Annals of Laboratory Medicine

Korean Society for Laboratory Medicine (대한진단검사의학회)
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Evaluation of a Multiplex PCR Kit Used for Detecting Y Chromosome Microdeletions

  • Park, Mi-Young (Department of Laboratory Medicine, Pusan National University School of Medicine) ;
  • Kang, Hye-Min (Department of Medicine, Pusan National University School of Medicine) ;
  • Hwang, Sang-Hyun (Department of Laboratory Medicine, Pusan National University School of Medicine) ;
  • Park, Hyun-Jun (Department of Urology, Pusan National University School of Medicine) ;
  • Park, Nam-Cheol (Department of Urology, Pusan National University School of Medicine) ;
  • Choi, Kyung-Un (Department of Pathology, Pusan National University School of Medicine) ;
  • Kim, Hyung-Hoi (Department of Laboratory Medicine, Pusan National University School of Medicine) ;
  • Chang, Chul-Hun L. (Department of Laboratory Medicine, Pusan National University School of Medicine) ;
  • Lee, Eun-Yup (Department of Laboratory Medicine, Pusan National University School of Medicine)
  • Received : 2010.02.24
  • Accepted : 2010.04.28
  • Published : 2010.08.31
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Abstract

Background : In addition to Klinefelter's syndrome, microdeletion of Yq is the most common genetic cause of male infertility; 15% of azoospermic or 5-10% of oligozoospermic males have Yq deletions. We evaluated a Yq microdeletion kit (LG Life Sciences, Korea) for identifying microdeletions in the azoospermic factor (AZF) regions of the Yq. Methods : The kit was designed to amplify 3 regions of the AZF gene (AZFa, AZFb, and AZFc) using 15 sequence-tagged sites. We evaluated the preclinical performance of the kit. For clinical validation, 58 patients including 25 idiopathic azoospermic or oligozoospermic patients were examined. Results : We observed clear bands on electrophoresis of DNA, up to a DNA concentration of 3.12 ng/${\mu}L$; the known microdeletion regions of all 6 reference cell-lines (Coriell, USA) were accurately detected and no false positive/negative results showed with normal female (n=11) and fertile male (n=15) specimens. This kit could identify the same microdeletions in the common regions, similar to another commercial kit. Among the 58 male infertile patients, 7 (12.1%) had microdeletions of the Yq. Among the idiopathic azoospermic (n=22) and oligozoospermic (n=3) patients, 3 (12.0%) had microdeletions. Further, 2 of 21 varicocele patients (9.5%), 1 of 4 patients with testicular failure, and 1 patient with a 45,X/46,XY mosaic had microdeletions. Conclusions : The kit was effective for detecting microdeletions of the Yq. We identified microdeletions in 12% of the infertile patients. This Y chromosome microdeletion detection kit is useful for screening Yq microdeletions in infertile patients.

배경 : Klinefelter 증후군과 더불어 남성불임에 있어 Y 염색체 장완의 미세결손은 가장 흔한 유전적 원인으로, 비폐쇄성 무정자증의 약 15%, 정자부족증의 약 5-10%에서 Yq 결손이 발견된다. 저자들은 Y 염색체의 azoospermic factor (AZF) 부위의 미세결손을 검출하는 Yq 미세결손 검출 키트(LG Life Sciences, Korea)의 성능을 평가하고자 하였다. 방법 : 이 키트는 세 개의 AZF 부위(AZFa, AZFb, AZFc)의 15개 sequence-tagged sites를 증폭시켜 결손 유무를 확인한다. 임상전 성능 평가와 함께 원인이 불명확한 무정자증/정자부족증 남성 25명을 포함한 58명을 대상으로 임상평가를 시행하였다. 결과 : 평가 대상 키트는 3.12 ng/${\mu}L$의 DNA 농도까지 명확한 밴드를 보였다. 6개의 표준 세포주(Coriell, USA)의 결손 부위를 정확하게 검출하였고, 여성 11명과 정상 가임력을 지닌 남성 15명의 검체에서는 위양성/위음성 결과를 보이지 않았다. 타키트와의 비교 검사에서 두 키트의 공통된 부위에서 모두 일치하는 결손 결과를 보였다. 남성 불임 환자 58명 중 7명(12.1%)에서 Y 염색체 미세결손이 관찰되었다. 원인이 없는 무정자증(n=22)/정자부족증 환자 (n=3) 중 3명(12.0%)에서, 덩굴정맥류환자 21명 중 2명(9.5%), 고환부전 환자 4명 중 1명 그리고 45,X/46,XY 모자이시즘 1명에서 Y 염색체 미세결손이 발견되었다. 결론 : 이 키트는 Yq 미세결실을 검출하는데 효과적이었다. 불임 남성을 대상으로 시행한 임상 성능평가에서 약 12%의 미세결손을 검출하였다. Y 염색체 미세결손 검출 키트(LG Life Sciences)는 Y 염색체 미세결손의 선별검사로 유용할 것으로 판단되었다.

