CASE REPORT
Ring chromosome 21 and monosomy 21 mosaicism in a patient with azoospermia Z. Cetin1, O. Altiok-Clark2, M. Sevuk3 & S. Berker Karauzum1 1 Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkey; 2 Department of Medical Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkey; 3 Department of Urology, Faculty of Medicine, Akdeniz University, Antalya, Turkey
Keywords Azoospermia—male infertility—monosomy 21—ring chromosome 21 Correspondence Prof. Dr S. Berker Karauzum, Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya, Dumlupınar Bulvarı 07058, Turkey. Tel.: +90 242 249 69 70; Fax: +90 242 227 44 95; E-mail: [email protected] Accepted: December 3, 2013 doi: 10.1111/and.12232
Summary In this report, we describe a patient with azoospermia in conjection with de novo ring chromosome 21 and monosomy 21 mosaicism. Inter-phase fluorescence in situ hybridisation (FISH) studies on uncultured peripheral blood and epithelial cells obtained by buccal smear revealed that 25% of the uncultured blood cells and 11% of the epithelial cells were monosomic for chromosome 21. Y chromosome microdeletion analysis ruled out the presence of any genomic deletions in the azoospermic factor a,b,c regions on the long arm of chromosome Y. Additionally, through subtelomeric FISH analysis, it was found that there was no deletion in the subtelomeric region of ring chromosome 21. Our results indicate that ring chromosome 21 is a rare, but recurrent chromosomal abnormality in male factor infertility. Furthermore, in individuals with ring chromosome 21, defective spermatogenesis is not associated with the deletion of any gene or genes located in the subtelomeric region of chromosome 21.
Introduction Infertility is an important health problem which affects approximately 10–15% of couples, and in 30–50% of couples, this infertility is due to male factor infertility (Akin et al., 2011). Hofherr et al. (2011) performed a meta-analysis by reviewing previously published reports in the literature to detect the cumulative frequencies of chromosomal abnormalities and chromosome Y microdeletions in infertile males. According to their results, the 47,XXY karyotype, which is associated with Klinefelter syndrome, and its variants were found to be the most frequent chromosomal abnormalities with a frequency of 4.9%, followed by autosomal chromosome abnormalities (3.5%) and other sex chromosomal abnormalities (1.8%). Furthermore, they estimated that the frequency of Y chromosome microdeletions in infertile males was 3.5% (Hofherr et al., 2011). Amongst the autosomal chromosome abnormalities, ring chromosomes were frequently associated with spermatogenetic failure, probably as a result of gamete instability at meiosis due to the ring chromosome (Rajesh et al., 2011). To the best of our knowledge, there have been only four cases reported in the literature in which male factor infertility was found in association with a ring chromosome that originates from 112
chromosome 21. In none of these prior instances, however, were detailed molecular genetic analyses performed. In this report, we describe the first male patient with azoospermia in conjunction with mosaicism for monosomy 21 and ring chromosome 21 and, on whom, detailed molecular cytogenetics and Y chromosome microdeletion analyses were performed. Case report A 33-year-old man was admitted to our clinic due to male factor infertility. Neither his previous medical nor family history contained any information of relevance to his case. On physical examination, both testes were soft and 11 cc in size. The ductus deferens was palpable and normal in shape bilaterally, and there was no sign of varicocele on either side. An analysis of the patient’s semen revealed azoospermia. The patient’s hormone profile was found as follows: luteinising hormone, 2.930 mIU ml 1 (1.70–8.60 mIU ml 1); follicle stimulating hormone, 5.830 mIU ml 1 (1.50–12.40 mIU ml 1); prolactin, 8.420 ng ml 1 (4.10–18.40 ng ml 1); total testosterone, 4.520 ng ml 1 (2.80–8.00 ng ml 1) and free testosterone, 9.680 pg ml 1 (8.90–42.50 pg mL 1). As is standard procedure with men found to have azoospermia, © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 112–115
