Journal of Genetic Medicine

Korean Society of Medical Genetics and Genomics (대한의학유전학회)
권호 표지

Validation of QF-PCR for Rapid Prenatal Diagnosis of Common Chromosomal Aneuploidies in Korea

  • Han, Sung-Hee (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories) ;
  • Ryu, Jae-Song (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories) ;
  • An, Jeong-Wook (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories) ;
  • Park, Ok-Kyoung (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories) ;
  • Yoon, Hye-Ryoung (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories) ;
  • Yang, Young-Ho (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories) ;
  • Lee, Kyoung-Ryul (Division of Molecular Genetics and Cytogenetics, Department of Laboratory Medicine Seoul Medical Science Institute, Seoul Clinical Laboratories)
  • Received : 2010.05.24
  • Accepted : 2010.06.18
  • Published : 2010.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: Quantitative fluorescent polymerase chain reaction (QF-PCR) allows for the rapid prenatal diagnosis of common aneuploidies. The main advantages of this assay are its low cost, speed, and automation, allowing for large-scale application. However, despite these advantages, it is not a routine method for prenatal aneuploidy screening in Korea. Our objective in the present study was to validate the performance of QF-PCR using short tandem repeat (STR) markers in a Korean population as a means for rapid prenatal diagnosis. Material and Methods: A QF-PCR assay using an Elucigene kit (Gen-Probe, Abingdon, UK), containing 20 STR markers located on chromosomes 13, 18, 21, X and Y, was performed on 847 amniotic fluid (AF) samples for prenatal aneuploidy screening referred for prenatal aneuploidy screening from 2007 to 2009. The results were then compared to those obtained using conventional cytogenetic analysis. To evaluate the informativity of STR markers, the heterozygosity index of each marker was determined in all the samples. Results: Three autosomes (13, 18, and 21) and X and Y chromosome aneuploidies were detected in 19 cases (2.2%, 19/847) after QF-PCR analysis of the 847 AF samples. Their results are identical to those of conventional cytogenetic analysis, with 100% positive predictive value. However, after cytogenetic analysis, 7 cases (0.8%, 7/847) were found to have 5 balanced and 2 unbalanced chromosomal abnormalities that were not detected by QF-PCR. The STR markers had a slightly low heterozygosity index (average: 0.76) compared to those reported in Caucasians (average: 0.80). Submicroscopic duplication of D13S634 marker, which might be a unique finding in Koreans, was detected in 1.4% (12/847) of the samples in the present study. Conclusion: A QF-PCR assay for prenatal aneuploidy screening was validated in our institution and proved to be efficient and reliable. However, we suggest that each laboratory must perform an independent validation test for each STR marker in order to develop interpretation guidelines of the results and must integrate QF-PCR into the routine cytogenetic laboratory workflow.

목 적: QF-PCR법은 흔한 염색체 이수성에 대한 빠른 산전 진단을 가능하게 하는데, 낮은 가격, 빠른 속도, 그리고 자동화가 가능하여 한꺼번에 많은 검체에 대해 적용할 수 있다는 장점들이 있다. 하지만 아직까지 국내에서 QF-PCR법은 산전 염색체 이수성 선별검사로 주로 사용되는 방법이 아니다. 본 연구에서는 한국인에서 빠른 산전 진단을 목적으로 시행하는 짧은 염기서열 반복(short tandem repeats, STR) 표지자를 이용한 QF-PCR법의 수행능을 검증하고자 한다. 대상 및 방법: 2007년에서 2009년까지 산전 염색체 이수성 선별을 목적으로 의뢰된 847개의 양수 검체에 대해 QF-PCR법을 시행하였는데 13번, 18번, 21번, X, Y염색체에 위치한 총 20개의 STR 표지자로 구성된 Elucigene kit (Gen-Probe, Abingdon, UK)를 사용하였다. 총 847개의 양수 검체에 대한QF-PCR 결과는 염색체 검사 결과와 비교하였고, STR 표지자의 정보력을 평가하기 위해서 각 표지자에 대해 이형접합체 지수(heterozygosity index)를 구하였다. 결 과: 총 847개 양수 검체에 대한 QF-PCR 검사 결과 19개(2.2%, 19/847)에서 13, 18, 21번 염색체와 X, Y염색체의 수적 이상이 관찰되었는데 염색체 검사에서도 동일한 결과를 보여100% 양성 예측율을 나타냈다. 하지만 염색체 검사 결과 7개(0.8%, 7/847) 검체에서 5개의 균형전좌와 2개의 불균형 염색체 이상이 관찰되었으나 QF-PCR에서는 진단되지 않았다. STR 표지자의 평균 이형접합체 지수(he-terozygosity index)는 0.76으로 서양인에서 보고된 0.8에 비해 다소 낮았다. 본 연구에서 D13S634표지자의 미세수준의 중복(submicroscopic duplication)이 1.4% (12/847)에서 관찰되었는데 이는 한국인에서 특징적인 소견으로 생각된다. 결 론: 본 기관에서는 산전 염색체 이수성 선별을 위한 QF-PCR법을 검증하였으며 효율적이고 신뢰할 수 있는 방법임이 입증되었다. 하지만 QF-PCR결과를 해석하기 위한 지침을 만들기 위해서 검사실마다 독립적으로 각각의 STR표지자에 대한 검증이 필요하며, 또한 QF-PCR법을 통상적인 염색체 검사 업무흐름에 통합하는 것이 필요하다고 사료된다.

