Document Type: Final File

Authors

1 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran.

2 Department of Medical Genetics, school of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Islamic Republic of Iran.

3 Department of Medical Genetics, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran.

Abstract

The aim of this study was to examine the feasibility of using an economic and practical method in order to perform non-invasive prenatal testing of thalassemia as a sing gene disorder.Sixteen (16) pregnant mothers in the 11th week of pregnancy who were referred for prenatal diagnosis of thalassemia were selected. The parents had one of IVSII-1, IVSI-5 or FR codon 8/9 mutations. Enrichment of cffDNA was performed by a modified whole genome amplification. Based on the relative mutation dosage assay, wild and mutant alleles were compared by allele specific and Taqman allele specific real time PCR. The results obtained were compared with the results of invasive CVS. When both paternal and maternal mutations were identical IVSII-1 or FR codon 8/9, all three major thalassemic fetuses were detected by significant minus ∆Cts (Ct M-CtW) but no different ∆Cts was observed in seven cases in which fetuses were normal or carrier. In two cases with identical IVSI-5 parental mutations, the two major thalassemic fetuses could not be detected. In four cases with different paternal and maternal mutations, all three carrier fetuses were detected and in one major fetus, only paternal mutation was detected.This innovative method showed the detection of three of the five major thalassemic fetuses when the parental mutations were identical. Furthermore, paternal mutation inheritance could be determined in carrier or major thalassemic fetuses when the parental mutations were different. Further studies on fetuses in late gestational age may have more successful results.
 

Keywords

Main Subjects

1. Allen S., Young E., Bowns B. Noninvasive prenatal diagnosis for single gene disorders. Curr.Opin.Obstet.Gynecol. 29(2):73-79(2017).

2. Hudecova I. and Chiu R.W. Non-invasive prenatal diagnosis of thalassemias using maternal plasma cell free DNA. Best.Pract.Res.Clin.Obstet.Gynaecol. 39:63-73(2017).

3. Chen J.J., Tan J.A.M.A., Chua K.H., Tan P.C., George E. Non-invasive prenatal diagnosis using fetal DNA in maternal plasma: a preliminary study for identification of paternally-inherited alleles using single nucleotide polymorphisms. BMJ.Open. 5: e007648(2015).

4. Perlado S., Bustamante-Aragones A., Donas M., Lorda-Sánchez I., Plaza J., Rodríguez de Alba M. Fetal Genotyping in Maternal Blood by Digital PCR: Towards NIPD of Monogenic Disorders Independently of Parental Origin. PloS.One. 11(4): e0153258(2016).

5. Lam K.W., Jiang P., Liao G. J., Chan K.C., Leung T.Y., Chiu R.W., Lo Y.M. Noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of maternal plasma: application to beta-thalassemia. Clin.Chem.58(10): 1467-75(2012).

6. Bischoff  F.Two stage enrichment of cell-free fetal dna in maternal plasma. Google .Patents; (2009).

7. Jorgez C.J. and Bischoff F. Improving Enrichment of Circulating Fetal DNA for Genetic Testing: Size Fractionation Followed by Whole Gene Amplification. Fetal.Diagn.Ther.25(3):314-319(2009).

8. Ramezanzadeh M., Salehi M., Farajzadegan Z., Kamali S., Salehi R. Detection of paternally inherited fetal point mutations for beta-thalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment. J.Matern.Fetal.Neonatal.Med.29(16):2645-2649(2016).

9. Debrand E., Lykoudi A., Bradshaw E., Allen S. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity. PloS.One. 10(11):e0142729(2015).

10. De Franco E., Caswell R., Houghton J.A.L., Iotova V., Hattersley A.T., Ellard S. Analysis of cell-free fetal DNA for non-invasive prenatal diagnosis in a family with neonatal diabetes.Diabet.Med. 34(4):582-585(2015).

11. Zafari M., Gill P., Kowsaryan M., Alipour A., Banihashemi A. High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers. J. Matern. Fetal. Neonatal. Med.29(20):3323-3328(2016).

12. Chen M., Lu S., Lai Z.F., Chen C., Luo K., Yuan K., Wang Y.S., Li S.Q., Gao Y., Chen F., Asan, Chen D.J.. Targeted sequencing of maternal plasma for haplotype-based non-invasive prenatal testing of spinal muscular atrophy. Ultrasound. Obstet. Gynecol. 49(6):799-802(2017).

13. Xiong W., Wang D., Gao Y., Wang H., Guan J., Lan L., Yan J., Zong L., Yuan Y., Dong W., Huang S., Wu K., Wang Y. Reproductive management through integration of PGD and MPS-based noninvasive prenatal screening/diagnosis for a family with GJB2-associated hearing impairment.Sci. China. Life. Sci. 58(9):829-838(2015).

 

14. Chitty L.S., Mason S., Barrett A.N., McKay F., Lench N., Daley R., Jenkins L.A. Non-invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: next-generation sequencing allows for a safer, more accurate, and comprehensive approach. Prenat. Diagn. 35(7):656-662(2015).

15. Webb A., Madgett T.E., Miran T., Sillence K., Kaushik N., Kiernan M., Avent N. Non Invasive Prenatal Diagnosis of Aneuploidy: Next Generation Sequencing or Fetal DNA Enrichment? Balkan. J. Med. Genet. 15(Suppl):17-26(2012).

16. Galbiati S., Brisci A., Lalatta F., Seia M., Makrigiorgos G.M., Ferrari M., Cremonesi L. Novel use of Full COLD-PCR protocol for noninvasive prenatal diagnosis of genetic diseases. Clin. chem. 57(1):136-138(2012).

17. Yap E.P. and McGee J.O. Short PCR product yields improved by lower denaturation temperatures. Nucleic. Acids. Res. 19(7):1713(1991).

18. Yang Q., Du Z., Song Y., Gao S., Yu S., Zhu H., Ren M., Zhang G. Size-selective separation and overall-amplification of cell-free fetal DNA fragments using PCR-based enrichment. Sci. Rep. 7:40936(2017).

19. Kotsopoulou L., Tsoploua P., Mavrommatis K., C. Kroupis. Non-invasive prenatal testing (NIPT): limitations on the way to become diagnosis. Diagnosis (Berl). 2(3): 141–158(2015).

20. Wang E., Batey A., Struble C., Musci T., Song K., Oliphant A. Gestational age and maternal weight effects on fetal cell‐free DNA in maternal plasma. Prenat. Diagn. 33(7):662-666(2013).