Ring chromosome 21 in a patient with azoospermia
Z. Cetin et al.
the patient was referred to the Medical Genetic Department for both chromosomal karyotyping and Y chromosome microdeletion analysis. Short-term phytohemagglutinin (PHA) stimulated peripheral blood lymphocyte cultures of the proband, and his parents were performed according to standard procedures. Karyotyping was performed using the image analyser (Applied Imaging, San Jose, CA, USA). At least 20 metaphase plates were evaluated from each individual using G-banding (GTG) over the 550 band level. The centromeric heterochromatin and the nucleolus organiser regions were analysed by C-banding and Ag/NOR-banding according to standard procedures, respectively. Conventional cytogenetic analysis of the patients’ peripheral blood lymphocytes revealed mosaicism for monosomy 21, ring chromosome 21 and dicentric ring chromosome 21 (Fig. 1a). C-banding and Ag/NOR staining showed that ring chromosome 21 was monocentric and had satellite (Fig 1b,c). Therefore, the breakpoint on the short arm of chromosome 21 was thought to be 21p13. The karyotypes of the parents were found to be normal. To evaluate the positions of the subtelomeric sequences on ring chromosome 21, subtelomeric fluorescence in situ hybridisation (FISH) analyses were performed on metaphase plates for the proband using the ToTelVysion probe kit Mix 4 (Abbott Molecular/Vysis, Des Plaines, IL, USA) according to the manufacturer’s instructions. The VIJyRM2029 locus located in the subtelomeric region on the long arm of chromosome 21 was labelled with both SpectrumOrange and SpectrumGreen dyes. Additionally, the D4S3359 and D4S2930 loci located on the short and long
Fig. 1 (a) Partial karyotype of the patient showing normal chromosome and ring chromosome 21. (b) CBG-banded metaphase plate of the patient with a black arrow indicating ring chromosome 21 having centromeric heterochromatin. (c) NOR-banded metaphase plate of the patient with a black arrow indicating the satellite regions of ring chromosome 21. (d) Representative picture from subtelomeric fluorescence in situ hybridisation (FISH) analysis showing that the subtelomeric region of ring chromosome 21 was not deleted. SpectrumGreen and SpectrumRed labelled FISH probes were specific for the subtelomeric regions of the short and long arms of chromosome 4, respectively. The VIJyRM2029 locus located at the subtelomeric region of chromosome 21 was labelled with both SpectrumOrange and SpectrumGreen dyes.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 112–115
arm of chromosome 4 were labelled with SpectrumGreen and SpectrumOrange dyes, respectively. In each instance, five metaphase plates of the proband were analysed. To determine the exact percentage of the cells with monosomy 21, FISH analysis of uncultured peripheral blood and buccal smear cells was performed using the AML1(21q22)/ ETO(8q21) dual-colour probe (AquariusTMprobes-Cytocellâ, Oxon, UK). The 21q22 and 8q21 loci were painted with Texas Red and fluorescein isothiocyanate fluorescence dyes, respectively. All images were recorded using a Zeiss Axioplan epifluorescence microscope equipped with a CCD camera (Photometrics Sensys, Tucson, AZ, USA) and analysed using MacProbe v4.3 software (Applied Imaging, Santa Clara, CA, USA). Subtelomeric FISH analysis showed that the VIJyRM2029 locus located in the subtelomeric region of chromosome 21 was intact on ring chromosome 21 (Fig. 1d). This result indicates that ring chromosome 21 did not have any subtelomeric deletions. Based on the FISH analysis, the patient’s final karyotype was designated as follows: mos 45,XY,-21[52]/46,XY,dic r(21)[4]/46,XY,r (21)(p13q22.3)[44]. ish r(21)(p13q22.3)(VIJyRM2029+, AML1+). Inter-phase FISH analysis with the AML1 (21q22)/ETO(8q21) dual-colour probe set revealed the presence of 25 cells (25%) with monosomy 21 and 75 cells (75%) with disomy for chromosome 21 in the uncultured peripheral blood sample and the presence of 11 cells (11%) with monosomy 21 and 89 cells (89%) with disomy for chromosome 21 in buccal smear sample. The patient was also evaluated for microdeletions of azoospermic factor (AZF) regions on the long arm of the Y chromosome. Genomic DNA was extracted from
(a)
(b)
(c)
(d)
113
Ring chromosome 21 in a patient with azoospermia
