicated that the inversion couples had a divergent effect on the reproductive fitness. To determine the meiotic segregation behavior in spermatozoa in pericentric inversion carriers, we used sperm-FISH in five cases of a pericentric inversion of chromosome 1 with different breakpoints: inv(1)(p11;q12), inv(1) (p32q21), inv(1)(p36.3q21), inv(1)(p32q32), and inv(1) (p36.2q42). As far as we know, this is the first report which analyzes the segregation pattern in spermatozoa from five carriers of a pericentric inversion involving the same chromosome with different meiotic segregation. There has yet to be a statistical study that analyzes the correlation between the frequency of the recombinants and the inverted segment size for the same chromosome. This primarily reflects the absence of the carrier of a pericentric inversion within the same chromosome with different breakpoints.
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Results Five inv(1) patients between 32 and 41 years of age were consulted for primary infertility (n ¼ 4) or repeated firsttrimester spontaneous abortions (n ¼ 1). The same sperm sample was analyzed for sperm parameters and FISH. According to the criteria of the World Health Organization [WHO 1999], the inv (1) carriers revealed abnormal sperm parameters with oligoasthenospermia or teratozoospermia. Seminal parameters are shown in Table 1. FISH was performed on metaphase spreads obtained from peripheral blood lymphocytes revealing a normal localization of the signals (Figure 1A) except an inv(1) (p36.3q21) carrier (Figure 2). The size of the inverted segment represented 8.54%, 36.9%, 59.9%, 62.3%, and 91.5% of the whole chromosome 1. The 1p subtelomere probe (Spectrum Green) was shown near the centromere of the inv(1) (p36.3q21) chromosome (Figure 1B). In the segregation analysis, 1438 spermatozoa from Patient 1, 1630 spermatozoa from Patient 2, 654 spermatozoa from
Patient 3, 867 spermatozoa from Patient 4, and 1858 spermatozoa from Patient 5 were analyzed. The frequencies of the non-recombinant products were 98.7%, 89.2%, 82.9%, 80.7%, and 56.6% for Patients 1, 2, 3, 4, and 5, respectively. Recombination gametes were absent in Patient 1, whereas Patients 2, 3, 4, and 5 had recombination gamete percentages of 9.2%, 15.3%, 17.3%, and 40.9%, respectively. A significant difference was presented for the frequencies of the recombinant spermatozoa among the five patients (p50.001). The remaining ratio of spermatozoa was 1.3%, 1.6%, 1.8%, 2.0%, and 2.5%, which had other signal combinations that probably resulted from incomplete hybridization or were classical recombinants with incomplete hybridization. The frequency of the dup(1p)/del(1q) recombinant sperm was 0% (Patient 1), 4.4% (Patient 2), 6.8% (Patient 3), 9.4% (Patient 4), and 19.7% (Patient 5), while the frequency of the del(1p)/dup(1q) recombinants was 0%, 4.8%, 8.5%, 7.9%, and 21.2% for Patients 1, 2, 3, 4, and 5, respectively. For each patient, the frequency of the two types of recombinant
Table 1. Patients, cytogenetic analysis, and sperm parameters. Patient 1 2 3 4 5
Karyotype
Ascertainment
Age (years)
Sperm concentration (106/ml)
Motility (%)
Abnormal forms (%)
inv(1)(p11q12) inv(1)(p32q21) inv(1)(p36.3q21) inv(1)(p32q32) inv(1)(p36.2q42)
infertility infertility infertility infertility repeated abortion
34 34 35 32 41
10 1.8 101 2 103 3 103 40
25 15 1 5 51
70 52 96 99 85
Figure 1. FISH analysis of five inversion carriers. Hybridization of the green chromosome 1p subtelomeric probe, red chromosome 1q subtelomeric probe, and aqua chromosome 18 centromeric probe are shown. (A) Hybridization of lymphocyte metaphase from carrier with inv(1)(p32q21). (B) Hybridization of lymphocyte metaphase from carrier with inv(1)(p36.3q21). (C) Sperm with normal or inv(1) chromosome (aqua, green, and red). (D) One dup(1q)/del(1p) sperm (two red and aqua). (E) Two dup(1p)/del(1q) sperm (two green and aqua).