Keywords

Acknowledgement

Supported by : Pusan National University Hospital

References

  1. Foresta C, Moro E, Ferlin A. Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev 2001;22:226-39. https://doi.org/10.1210/er.22.2.226
  2. Korea Institute for Health and Social Affairs. http://www.kihasa.re.kr (Accessed on April 2010).
  3. Dada R, Gupta NP, Kucheria K. Yq microdeletions--azoospermia factor candidate genes and spermatogenic arrest. J Biomol Tech 2004; 15:176-83.
  4. Tiepolo L and Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119-24. https://doi.org/10.1007/BF00278879
  5. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933-43. https://doi.org/10.1093/hmg/5.7.933
  6. Land JA and Evers JL. Risks and complications in assisted reproduction techniques: Report of an ESHRE consensus meeting. Hum Reprod 2003;18:455-7. https://doi.org/10.1093/humrep/deg081
  7. Mitra A, Dada R, Kumar R, Gupta NP, Kucheria K, Gupta SK. Screening for Y-chromosome microdeletions in infertile Indian males: utility of simplified multiplex PCR. Indian J Med Res 2008;127:124-32.
  8. Simoni M, Bakker E, Krausz C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art 2004. Int J Androl 2004;27:240-9. https://doi.org/10.1111/j.1365-2605.2004.00495.x
  9. Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, et al. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 2001;29:279-86. https://doi.org/10.1038/ng757
  10. Repping S, Skaletsky H, Brown L, van Daalen SK, Korver CM, Pyntikova T, et al. Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 2003;35:247-51. https://doi.org/10.1038/ng1250
  11. Y chromosome microdeletion detection kit. Seoul, Korea: LG Life Sciences, Ltd.; 2009 Sep. Package insert.
  12. Simoni M, Bakker E, Eurlings MC, Matthijs G, Moro E, Muller CR, et al. Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl 1999;22:292-9. https://doi.org/10.1046/j.1365-2605.1999.00193.x
  13. Ferlin A, Arredi B, Foresta C. Genetic causes of male infertility. Reprod Toxicol 2006;22:133-41. https://doi.org/10.1016/j.reprotox.2006.04.016
  14. Cram DS, Osborne E, McLachlan RI. Y chromosome microdeletions: implications for assisted conception. Med J Aust 2006;185:433-4.
  15. Krausz C, Forti G, McElreavey K. The Y chromosome and male fertility and infertility. Int J Androl 2003;26:70-5. https://doi.org/10.1046/j.1365-2605.2003.00402.x
  16. Sadeghi-Nejad H and Farrokhi F. Genetics of azoospermia: current knowledge, clinical implications, and future directions. Part II: Y chromosome microdeletions. Urol J 2007;4:192-206.
  17. Krausz C, Quintana-Murci L, McElreavey K. Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod 2000;15:1431-4. https://doi.org/10.1093/humrep/15.7.1431
  18. Ferlin A, Moro E, Garolla A, Foresta C. Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum Reprod 1999;14:1710-6. https://doi.org/10.1093/humrep/14.7.1710
  19. Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, Schlegel PN. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod 2003;18:1660-5. https://doi.org/10.1093/humrep/deg348
  20. Peterlin B, Kunej T, Sinkovec J, Gligorievska N, Zorn B. Screening for Y chromosome microdeletions in 226 Slovenian subfertile men. Hum Reprod 2002;17:17-24.
  21. Oates RD, Silber S, Brown LG, Page DC. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod 2002;17:2813-24. https://doi.org/10.1093/humrep/17.11.2813
  22. Bhasin S. Approach to the infertile man. J Clin Endocrinol Metab 2007;92:1995-2004. https://doi.org/10.1210/jc.2007-0634
  23. Enciso M, Muriel L, Fernandez JL, Goyanes V, Segrelles E, Marcos M, et al. Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test. J Androl 2006;27:106-11. https://doi.org/10.2164/jandrol.05115
  24. Rao L, Babu A, Kanakavalli M, Padmalatha V, Singh A, Singh PK, et al. Chromosomal abnormalities and y chromosome microdeletions in infertile men with varicocele and idiopathic infertility of South Indian origin. J Androl 2004;25:147-53. https://doi.org/10.1002/j.1939-4640.2004.tb02770.x
  25. Dada R, Kumar R, Shamsi MB, Sidhu T, Mitra A, Singh S, et al. Azoospermia factor deletions in varicocele cases with severe oligozoospermia. Indian J Med Sci 2007;61:505-10. https://doi.org/10.4103/0019-5359.34519