peripheral blood using the AxyprepTM Multiprobe Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA). The ZFX/ZFY and SRY control loci and 11 different short tandem sequence markers in the AZFa, b and c regions (sY84, sY86, sY95, sY255, DFFRY, DBY, sY127, sY117, sY125, sY134, sY254) were analysed by polymerase chain reaction (PCR) using the Genequality AZF-MX-COMPLETE kit according to the manufacturer’s instructions (AB Analitica, Padova, Italy). The PCR products were electrophoresed on 2% agarose gel and visualised under ultraviolet light. Y chromosome microdeletion analysis showed that the patient did not have any genomic deletions in the AZFa, AZFb and AZFc regions on the long arm of the Y chromosome. Discussion Guilherme et al. (2011) have proposed several possible mechanisms for ring formation, including: breaks in both chromosome arms followed by end-to-end reunion, a break in one chromosome arm followed by fusion with the subtelomeric region of the other, a break in one chromosome arm followed by fusion with the opposite telomeric region, complex formation of mechanisms resulted in partial deletion and duplications in chromosome arms, fusion of two subtelomeric regions and telomere–telomere fusion. The latter two mechanisms lead to complete rings, as they do not result in a loss of genomic material in the chromosome arms. Likewise, in our case, subtelomeric FISH analysis showed that the presence of ring chromosome 21 did not incur a loss of any genetic material. In patients with ring chromosomes, sister chromatid exchange events during mitosis usually result in formation of secondary chromosomal abnormalities including dicentric rings, inter-locked rings and other structural rear-
Z. Cetin et al.
rangements. Also, loss of these unstable ring chromosomes can lead to formation of monosomic cell lines (Kosztolanyi, 2009; Sodre et al., 2010). Ring chromosome 21 instability was previously reported in patients with phenotypic abnormalities or phenotypically normal infertile cases (Bertini et al., 2008). Dicentric ring chromosomes and loss of ring chromosome 21 were observed in 4 and 54 cultivated cells in our patient. However, inter-phase FISH analysis showed that 25% and 11% of the uncultured peripheral blood and buccal smear cells were monosomic for chromosome 21. Discrepancy in the frequency of cells with monosomy 21 between cultured and uncultured peripheral blood cells might be resulted from selective proliferation of the cells with monosomy 21 in cultivation conditions. Lower percentage of the cells with monosomy 21 in the buccal smear then the peripheral blood is compatible with the presence of infertility without any phenotypic abnormalities in our case and indicates the importance of the tissue-specific mosaicism. The clinical consequences of the presence of ring chromosome 21 in patients are variable, ranging from epilepsy, intellectual impairment and dysmorphic features to spontaneous abortions in female carriers and azoospermia in male carriers (Bertini et al., 2008; Specchio et al., 2011). At the time of writing, only four prior patients with male factor infertility that is associated with ring chromosome 21 have been reported in the literature (McIlree et al., 1966; Huret et al., 1985; Dallapiccola et al., 1986; Hammoud et al., 2009). As with our case, three of these patients had azoospermia, whereas the patient described by Hammoud et al. (2009) had cryptozoospermia. Y chromosome microdeletion analysis was not performed in any of these four previously reported patients. In this study, we performed Y chromosome microdeletion analysis, the results of which ruled out the
Table 1 Previously reported cases with ring chromosome 21 and defective spermatogenesis
Karyotype by GTG chromosome analysis
Y chromosome deletion analysis
Subtelomeric FISH analysis in peripheral blood
Buccal smear FISH
Age (years)
Spermiogram
References
NP
NP
NP
27
Azoospermia
Huret et al. (1985)
2
mos 45,XY,-21[6]/46,XY dic r(21)[4]/46,XY, r(21)(p11q22.3)[90] 46,XY,r(21)(pll.2;q22.3)
NP
NP
NP
34
Azoospermia
3
46,XY,r(21)
NP
NP
NP
28
Azoospermia
4
mos 45,XY,-21[3]/46,XY, r(21)[95]/46,XY[2] mos 45,XY,-21[52]/46,XY, dic r(21)[4]/46,XY, r(21)(p13q22.3)[44]
NP
DSCR1+, 21q
NR
Cryptozoospermia
No deletion
AML1+, 21q+
mos 45,XY,-21[5]/46,XY, r(21)[88]/46,XY[7] mos 45,XY,-21[11]/46, XY,r(21)[89]/
33
Azoospermia
Dallapiccola et al. (1986) McIlree et al. (1966) Hammoud et al. (2009) Our case
Case 1
5
NP, not performed; NR, not recorded; FISH, fluorescence in situ hybridisation.