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Figure 2. Idiograms and GTG banding. The idiograms for the normal chromosome 1 and five inversion carriers are shown. Breakpoints are indicated.
gametes (dup(1p)/del(1q) or del(1p)/dup(1q)) did not exhibit a statistically significant difference in comparison with the expected 1:1 ratio (p40.05).
Discussion In this study, sperm-FISH was used to analyze the production of recombinant gametes of five carriers due to a pericentric inversion of chromosome 1. The frequencies of the recombinant products were significantly different among the five male patients. No significant difference was discovered between the frequencies of the two types of recombinant gametes and the expected 1:1 ratio, therefore, genotypic selection was not apparent. The inverted segment size corresponds to only 8.54% of chromosome 1 for the inv(1)(p11q12) patient, and no recombinant spermatozoa were discovered. The heterochromatic region of chromosome 1 was always considered a nonpathological polymorphism, and this inversion is not linked to an increased risk of abnormal progeny in comparison with the general population. This group of patients would not benefit from preimplantation genetic diagnosis or amniocentesis and chorion villus sampling. For the inv(1)(p32q21) carrier, the inverted segment is approximately one-third of the chromosome length, and both breakpoints are in the G light bands. The rate of spermatozoa exhibiting a chromosomal imbalance was 9.2%. In comparison, Jaarola et al. [1998] discovered that few recombinant spermatozoa appeared as inv(1)(p31q12) carriers, and the inversion size was approximately 30% of the total chromosomal length. The breakpoint is present at the 1q12 of the heterochromatic region where recombination is restrained, which may explain this low frequency of recombinant spermatozoa. For Patients 3 and 4, the inversion corresponded to more than half of the total chromosome length (59.9 and 62.3%,
respectively), and the imbalanced spermatozoa were 15.3% (Patient 3) and 17.3% (Patient 4). In addition, two similar cases of pericentric inversion of chromosome 1 have been reported. In one study, Yakut et al. [2003] discovered that the rate of the recombinant gametes was 16% when the inversion size was 47.97% of the total chromosomal length. In another study, Chantot-Bastaraud et al. [2007] reported that the frequency of the recombinant products was 14.8% while the size of the inverted segment represented 52.2% in an inv(1)(p22q42) carrier. A high level of recombinant product (40.9%) was discovered for the inv(1) (p36.2q42) carrier, and the inverted segment size was 91.5% of the total length of chromosome 1. A similar result was observed in a inv(1) (p36.3q43) carrier with a large chromosome inversion. The inverted segment size was 95%, and the percentage of imbalanced gametes was 30.43% [Morel et al. 2007]. Thus, the risk of recombination aneusomy in the offspring was high, consistent with two early miscarriages. The high risk of miscarriage (13/38) was observed in the same inversion inv(1)(p36.2q42) in five generational pedigrees [Honeywell et al. 2012]. A disequilibrium recombinant can lead to repeated spontaneous abortions in some conceptuses because of an increase or decrease in the size of the distal segment. In this study, the range of the recombinant spermatozoa varied from 0 to 40.9% causing the variable reproductive risks for inv (1). The rate of imbalanced recombinants seemed to be influenced by many factors, such as the chromosome involved, the region affected, the position of the breakpoints, or the size of the inverted segment [Anton et al. 2005; Morel et al. 2007]. The frequency of the recombinant gametes was plotted according to the relative length of the inverted segment of nine male carriers of a pericentric inversion of -chromosome 1. The meiotic segregation of nine inv(1) carriers (including those presented in the paper) is now available and summarized in Table 2. A significant
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Table 2. Frequencies of non-recombinant and recombinant sperm in nine men heterozygous for a pericentric inversion of chromosome 1 by spermFISH analysis.
