Direct PCR-based determination of fetal gender from maternal serum

Öz

Aim: Current analyses based on the examination of cell-free fetal DNA in maternal circulation are widely preferred to minimize complications associated with invasive methods, particularly for detecting aneuploidies and genetic disorders with sex-dependent inheritance that could significantly affect the fetal health. One of the primary factors influencing the reliability of these analyses is the availability of analyzable amounts of fetal DNA. During the isolation of cell-free DNA in maternal circulation, it is not always possible to isolate the entirety of the DNA present in the source material. In such cases, an alternative approach is to directly analyze the source material without any isolation process by using a direct PCR method. In our study, the feasibility of using maternal serum directly in PCR analysis for determining fetal sex was tested. Method: PCR reactions specific to the DYS14 and SRY genes on the Y chromosome were designed to determine the fetal sex. Serum samples from 40 pregnant women were included in the study for analysis. In the reactions designed for the Y chromosome, the sex of the fetus was assumed to be male if specific amplification was observed and female if not. In cases where specific amplification was not observed, the presence of DNA was confirmed with PCR specific to GAPDH. Results: When comparing the results obtained from PCR analysis with clinical outcomes, it was determined that the sensitivity, specificity, and positive predictive value (PPV) of determining fetal sex via direct-PCR analysis were 87.5%, 81.8%, and 91.3%, respectively. Conclusion: The obtained results demonstrate that serum samples from pregnant women can be directly utilized in molecular analyses to reveal the genetic status of the fetus.

Anahtar Kelimeler

• Cell-free fetal DNA
• maternal serum
• direct-PCR
• DYS14
• SRY

Maternal serumdan doğrudan-PCR ile fetüsün cinsiyetinin belirlenmesi

Öz

Amaç: Fetüsün sağlık durumunu ciddi ölçüde etkileyebilecek anöploidiler ve cinsiyete bağlı geçiş gösteren genetik hastalıklar için, günümüzde maternal dolaşımdaki fetüse ait serbest DNA’nın incelenmesine dayanan analizler, girişimsel yöntemlere bağlı komplikasyonları en aza indirmek amacıyla yaygın olarak tercih edilmektedir. Bu analizlerin güvenirliğini etkileyen başlıca etken analiz edilebilir miktarda fetüse ait DNA’nın elde edilebilirliğidir. Maternal dolaşımdaki serbest DNA’nın izolasyonu sırasında kaynaktaki miktara göre her zaman bir kayıp yaşanmaktadır. Bu gibi durumlarda alternatif bir yaklaşım da kaynağın herhangi bir izolasyon işlemi yapılmadan kalıp olarak kullanıldığı doğrudan-PCR yöntemi ile analiz edilmesidir. Çalışmamızda PCR analizi ile fetüsün cinsiyetini belirlemede maternal serumun doğrudan kullanılabilirliği test edilmiştir. Yöntem: Fetüsün cinsiyetini belirlemek amacıyla Y kromozomu üzerindeki DYS14 ve SRY genlerine özgü PCR reaksiyonları tasarlanmıştır. Analiz edilmek üzere 40 gebe kadının serum örneği araştırmaya dahil edilmiştir. Serum örneklerinin Y kromozomu için tasarlanan reaksiyonlarında özgün amplifikasyon görülmesi durumunda fetüsün cinsiyeti erkek, görülmemesi durumunda dişi olarak kabul edilmiştir. Özgün amplifikasyonun görülmediği durumlarda DNA’nın varlığı GAPDH’e özgü PCR ile teyit edilmiştir. Bulgular: PCR analizi ile elde edilen sonuçlar ile klinik sonuçlar karşılaştırıldığında, doğrudan-PCR analizi ile fetal cinsiyeti belirleme duyarlılığının %87.5, özgünlüğünün %81.8 ve pozitif tahmin değerinin (PPV) %91.3 olduğunu ortaya koymuştur. Sonuç: Elde edilen sonuçlar, fetüsün genetik durumunu ortaya koymak amacıyla, gebe kadınların serum örneklerinin doğrudan moleküler analizlerde kullanılabileceğini göstermektedir.