114
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 112–115
Z. Cetin et al.
contribution of AZF deletions in the pathogenesis of the patient’s infertility. Furthermore, only Hammoud et al. performed a subtelomeric FISH analysis to determine the presence of a possible deletion at the subtelomeric region of ring chromosome 21, and they found that the subtelomeric region of ring chromosome 21 was deleted during ring chromosome formation. In our case, however, the subtelomeric region of ring chromosome 21 was not deleted, indicating that the azoospermia was not associated with haploinsufficiency of a gene or genes located in the subtelomeric region of chromosome 21. Defective spermatogenesis has been found in association with various structural chromosome aberrations, including Robertsonian and reciprocal translocations, pericentric inversions and ring chromosomes. Different possible mechanisms for defective spermatogenesis have been proposed, including position effect, low chiasma count, impaired synapsis and perturbed inactivation of the XY bivalent (Dallapiccola et al., 1986; Zuccarello et al., 2010). Furthermore, it has been hypothesised that a pachytene checkpoint during meiosis I detects failures in chromosome synapsis and recombination, leading to spermatogenetic arrest. It is thought that in the pachytene cells of infertile male carriers of a chromosomal rearrangement, failures in chromosome synapsis and recombination lead to the activation of a pachytene checkpoint, spermatogenetic arrest and, finally, apoptosis (Roeder & Bailis, 2000; Joly-Helas et al., 2007). These mechanisms could also be responsible for the spermatogonial arrest found in the present patient. As a result, ring chromosome 21 is a rare, but recurrent chromosomal abnormality in male factor infertility, Underlying cellular mechanisms are not explained yet, but defective spermatogenesis in cases with ring chromosome 21 is not associated with a deletion of any gene or genes on chromosome 21 (Table 1). However, possible contribution of currently uncharacterised autosomal genes on male factor infertility cannot be excluded. Anyway, it is very important that phenotypically normal prepubertal males with ring chromosome 21 be followed up with regard to primary male sterility. Acknowledgement This work was supported by the Akdeniz University Scientific Projects Management Unit. References Akin H, Onay H, Turker E, Ozkinay F (2011) Primary male infertility in Izmir/Turkey: a cytogenetic and molecular study of 187 infertile Turkish patients. J Assist Reprod Genet 28:419–423.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 112–115
Ring chromosome 21 in a patient with azoospermia
Bertini V, Valetto A, Uccelli A, Tarantino E, Simi P (2008) Ring chromosome 21 and reproductive pattern: a familial case and review of the literature. Fertil Steril 90:2004. e1–5. Dallapiccola B, De Filippis V, Notarangelo A, Perla G, Zelante L (1986) Ring chromosome 21 in healthy persons: different consequencies in females and in males. Hum Genet 73:218– 220. Guilherme RS, Meloni VF, Kim CA, Pellegrino R, Takeno SS, Spinner NB, Conlin LK, Christofolini DM, Kulikowski LD, Melaragno MI (2011) Mechanisms of ring chromosome formation, ring instability and clinical consequences. BMC Med Genet 12:171. Hammoud I, Gomes DM, Bergere M, Wainer R, Selva J, Vialard F (2009) Sperm chromosome analysis of an infertile patient with a 95% mosaic r(21) karyotype and normal phenotype. Fertil Steril 91:930.e13–15. Hofherr