Patient 1 2 3 4 5 [Jaarola et al. 1998] [Yakut et al. 2003] [Chantot-Bastaraud et al. 2007] [Morel et al. 2007]
Inversion inv(1) inv(1) inv(1) inv(1) inv(1) inv(1) inv(1) inv(1)
Inverted segment (%)
Total Spermatozoa
Nonrecombinants (%)
Recombinants (%)
dup(1p)/ del(1q)
del(1p)/ dup(1q)
Others
8.54 36.9 59.9 62.3 91.5 30.49 47.97 52.2
1438 1630 654 867 1858 6006 1636 2133
98.7 89.2 82.9 80.7 56.6 99.33 82.5 80.1
0 9.2 15.3 17.3 40.9 0.38 16.0 14.8
0 4.4 6.8 9.4 19.7 0.25 8.7 7.6
0 4.8 8.5 7.9 21.2 0.13 7.3 7.2
1.3 1.6 1.8 2.0 2.5 0.28 1.5 5.1
69
68.12
30.43
11.59
18.84
1.45
(p11q12) (p32q21) (p36.3q21) (p32q32) (p36.2q42) (p31q12) (p36q32) (p22q42)
inv(1) (p36.3q43)
95
Figure 3. Relative size of inversions observed. The distribution of the percentages of the recombinant gametes as a function of the relative size of the inversion is shown. Individual data points are shown as well as the regression line (R ¼ 0.9435).
correlation (R ¼ 0.9435, p50.001) was discovered between the rate of recombination and the proportion of the chromosome implicated in the inversion (Figure 3). To our knowledge, this is the first report to analyze the relation between the frequency of the recombinants and the inverted segment size in the case of pericentric inversion for the same chromosome. The frequency of the recombinants was strongly associated with the inverted segment size, but not with the affected chromosome. This study sheds new light on the role of the proportion of the inverted segment on the final outcome of recombinant gametes. The risk of unbalanced gametes must be considered with care as a function of the increase of the inverted segment proportion. If the inverted segment was nearly the total chromosome size, approximately 50% of recombinant chromosomes could be produced. Sperm-FISH provided a personalized risk assessment of unbalanced spermatozoa analyzing the patterns of meiotic segregation. The inv (1) cases appeared to be at a special risk because four of the five carriers were infertile. Chromosome
1 harbors a critical chromosomal domain, the integrity of which was essential for normal spermatogenesis [Bache et al. 2004]. Spermatogenic failure associated with rearrangements on chromosome 1 can be correlated with spermatogenic failure 2 (SPGF2;OMIM 108420). The detection of particular breakpoint sites can be correlated with a degree of spermatogenic failure in male carriers. The inv(1) carrier displayed synapsis impairment for all unsynapsed bivalents exhibiting markers of transcriptional silencing, which may cause impaired spermatogenesis [Kirkpatrick et al. 2012]. A meiotic failure could be due to a reduction in recombination events with an absence of a full loop formation and the rare occurrence of a partial loop leading [Chandley et al. 1987]. In conclusion, the frequency of the recombinant gametes was directly related to the proportion of the chromosome inverted. Sperm-FISH provided additional understanding of the patterns of meiotic segregation which should aid accurate genetic counseling. The inv(1) carriers appeared to be at high risk for male infertility.