Anahtar Kelimeler

• Serbest fetal DNA
• maternal serum
• doğrudan-PCR
• DYS14
• SRY

Kaynakça

1. Alfirevic Z, Navaratnam K, Mujezinovic F. Amniocentesis and chorionic villus sampling for prenatal diagnosis. Cochrane Database Syst Rev. Sep 4 2017;9(9):CD003252. doi:10.1002/14651858.CD003252.pub2
2. Evans MI, Wapner RJ. Invasive prenatal diagnostic procedures 2005. Semin Perinatol. Aug 2005;29(4):215-8. doi:10.1053/j.semperi.2005.06.004
3. Nassr AA, Hessami K, D'Alberti E, et al. Obstetrical outcomes following amniocentesis performed after 24 weeks of gestation: A systematic review and meta-analysis. Prenat Diagn. Oct 2023;43(11):1425-1432. doi:10.1002/pd.6435
4. Society for Maternal-Fetal M, Berry SM, Stone J, Norton ME, Johnson D, Berghella V. Fetal blood sampling. Am J Obstet Gynecol. Sep 2013;209(3):170-80. doi:10.1016/j.ajog.2013.07.014
5. Carlson LM, Vora NL. Prenatal Diagnosis: Screening and Diagnostic Tools. Obstet Gynecol Clin North Am. Jun 2017;44(2):245-256. doi:10.1016/j.ogc.2017.02.004
6. Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. Aug 16 1997;350(9076):485-7. doi:10.1016/S0140-6736(97)02174-0
7. Minear MA, Alessi S, Allyse M, Michie M, Chandrasekharan S. Noninvasive Prenatal Genetic Testing: Current and Emerging Ethical, Legal, and Social Issues. Annu Rev Genomics Hum Genet. 2015;16:369-98. doi:10.1146/annurev-genom-090314-050000
8. Gupta AK, Holzgreve W, Huppertz B, Malek A, Schneider H, Hahn S. Detection of fetal DNA and RNA in placenta-derived syncytiotrophoblast microparticles generated in vitro. Clin Chem. Nov 2004;50(11):2187-90. doi:10.1373/clinchem.2004.040196