SE, Wiktor AE, Kipp BR, Dawson DB, Van Dyke DL (2011) Clinical diagnostic testing for the cytogenetic and molecular causes of male infertility: the Mayo Clinic experience. J Assist Reprod Genet 28:1091–1098. Huret JL, Leonard C, Kanoui V (1985) Ring chromosome 21 in a phenotypically normal but infertile man. Clin Genet 28:541–545. Joly-Helas G, de La Rochebrochard C, Mousset-Simeon N, Moirot H, Tiercin C, Romana SP, Le Caignec C, Clavier B, Mace B, Rives N (2007) Complex chromosomal rearrangement and intracytoplasmic sperm injection: a case report. Hum Reprod 22:1292–1297. Kosztolanyi G (2009) The genetics and clinical caracteristics of constitutional ring chromosomes. J Assoc Genet Technol 35:44–48. McIlree ME, Price WH, Brown WM, Tulloch WS, Newsam JE, Maclean N (1966) Chromosome studies on testicular cells from 50 subfertile men. Lancet 2:69–71. Rajesh H, Freckmann ML, Chapman M (2011) Azoospermia and paternal autosomal ring chromosomes: case report and literature review. Reprod Biomed Online 23:466–470. Roeder GS, Bailis JM (2000) The pachytene checkpoint. Trends Genet 16:395–403. Sodre CP, Guilherme RS, Meloni VF, Brunoni D, Juliano Y, Andrade JA, Belangero SI, Christofolini DM, Kulikowski LD, Melaragno MI (2010) Ring chromosome instability evaluation in six patients with autosomal rings. Genet Mol Res 26:134–143. Specchio N, Carotenuto A, Trivisano M, Cappelletti S, Digilio C, Capolino R, Di Capua M, Fusco L, Vigevano F (2011) Ring 21 chromosome presenting with epilepsy and intellectual disability: clinical report and review of the literature. Am J Med Genet A 155A:911–914. Zuccarello D, Dallapiccola B, Novelli A, Foresta C (2010) Azoospermia in a man with a constitutional ring 22 chromosome. Eur J Med Genet 53:389–391.
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Ring chromosome 21 and monosomy 21 mosaicism in a patient with azoospermia Z. Cetin1, O. Altiok-Clark2, M. Sevuk3 & S. Berker Karauzum1 1 Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkey; 2 Department of Medical Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkey; 3 Department of Urology, Faculty of Medicine, Akdeniz University, Antalya, Turkey
Keywords Azoospermia—male infertility—monosomy 21—ring chromosome 21 Correspondence Prof. Dr S. Berker Karauzum, Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya, Dumlupınar Bulvarı 07058, Turkey. Tel.: +90 242 249 69 70; Fax: +90 242 227 44 95; E-mail: [email protected] Accepted: December 3, 2013 doi: 10.1111/and.12232
Summary In this report, we describe a patient with azoospermia in conjection with de novo ring chromosome 21 and monosomy 21 mosaicism. Inter-phase fluorescence in situ hybridisation (FISH) studies on uncultured peripheral blood and epithelial cells obtained by buccal smear revealed that 25% of the uncultured blood cells and 11% of the epithelial cells were monosomic for chromosome 21. Y chromosome microdeletion analysis ruled out the presence of any genomic deletions in the azoospermic factor a,b,c regions on the long arm of chromosome Y. Additionally, through subtelomeric FISH analysis, it was found that there was no deletion in the subtelomeric region of ring chromosome 21. Our results indicate that ring chromosome 21 is a rare, but recurrent chromosomal abnormality in male factor infertility. Furthermore, in individuals with ring chromosome 21, defective spermatogenesis is not associated with the deletion of any gene or genes located in the subtelomeric region of chromosome 21.