Keywords

References

  1. Kozlowski P, Grund I, Hickmann G, Stressig R, Knippel AJ. Quantitative fluorescent polymerase chain reaction versus cytogenetics: risk-related indication and clinical implication of nondetected chromosomal disorders. Fetal Diagn Ther 2006;21:217-23. https://doi.org/10.1159/000089306
  2. Pertl B, Pieber D, Lercher-Hartlieb A, Orescovic I, Haeusler M, Winter R, et al. Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders. Mol Hum Reprod 1999;5:1176-9. https://doi.org/10.1093/molehr/5.12.1176
  3. Hult n MA, Dhanjal S, Pertle B. Rapid and simple prenatal diagnosis of common chromosome disorders: advantages and disadvantages of the molecular methods FISH and QF-PCR. Reproduction 2003;126: 279-97. https://doi.org/10.1530/rep.0.1260279
  4. Nicolini U, Lalatta F, Natacci F, Curcio C, Bui TH. The introduction of QF-PCR in prenatal diagnosis of fetal aneuploidies: time for reconsideration. Hum Reprod Update 2004;10:541-8. https://doi.org/10.1093/humupd/dmh046
  5. Cirigliano V, Voglino G, Ordonez E, Marongiu A, Paz Canadas M, Ejarque M, et al. Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR, results of 9 years of clinical experience. Prenat Diagn 2009;29:40-9. https://doi.org/10.1002/pd.2192
  6. Lee MH, Kim DJ, Yang JH, Cho YH, Park SY, Ryu HM. A large-scale validation for QF-PCR aneuploidy testing in Korean. Korean J Genet 2008;30:35-8.
  7. Sacchetti L, Calcagno G, Coto I, Tinto N, Vuttariello E, Salvatore F. Efficiency of two different nine-loci short tandem repeat systems for DNA typing purposes. Clin Chem 1999;45:178-83.
  8. Quaife R, Wong LF, Tan SY, Chua WY, Lim SS, Hammersley CJ, et al. QF-PCR-based prenatal detection of aneuploidy in a southeast Asian population. Prenat Diagn 2004;24:407-13. https://doi.org/10.1002/pd.826
  9. Han SH, An JW, Jeong GY, Yoon HR, Lee A, Yang YH, et al. Clinical and Cytogenetic Findings on 31,615 Mid-trimester Amniocenteses. Korean J Lab Med 2008;28:378-85. https://doi.org/10.3343/kjlm.2008.28.5.378
  10. Mansfield ES. Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. Hum Mol Genet 1993;2:43-50. https://doi.org/10.1093/hmg/2.1.43
  11. Cirigliano V, Ejarque M, Canadas MP, Lloveras E, Plaja A, Perez MM, et al. Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies. Mol Hum Reprod 2001;7: 1001-6. https://doi.org/10.1093/molehr/7.10.1001
  12. Mann K, Fox SP, Abbs SJ, Yau SC, Scriven PN, Docherty Z, et al. Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis. Lancet 2001;358:1057-61. https://doi.org/10.1016/S0140-6736(01)06183-9
  13. Tepperberg J, Pettenati MJ, Rao PN, Lese CM, Rita D, Wyandt H, et al. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature. Prenat Diagn 2001;21:293-301. https://doi.org/10.1002/pd.57
  14. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57. https://doi.org/10.1093/nar/gnf056