9. Chan KC, Zhang J, Hui AB, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem. Jan 2004;50(1):88-92. doi:10.1373/clinchem.2003.024893
10. La Verde M, De Falco L, Torella A, et al. Performance of cell-free DNA sequencing-based non-invasive prenatal testing: experience on 36,456 singleton and multiple pregnancies. BMC Med Genomics. Mar 30 2021;14(1):93. doi:10.1186/s12920-021-00941-y
11. Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. Apr 1998;62(4):768-75. doi:10.1086/301800
12. Lo YM, Chan KC, Sun H, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med. Dec 8 2010;2(61):61ra91. doi:10.1126/scitranslmed.3001720
13. Lun FM, Chiu RW, Chan KC, Leung TY, Lau TK, Lo YM. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem. Oct 2008;54(10):1664-72. doi:10.1373/clinchem.2008.111385
14. Vora NL, Johnson KL, Basu S, Catalano PM, Hauguel-De Mouzon S, Bianchi DW. A multifactorial relationship exists between total circulating cell-free DNA levels and maternal BMI. Prenat Diagn. Sep 2012;32(9):912-4. doi:10.1002/pd.3919
15. Zhou Y, Zhu Z, Gao Y, et al. Effects of Maternal and Fetal Characteristics on Cell-Free Fetal DNA Fraction in Maternal Plasma. Reprod Sci. Nov 2015;22(11):1429-35. doi:10.1177/1933719115584445
16. Illanes S, Denbow M, Kailasam C, Finning K, Soothill PW. Early detection of cell-free fetal DNA in maternal plasma. Early Hum Dev. Sep 2007;83(9):563-6. doi:10.1016/j.earlhumdev.2006.11.001
17. Wright CF, Burton H. The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis. Hum Reprod Update. Jan-Feb 2009;15(1):139-51. doi:10.1093/humupd/dmn047
18. Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. Apr 2017;17(4):223-238. doi:10.1038/nrc.2017.7
19. Beranek M, Sirak I, Vosmik M, Petera J, Drastikova M, Palicka V. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer. Acta Medica (Hradec Kralove). 2016;59(2):54-8. doi:10.14712/18059694.2016.54
20. Breitbach S, Tug S, Helmig S, et al. Direct quantification of cell-free, circulating DNA from unpurified plasma. PLoS One. 2014;9(3):e87838. doi:10.1371/journal.pone.0087838
21. Abe K. Direct PCR from serum: application to viral genome detection. Methods Mol Biol. 2003;226:161-6. doi:10.1385/1-59259-384-4:161
22. Bachofen C, Willoughby K, Zadoks R, Burr P, Mellor D, Russell GC. Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus. J Virol Methods. Jun 2013;190(1-2):1-3. doi:10.1016/j.jviromet.2013.03.015
23. Nanayakkara IA, Cao W, White IM. Simplifying Nucleic Acid Amplification from Whole Blood with Direct Polymerase Chain Reaction on Chitosan Microparticles. Anal Chem. Mar 21 2017;89(6):3773-3779. doi:10.1021/acs.analchem.7b00274
24. Templeton JE, Taylor D, Handt O, Skuza P, Linacre A. Direct PCR Improves the Recovery of DNA from Various Substrates. J Forensic Sci. Nov 2015;60(6):1558-62. doi:10.1111/1556-4029.12843
25. Shinohara M, Niba ETE, Wijaya YOS, et al. A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study. Int J Neonatal Screen. Dec 2019;5(4):41. doi:10.3390/ijns5040041
26. Wagner FF, Flegel WA, Bittner R, Doscher A. Molecular typing for blood group antigens within 40 min by direct polymerase chain reaction from plasma or serum. Br J Haematol. Mar 2017;176(5):814-821. doi:10.1111/bjh.14469
27. Bianchi DW. Pregnancy: Prepare for unexpected prenatal test results. Nature. Jun 4 2015;522(7554):29-30. doi:10.1038/522029a
28. Elgun T, Musteri Oltulu Y, Gok Yurttas A, Agyuz U, Ozkal Molla F, Kilic U. Determination of Rh type and gender using circulating cell-free fetal DNA in early pregnancy of Rh negative women in turkey. Transfus Clin Biol. Aug 2023;30(3):324-328. doi:10.1016/j.tracli.2023.04.004
29. Aykut A, Onay H, Sagol S, Gunduz C, Ozkinay F, Cogulu O. Determination of fetal rhesus d status by maternal plasma DNA analysis. Balkan J Med Genet. Dec 2013;16(2):33-8. doi:10.2478/bjmg-2013-0029
30. Zeybek YG, Gunel T, Benian A, Aydinli K, Kaleli S. Clinical evaluations of cell-free fetal DNA quantities in pre-eclamptic pregnancies. J Obstet Gynaecol Res. Mar 2013;39(3):632-40. doi:10.1111/j.1447-0756.2012.02011.x
31. Yenilmez ED, Tuli A, Evruke IC. Noninvasive prenatal diagnosis experience in the Cukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and beta-thalassemia in cell-free fetal DNA using high-resolution melting analysis. Prenat Diagn. Nov 2013;33(11):1054-62. doi:10.1002/pd.4196
32. Adiyaman D, Kuyucu M, Atakul BK, et al. Can the Cell-free DNA Test Predict Placenta Accreta Spectrum or Placenta Previa Totalis? Z Geburtshilfe Neonatol. Apr 2022;226(2):92-97. doi:10.1055/a-1579-1338
33. Zolotukhina TV, Shilova NV, Voskoboeva EY. Analysis of cell-free fetal DNA in plasma and serum of pregnant women. J Histochem Cytochem. Mar 2005;53(3):297-9. doi:10.1369/jhc.4B6398.2005
34. Aghanoori MR, Vafaei H, Kavoshi H, Mohamadi S, Goodarzi HR. Sex determination using free fetal DNA at early gestational ages: a comparison between a modified mini-STR genotyping method and real-time PCR. Am J Obstet Gynecol. Sep 2012;207(3):202 e1-8. doi:10.1016/j.ajog.2012.06.026
35. Dar P, Jacobsson B, MacPherson C, et al. Cell-free DNA screening for trisomies 21, 18, and 13 in pregnancies at low and high risk for aneuploidy with genetic confirmation. Am J Obstet Gynecol. Aug 2022;227(2):259 e1-259 e14. doi:10.1016/j.ajog.2022.01.019
36. Lu X, Wang C, Sun Y, Tang J, Tong K, Zhu J. Noninvasive prenatal testing for assessing foetal sex chromosome aneuploidy: a retrospective study of 45,773 cases. Mol Cytogenet. Jan 6 2021;14(1):1. doi:10.1186/s13039-020-00521-2
37. Wright D, Wright A, Nicolaides KH. A unified approach to risk assessment for fetal aneuploidies. Ultrasound Obstet Gynecol. Jan 2015;45(1):48-54. doi:10.1002/uog.14694
38. Canick JA, Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn. Jul 2013;33(7):667-74. doi:10.1002/pd.4126
39. Chiu RW, Lo YM. Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age. Semin Fetal Neonatal Med. Apr 2011;16(2):88-93. doi:10.1016/j.siny.2010.10.003
40. Nishimori A, Konnai S, Ikebuchi R, et al. Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection. J Vet Med Sci. Jun 1 2016;78(5):791-6. doi:10.1292/jvms.15-0577
41. Imai H, Watanabe Y, Shimada D, et al. Utility of a Cell-Direct Polymerase Chain Reaction-Based Nucleic Acid Lateral Flow Immunoassay for Detection of Bacteria in Peripheral Blood Leukocytes of Suspected Sepsis Cases. Infect Drug Resist. 2021;14:5137-5144. doi:10.2147/IDR.S345361
42. Tjhie JH, van Kuppeveld FJ, Roosendaal R, et al. Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J Clin Microbiol. Jan 1994;32(1):11-6. doi:10.1128/jcm.32.1.11-16.1994
43. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. Jan 1999;64(1):218-24. doi:10.1086/302205