Introduction Infertility is an important health problem which affects approximately 10–15% of couples, and in 30–50% of couples, this infertility is due to male factor infertility (Akin et al., 2011). Hofherr et al. (2011) performed a meta-analysis by reviewing previously published reports in the literature to detect the cumulative frequencies of chromosomal abnormalities and chromosome Y microdeletions in infertile males. According to their results, the 47,XXY karyotype, which is associated with Klinefelter syndrome, and its variants were found to be the most frequent chromosomal abnormalities with a frequency of 4.9%, followed by autosomal chromosome abnormalities (3.5%) and other sex chromosomal abnormalities (1.8%). Furthermore, they estimated that the frequency of Y chromosome microdeletions in infertile males was 3.5% (Hofherr et al., 2011). Amongst the autosomal chromosome abnormalities, ring chromosomes were frequently associated with spermatogenetic failure, probably as a result of gamete instability at meiosis due to the ring chromosome (Rajesh et al., 2011). To the best of our knowledge, there have been only four cases reported in the literature in which male factor infertility was found in association with a ring chromosome that originates from 112
chromosome 21. In none of these prior instances, however, were detailed molecular genetic analyses performed. In this report, we describe the first male patient with azoospermia in conjunction with mosaicism for monosomy 21 and ring chromosome 21 and, on whom, detailed molecular cytogenetics and Y chromosome microdeletion analyses were performed. Case report A 33-year-old man was admitted to our clinic due to male factor infertility. Neither his previous medical nor family history contained any information of relevance to his case. On physical examination, both testes were soft and 11 cc in size. The ductus deferens was palpable and normal in shape bilaterally, and there was no sign of varicocele on either side. An analysis of the patient’s semen revealed azoospermia. The patient’s hormone profile was found as follows: luteinising hormone, 2.930 mIU ml 1 (1.70–8.60 mIU ml 1); follicle stimulating hormone, 5.830 mIU ml 1 (1.50–12.40 mIU ml 1); prolactin, 8.420 ng ml 1 (4.10–18.40 ng ml 1); total testosterone, 4.520 ng ml 1 (2.80–8.00 ng ml 1) and free testosterone, 9.680 pg ml 1 (8.90–42.50 pg mL 1). As is standard procedure with men found to have azoospermia, © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 112–115
Ring chromosome 21 in a patient with azoospermia
Z. Cetin et al.
the patient was referred to the Medical Genetic Department for both chromosomal karyotyping and Y chromosome microdeletion analysis. Short-term phytohemagglutinin (PHA) stimulated peripheral blood lymphocyte cultures of the proband, and his parents were performed according to standard procedures. Karyotyping was performed using the image analyser (Applied Imaging, San Jose, CA, USA). At least 20 metaphase plates were evaluated from each individual using G-banding (GTG) over the 550 band level. The centromeric heterochromatin and the nucleolus organiser regions were analysed by C-banding and Ag/NOR-banding according to standard procedures, respectively. Conventional cytogenetic analysis of the patients’ peripheral blood lymphocytes revealed mosaicism for monosomy 21, ring chromosome 21 and dicentric ring chromosome 21 (Fig. 1a). C-banding and Ag/NOR staining showed that ring chromosome 21 was monocentric and had satellite (Fig 1b,c). Therefore, the breakpoint on the short arm of chromosome 21 was thought to be 21p13. The karyotypes of the parents were found to be normal. To evaluate the positions of the subtelomeric sequences on ring chromosome 21, subtelomeric fluorescence in situ hybridisation (FISH) analyses were performed on metaphase plates for the proband using the ToTelVysion probe kit Mix 4 (Abbott Molecular/Vysis, Des Plaines, IL, USA) according to the manufacturer’s instructions. The VIJyRM2029 locus located in the subtelomeric region on the long arm of chromosome 21 was labelled with both SpectrumOrange and SpectrumGreen dyes. Additionally, the D4S3359 and D4S2930 loci located on the short and long
