Volume 28, Issue 1 (3-2021)                   RJMS 2021, 28(1): 0-0 | Back to browse issues page

Research code: None
Ethics code: IR.MAZUMS.REC.1393.926
Clinical trials code: IR.MAZUMS.REC.1393.926

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Zafari M, Gill P, Kowsaryan M, saadati rad M T. Non Invasive Prenatal Diagnosis Of β-Thalassemia by detection paternally inherited mutation. RJMS. 2021; 28 (1)
URL: http://rjms.iums.ac.ir/article-1-6327-en.html
Faculty Member of Nursing Department, Faculty of Nursing and Midwifery, Islamic Azad University, Sari, Iran , mandana.zafari1762@gmail.com
Abstract:   (156 Views)
Background and purpose: Prenatal diagnose (PND) in carrier β-Thalassemia couples is very important. PND of Thalassemia, is a part of national prevention program. In all over the world, conventional prenatal diagnosis of Thalassemia is CVS or AC. These methods are invasive, we want to experience the new NIPD for β-Thalassemia with this hypothesis that 50% of invasive PND removed.
 Non invasive prenatal diagnose of Thalassemia is possible via detection of paternally mutation in maternal plasma. The history of fetal DNA (fDNA) in maternal circulation dates back to the 1948 by Mandel and Metais. The discovery of fDNA in maternal plasma and serum has opened up new opportunities in   non invasive prenatal diagnosis (NIPD). This method has been shown to be potentially useful for prenatal diagnosis of certain neurological disorders, fetal sex determination, sex – linked disease, RhD genotyping, and aneuploidies. Fetal DNA originated from trophoblast cells in the placenta, and it detectable very soon during pregnancy, it only comprised about 3 to 6% (although up to 10% has been reported).  Fetal DNA fragments are small in size (< 313 bp). It present during pregnancy until birth. They are rapidly cleared within 2 hours after delivery.
Taq- Man is one of the first methods introduced for Real-time sequence detection. A Taq-Man reaction involves 2 specific primers and probes. Each probe is complimentary to one of the alleles of a mutation and each of them is labeled with a different fluorophore. This method exploits the 5chr(chr('39')39chr('39')) endonucleolytic activity of Taq DNA polymerase to slice an oligonucleotide probe through PCR amplification thus generating a detectable signal.The aim of this study was non invasive prenatal diagnosis of β-Thalassemia and determination the sensitivity and specificity of this technique for detection paternally inherited IVS II-I (GàA) mutation in fetus.
Materials and methods:
Subjects were selected following ethical approval of study protocol from the ethical committee of Mazandaran University of Medical Science, Iran. The sample size of this study calculated according the related studies . Fifty Iranian pregnant women enrolled in this study, they were at risk of having fetuses with β-Thalassemia major. They attended in prenatal diagnosis of Thalassemia center (at 11-14 weeks of pregnancy, before performance of CVS), between May 2014 and Feb 2015, in Sari, Iran. All women assigned informed consent form. We focused exclusively on couples in which the father was carrier for IVS II-I GàA mutation and the mother had been genotyped to carry another β-globin gene mutation, (Of course, the mutation in each subject had been determined previously by conventional PCR and ARMS method, and followed by agarose gel electrophoresis). Also, 10 milliliters of maternal blood from each pregnant woman were collected into EDTA-containing tube. Maternal plasma was separated from cells by high –speed centrifugation (1900 g or 3000 rpm), and it stored at – 80 0 c until analysis. We were masked as to the identity the samples with numerical coding system, so, these were examined in a blinded manner. We designed two specific primers for IVSII-1(GàA) mutation (Accession No.141900) (www.hgvs.org), on the HBB DNA sequences (NCBI Reference Sequence: NG_000007.3 3 (Homo sapiens beta globin region (HBB)), the oligonucleotid primers of this study were; forward primer: 5chr(chr('39')39chr('39'))TCACCTGGACAACCTCAAG3chr(chr('39')39chr('39')) , and reverse primer: 5chr(chr('39')39chr('39'))CCCATTCTAAACTGTACCC3chr(chr('39')39chr('39')). The primers pair was optimized to particularly amplify a 200-bp segment of exon 2 of the human β-globin gene. Of course we consider the locus of maternal mutation in β-globin gene that doesnchr(chr('39')39chr('39'))t interfere to our PCR product. Also, for design the Taq-Man probes, we refer in Taq- Man design site (primer digital.com/fast PCR/m15.html FAST PCR manual). The characterics of probes were; GC content 45-65%, no dimer with primer, high Tm 60-65 oc, probe length 18-30 bp, and Tm of primer was 8-10 oc higher than Tm primer. Also, the kind of quencher and reporter were matched with kind of quencher and reporter in the kit. Probe for wild allele was; FAM-AGAACTTCAGGGTGAGTCTATGGG-BHQ1, and the Probe for mutant allele was; VIC-AGAACTTCAGGATGAGTCTATGGG-BHQ1. Circulatory DNA was extracted from 200 µl of peripheral blood plasma with QIAamp DNA mini and blood kit according to the manufacturerchr(chr('39')39chr('39'))s instructions. DNA was eluted in a final volume of 200 µl   AE buffer and stored at -20 0 c until used for additional analyzing. Also, the genomic DNA concentration was measured by NanoDrope system and electrophoresis. For detection of paternally inherited mutation Taq-Man Real time PCR was used and the result of Taq- man method compared with result of chorionic vilus sampling method (CVS) as a gold standard.
Results: The clinical performance of Taq-Man test was assayed by comparing with routine test. Included in this study were 50 pregnant mothers. In Taq-Man method 38 samples cited in scatter analysis graph as a negative control, because the concentration of DNA was very low and this method didn’t have enough sensitivity and specificity for detection, so, the final analysis for assessment of this method was done on 12 samples. We reported 5 cases true positive, 4 cases true negative, 2 cases false negative and 1 cases false positive. Also the sensitivity of Taq-Man was 71.43% with CI95% (60%-83.43%), and specificity 66.67% with CI 95% (54.67%-78.67%). LR+ and LR- were 2.15 and 0.5 respectively. Also NPV and PPN of Taq-Man test were 66.67%, 83.4% respectively.
Conclusion: non invasive prenatal diagnose can be detect paternally inherited mutation of thalassemia.
Type of Study: Research | Subject: Genetic

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