  15. Hochstenbach R, Meijer J, van de Brug J, Vossebeld-Hoff I, Jansen R, van der Luijt RB, et al. Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification (MLPA). Prenat Diagn 2005;25:1032-9. https://doi.org/10.1002/pd.1247
  16. Stojilkovic-Mikic T, Mann K, Docherty Z, Mackie Ogilvie C. Maternal cell contamination of prenatal samples assessed by QF-PCR genotyping. Prenat Diagn 2005;25:79-83. https://doi.org/10.1002/pd.1089
  17. Schmidt W, Jenderny J, Hecher K, Hackeloer BJ, Kerber S, Kochhan L, et al. Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF-PCR in 662 selected pregnancies at risk. Mol Hum Reprod 2000;6:855-60. https://doi.org/10.1093/molehr/6.9.855
  18. Levett LJ, Liddle S, Meredith R. A large-scale evaluation of amnio-PCR for the rapid prenatal diagnosis of fetal trisomy. Ultrasound Obstet Gynecol 2001;17: 115-8. https://doi.org/10.1046/j.1469-0705.2001.00340.x
  19. Donaghue C, Roberts A, Mann K, Ogilvie CM. Development and targeted application of a rapid QF-PCR test for sex chromosome imbalance. Prenat Diagn 2003; 23:201-10. https://doi.org/10.1002/pd.569
  20. Cirigliano V, Ejarque M, Fuster C, Adinolfi M. X chromosome dosage by quantitative fluorescent PCR and rapid prenatal diagnosis of sex chromosome aneuploidies. Mol Hum Reprod 2002;8:1042-5. https://doi.org/10.1093/molehr/8.11.1042
  21. Cirigliano V, Voglino G, Canadas MP, Marongiu A, Ejarque M, Ordonez E, et al. Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR. Assessment on 18,000 consecutive clinical samples. Mol Hum Reprod 2004;10:839-46. https://doi.org/10.1093/molehr/gah108
  22. Cirigliano V, Voglino G, Adinolfi M. Non-invasive screening and rapid QF-PCR assay can greatly reduce the need for conventional cytogenetic analyses in pre natal diagnosis. Reprod Biomed Online 2005;11:671-3. https://doi.org/10.1016/S1472-6483(10)61682-3
  23. Cirigliano V, Voglino G, Marongiu A, Canadas P, Ordonez E, Lloveras E, et al. Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications. Ann N Y Acad Sci 2006;1075:288-98. https://doi.org/10.1196/annals.1368.039
  24. Evans MI, Henry GP, Miller WA, Bui TH, Snidjers RJ, Wapner RJ, et al. International, collaborative assessment of 146,000 prenatal karyotypes: expected limitations if only chromosome-specific probes and fluorescent in-situ hybridization are used. Hum Reprod 1999;14:1213-6. https://doi.org/10.1093/humrep/14.5.1213
  25. Thein AT, Abdel-Fattah SA, Kyle PM, Soothill PW. An assessment of the use of interphase FISH with chromosome specific probes as an alternative to cytogenetics in prenatal diagnosis. Prenat Diagn 2000;20: 275-80. https://doi.org/10.1002/(SICI)1097-0223(200004)20:4<275::AID-PD799>3.0.CO;2-Z
  26. Mann K, Donaghue C, Fox SP, Docherty Z, Ogilvie CM. Strategies for the rapid prenatal diagnosis of chromosome aneuploidy. Eur J Hum Genet 2004;12: 907-15. https://doi.org/10.1038/sj.ejhg.5201224
  27. Cho EH, Park BY, Kang YS, Lee EH. Validation of QF-PCR in a Korean population. Prenat Diagn 2009; 29:213-6. https://doi.org/10.1002/pd.2190