Fig. 1 (a) Partial karyotype of the patient showing normal chromosome and ring chromosome 21. (b) CBG-banded metaphase plate of the patient with a black arrow indicating ring chromosome 21 having centromeric heterochromatin. (c) NOR-banded metaphase plate of the patient with a black arrow indicating the satellite regions of ring chromosome 21. (d) Representative picture from subtelomeric fluorescence in situ hybridisation (FISH) analysis showing that the subtelomeric region of ring chromosome 21 was not deleted. SpectrumGreen and SpectrumRed labelled FISH probes were specific for the subtelomeric regions of the short and long arms of chromosome 4, respectively. The VIJyRM2029 locus located at the subtelomeric region of chromosome 21 was labelled with both SpectrumOrange and SpectrumGreen dyes.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 112–115
arm of chromosome 4 were labelled with SpectrumGreen and SpectrumOrange dyes, respectively. In each instance, five metaphase plates of the proband were analysed. To determine the exact percentage of the cells with monosomy 21, FISH analysis of uncultured peripheral blood and buccal smear cells was performed using the AML1(21q22)/ ETO(8q21) dual-colour probe (AquariusTMprobes-Cytocellâ, Oxon, UK). The 21q22 and 8q21 loci were painted with Texas Red and fluorescein isothiocyanate fluorescence dyes, respectively. All images were recorded using a Zeiss Axioplan epifluorescence microscope equipped with a CCD camera (Photometrics Sensys, Tucson, AZ, USA) and analysed using MacProbe v4.3 software (Applied Imaging, Santa Clara, CA, USA). Subtelomeric FISH analysis showed that the VIJyRM2029 locus located in the subtelomeric region of chromosome 21 was intact on ring chromosome 21 (Fig. 1d). This result indicates that ring chromosome 21 did not have any subtelomeric deletions. Based on the FISH analysis, the patient’s final karyotype was designated as follows: mos 45,XY,-21[52]/46,XY,dic r(21)[4]/46,XY,r (21)(p13q22.3)[44]. ish r(21)(p13q22.3)(VIJyRM2029+, AML1+). Inter-phase FISH analysis with the AML1 (21q22)/ETO(8q21) dual-colour probe set revealed the presence of 25 cells (25%) with monosomy 21 and 75 cells (75%) with disomy for chromosome 21 in the uncultured peripheral blood sample and the presence of 11 cells (11%) with monosomy 21 and 89 cells (89%) with disomy for chromosome 21 in buccal smear sample. The patient was also evaluated for microdeletions of azoospermic factor (AZF) regions on the long arm of the Y chromosome. Genomic DNA was extracted from
(a)
(b)
(c)
(d)
113
Ring chromosome 21 in a patient with azoospermia
peripheral blood using the AxyprepTM Multiprobe Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA). The ZFX/ZFY and SRY control loci and 11 different short tandem sequence markers in the AZFa, b and c regions (sY84, sY86, sY95, sY255, DFFRY, DBY, sY127, sY117, sY125, sY134, sY254) were analysed by polymerase chain reaction (PCR) using the Genequality AZF-MX-COMPLETE kit according to the manufacturer’s instructions (AB Analitica, Padova, Italy). The PCR products were electrophoresed on 2% agarose gel and visualised under ultraviolet light. Y chromosome microdeletion analysis showed that the patient did not have any genomic deletions in the AZFa, AZFb and AZFc regions on the long arm of the Y chromosome. Discussion Guilherme et al. (2011) have proposed several possible mechanisms for ring formation, including: breaks in both chromosome arms followed by end-to-end reunion, a break in one chromosome arm followed by fusion with the subtelomeric region of the other, a break in one chromosome arm followed by fusion with the opposite telomeric region, complex formation of mechanisms resulted in partial deletion and duplications in chromosome arms, fusion of two subtelomeric regions and telomere–telomere fusion. The latter two mechanisms lead to complete rings, as they do not result in a loss of genomic material in the chromosome arms. Likewise, in our case, subtelomeric FISH analysis showed that the presence of ring chromosome 21 did not incur a loss of any genetic material. In patients with ring chromosomes, sister chromatid exchange events during mitosis usually result in formation of secondary chromosomal abnormalities including dicentric rings, inter-locked rings and other structural rear-