DOI: 10.3109/19396368.2014.948580
Materials and Methods Patients Five inv(1) patients (Figure 2) between 32 and 41 years old were consulted for primary infertility (n ¼ 4) or for two firsttrimester spontaneous abortions (n ¼ 1). The size of the inverted segment represented 8.54%, 36.9%, 59.9%, 62.3%, and 91.5% of the whole chromosome 1. Each patient provided their informed consent for the genetic investigation and sperm chromosomal evaluation as approved by our institutional ethics committee. None of the five patients had any positive familial history for congenital malformations or mental retardation. No AZF microdeletions were found using six sequence targeted sites (sY254, sY86, sY127, sY84, sY134, and sY255) in four inv(1) infertility carriers [Simoni et al. 2004]. Sperm parameters The same sperm sample was analyzed for sperm parameters and FISH. According to the criteria of the WHO [1999], the inv (1) carriers revealed abnormal sperm parameters with oligoasthenospermia or teratozoospermia. Seminal parameters are shown in Table 1. Sperm FISH analysis The sperm samples of the five carriers were analyzed by triple-FISH with the 1p subtelomeric probe (Tel1p, CEB108/ T7, Spectrum Green, Vysis, Downers Grove, IL, USA), the 1q subtelomeric probe (Tel1q, VIJ2YRM2123, Spectrum Orange, Vysis), and the chromosome 18 centromeric probe (CEP18, Spectrum Aqua, Vysis). The triple-FISH was performed on the blood cell metaphases in the five inv (1) carriers in order to avoid chromosome 1 translocation. The sperm sample was washed three times with phosphatebuffered saline (PBS) and was fixed in fresh fixative (3:1 methanol: acetic acid). The spermatozoa were then spread onto a clean glass slide and air-dried. The sperm heads were decondensed by incubation in 1 M NaOH for 2 min at room temperature. Probe hybridization and detection were performed in accordance with the manufacturer’s instructions (Vysis). The slides were examined using a Zeiss Imager. An A2 microscope (Zeiss, France) equipped with a FISH Imaging System (Isis, MetaSystems, Germany) and filter sets for DAPI, FITC, RBITC, Texas Red, and Aqua were used. Chromosome 1 segregation pattern In the segregation outcome analysis, the sperm heads containing one blue, one green, and one orange signal represented normal or inverted chromosome 1, while those containing one blue signal and two green signals were considered as a recombinant dup(1p)/del(1q) chromosome, and those with one blue signal and two orange signals were characterized as recombinant del(1p)/dup(1q) chromosome. Statistical analysis Data were statistically analyzed with the chi-square test to compare the frequencies of the recombinant products of both types and the non-recombinant spermatozoa for each patient.
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Pearson’s correlation coefficient was used to analyze the relation between the inversion size and the frequency of the recombinants.
Declaration of interest This work was supported by the National Basic Research Program of China (No. 2012CB944901), the Scientific Research Foundation of Zhejiang Medical and Health System (2010KYB065), and Grant of Science and Technology Plan of Zhejiang Province (2010C33096).The authors have no conflicts of interest to declare.
Author contributions Conceived and designed the experiments: Y-QL, FJ; Performed the experiments: Y-QL, C-MX; Analyzed the data: Y-XS, L-YW; Wrote the manuscript: Y-QL; Revised the manuscript: L-YW; S-CC; All authors approved the revisions to the manuscript.