Z. Cetin et al.
rangements. Also, loss of these unstable ring chromosomes can lead to formation of monosomic cell lines (Kosztolanyi, 2009; Sodre et al., 2010). Ring chromosome 21 instability was previously reported in patients with phenotypic abnormalities or phenotypically normal infertile cases (Bertini et al., 2008). Dicentric ring chromosomes and loss of ring chromosome 21 were observed in 4 and 54 cultivated cells in our patient. However, inter-phase FISH analysis showed that 25% and 11% of the uncultured peripheral blood and buccal smear cells were monosomic for chromosome 21. Discrepancy in the frequency of cells with monosomy 21 between cultured and uncultured peripheral blood cells might be resulted from selective proliferation of the cells with monosomy 21 in cultivation conditions. Lower percentage of the cells with monosomy 21 in the buccal smear then the peripheral blood is compatible with the presence of infertility without any phenotypic abnormalities in our case and indicates the importance of the tissue-specific mosaicism. The clinical consequences of the presence of ring chromosome 21 in patients are variable, ranging from epilepsy, intellectual impairment and dysmorphic features to spontaneous abortions in female carriers and azoospermia in male carriers (Bertini et al., 2008; Specchio et al., 2011). At the time of writing, only four prior patients with male factor infertility that is associated with ring chromosome 21 have been reported in the literature (McIlree et al., 1966; Huret et al., 1985; Dallapiccola et al., 1986; Hammoud et al., 2009). As with our case, three of these patients had azoospermia, whereas the patient described by Hammoud et al. (2009) had cryptozoospermia. Y chromosome microdeletion analysis was not performed in any of these four previously reported patients. In this study, we performed Y chromosome microdeletion analysis, the results of which ruled out the
Table 1 Previously reported cases with ring chromosome 21 and defective spermatogenesis
Karyotype by GTG chromosome analysis
Y chromosome deletion analysis
Subtelomeric FISH analysis in peripheral blood
Buccal smear FISH
Age (years)
Spermiogram
References
NP
NP
NP
27
Azoospermia
Huret et al. (1985)
2
mos 45,XY,-21[6]/46,XY dic r(21)[4]/46,XY, r(21)(p11q22.3)[90] 46,XY,r(21)(pll.2;q22.3)
NP
NP
NP
34
Azoospermia
3
46,XY,r(21)
NP
NP
NP
28
Azoospermia
4
mos 45,XY,-21[3]/46,XY, r(21)[95]/46,XY[2] mos 45,XY,-21[52]/46,XY, dic r(21)[4]/46,XY, r(21)(p13q22.3)[44]
NP
DSCR1+, 21q
NR
Cryptozoospermia
No deletion
AML1+, 21q+
mos 45,XY,-21[5]/46,XY, r(21)[88]/46,XY[7] mos 45,XY,-21[11]/46, XY,r(21)[89]/
33
Azoospermia
Dallapiccola et al. (1986) McIlree et al. (1966) Hammoud et al. (2009) Our case
Case 1
5
NP, not performed; NR, not recorded; FISH, fluorescence in situ hybridisation.
114
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contribution of AZF deletions in the pathogenesis of the patient’s infertility. Furthermore, only Hammoud et al. performed a subtelomeric FISH analysis to determine the presence of a possible deletion at the subtelomeric region of ring chromosome 21, and they found that the subtelomeric region of ring chromosome 21 was deleted during ring chromosome formation. In our case, however, the subtelomeric region of ring chromosome 21 was not deleted, indicating that the azoospermia was not associated with haploinsufficiency of a gene or genes located in the subtelomeric region of chromosome 21. Defective spermatogenesis has been found in association with various structural chromosome aberrations, including Robertsonian and reciprocal translocations, pericentric inversions and ring chromosomes. Different possible mechanisms for defective spermatogenesis have been proposed, including position effect, low chiasma count, impaired synapsis and perturbed inactivation of the XY bivalent (Dallapiccola et al., 1986; Zuccarello et al., 2010). Furthermore, it has been hypothesised that a pachytene checkpoint during meiosis I detects failures in chromosome synapsis and recombination, leading to spermatogenetic arrest. It is thought that in the pachytene cells of infertile male carriers of a chromosomal rearrangement, failures in chromosome synapsis and recombination lead to the activation of a pachytene checkpoint, spermatogenetic arrest and, finally, apoptosis (Roeder & Bailis, 2000; Joly-Helas et al., 2007). These mechanisms could also be responsible for the spermatogonial arrest found in the present patient. As a result, ring chromosome 21 is a rare, but recurrent chromosomal abnormality in male factor infertility, Underlying cellular mechanisms are not explained yet, but defective spermatogenesis in cases with ring chromosome 21 is not associated with a deletion of any gene or genes on chromosome 21 (Table 1). However, possible contribution of currently uncharacterised autosomal genes on male factor infertility cannot be excluded. Anyway, it is very important that phenotypically normal prepubertal males with ring chromosome 21 be followed up with regard to primary male sterility. Acknowledgement This work was supported by the Akdeniz University Scientific Projects Management Unit. References Akin H, Onay H, Turker E, Ozkinay F (2011) Primary male infertility in Izmir/Turkey: a cytogenetic and molecular study of 187 infertile Turkish patients. J Assist Reprod Genet 28:419–423.
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