References Anton, E., Blanco, J., Egozcue, J., and Vidal, F. (2002) Risk assessment and segregation analysis in a pericentric inversion inv6p23q25 carrier using FISH on decondensed sperm nuclei. Cytogenet Genome Res 97: 149–54. Anton, E., Blanco, J., Egozcue, J., and Vidal, F. (2005) Sperm studies in heterozygote inversion carriers: a review. Cytogenet Genome Res 111: 297–304. Anton, E., Vidal, F., Egozcue, J., and Blanco, J. (2006) Genetic reproductive risk in inversion carriers. Fertil Steril 85:661–6. Bache, I., Assche, E.V., Cingoz, S., Bugge, M., Tumer, Z., Hjorth, M., et al. (2004) An excess of chromosome 1 breakpoints in male infertility. Eur J Hum Genet 12:993–1000. Bernicot, I., Dechanet, C., Mace, A., Hedon, B., Hamamah, S., Pellestor, F., et al. (2010) Predictive value of sperm-FISH analysis on the outcome of preimplantation genetic diagnosis (PGD) for a pericentric inversion inv5(p15.3q11.2) carrier. Hum Reprod 25: 1818–23. Caer, E., Perrin, A., Douet-Guilbert, N., Amice, V., De Braekeleer, M., and Morel, F. (2008) Differing mechanisms of meiotic segregation in spermatozoa from three carriers of a pericentric inversion of chromosome 8. Fertil Steril 89:1637–40. Chandley, A.C., McBeath, S., Speed, R.M., Yorston, L., and Hargreave, T.B. (1987) Pericentric inversion in human chromosome 1 and the risk for male sterility. J Med Genet 24:325–34. Chantot-Bastaraud, S., Ravel, C., Berthaut, I., McElreavey, K., Bouchard, P., Mandelbaum, J., et al. (2007) Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46, XY, inv(1)(p22q42), associated with infertility. Mol Hum Reprod 13:55–9. Ferfouri, F., Clement, P., Gomes, D.M., Minz, M., Amar, E., Selva, J., et al. (2009) Is classic pericentric inversion of chromosome 2 inv(2)(p11q13) associated with an increased risk of unbalanced chromosomes? Fertil Steril 92:1491–7. Honeywell, C., Argiropoulos, B., Douglas, S., Blumenthal, A.L., Allanson, J., McGowan-Jordan, J., et al. (2012) Apparent transmission distortion of a pericentric chromosome one inversion in a large multigeneration pedigree. Am J Med Genet A 158A:1262–8. Jaarola, M., Martin, R.H., and Ashley, T. (1998) Direct evidence for suppression of recombination within two pericentric inversions in humans: a new sperm-FISH technique. Am J Hum Genet 63: 218–24. Kaiser, P. (1984) Pericentric inversions. Problems and significance for clinical genetics. Hum Genet 68:1–47. Kirkpatrick, G., Chow, V., and Ma, S. (2012) Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier. Reprod Biomed Online 24:91–100. Malan, V., Pipiras, E., Sifer, C., Kanafani, S., Cedrin-Durnerin, I., Martin-Pont, B., et al. (2006) Chromosome segregation in an infertile
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man carrying a unique pericentric inversion, inv(21)(p12q22.3), analysed using fluorescence in situ hybridization on sperm nuclei: Significance for clinical genetics. A case report. Hum Reprod 21: 2052–6. Mikhaail-Philips, M.M., Ko, E., Chernos, J., Greene, C., Rademaker, A., and Martin, R. H. (2004) Analysis of chromosome segregation in sperm from a chromosome 2 inversion heterozygote and assessment of an interchromosomal effect. Am J Med Genet A 127A: 139–43. Mikhaail-Philips, M.M., McGillivray, B.C., Hamilton, S.J., Ko, E., Chernos, J., Rademaker, A., et al. (2005) Unusual segregation products in sperm from a pericentric inversion 17 heterozygote. Hum Genet 117:357–65. Morel, F., Laudier, B., Guerif, F., Couet, M.L., Royere, D., Roux, C., et al. (2007) Meiotic segregation analysis in spermatozoa of pericentric inversion carriers using fluorescence in-situ hybridization. Hum Reprod 22:136–41.
Syst Biol Reprod Med, 2014; 60(6): 367–372
Nielsen, J., and Wohlert, M. (1991) Chromosome abnormalities found among 34,910 newborn children: Results from a 13-year incidence study in Arhus, Denmark. Hum Genet 87:81–3. Ravel, C., Berthaut, I., Bresson, J.L., and Siffroi, J.P. (2006) Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: Large-scale survey of over 10,000 sperm donor karyotypes. Hum Reprod 21:1484–9. Simoni, M., Bakker, E., and Krausz, C. (2004) EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art 2004. Int J Androl 27:240–9. World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction. Cambridge University Press; Cambridge, United Kingdom. Yakut, T., Acar, H., Egeli, U., and Kimya, Y. (2003) Frequency of recombinant and nonrecombinant products of pericentric inversion of chromosome 1 in sperm nuclei of carrier: By FISH technique. Mol Reprod Dev 66:67–71.
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