PB2411 DETECTION OF PATERNALLY INHERITED MUTATION (IVS‐II‐I G → A) OF THALASSEMIA IN MATERNAL PLASMA BY USING DHPLC METHODS

• Published in: HemaSphere 2019-06, Vol.3 (S1), p.1071-n/a
• Main Author: Zafari, M.
• Format: Article
• Language: English
• ISSN: 2572-9241; 2572-9241
• DOI: 10.1097/01.HS9.0000568108.15646.a1

Background: β‐ Thalassemia is the most common inheritable disease in the world. About 3% of populations (150 million persons) are β‐thalassemi carriers’, and they run 25% risk of having Thalassemia major baby in each pregnancy. Prenatal diagnosis (PND) if offered in many country for prevent Thalassemia, and it is a part of national prevention program by invasive methods, such as; chorionic villus sampling (CVS) at 11‐14 weeks gestation or amniocentesis (AC) from 15 weeks gestation. Aims: the purpose of this study was detection of paternally inherited mutation (IVS‐II‐I G → A) of Thalassemia in maternal plasma by using DHPLC methods with its assessments via sensitivity and specificity determinations for prenatal diagnosis of thalassemia major. Methods: Subjects included Iranian pregnant women who at risk of having fetuses with β‐thalassemia major. We focused exclusively on samples in which the father was carrier for IVS II‐I G → A mutation and the mother had been genotyped to carry another β‐globin gene mutation. 10 ml peripheral blood sample were obtained from 50 pregnant women. The CVS sample was obtained Trans abdominal puncture with a 23‐gauge needle under ultrasound guidance. The samples were analyzed by reveres dot blot analysis. We were masked as to the identity the samples with numerical coding system, so, these were examined in a blinded manner. Circulatory DNA was extracted from 200 μl of peripheral blood plasma with QIAamp DNA mini and blood kit (QIAGEN‐USA) according to the manufacture s instructions. The genomic DNA concentration was measured by NanoDrpe system (ND1000, USA) and electrophoresis. They stored at ‐20 c until used. At firs beta‐Globin gene fragments containing IVSII‐1(G > A) mutation was amplified with HRM real time PCR, also by using specific primers. DHPLC was performed on the WAVW fragment analysis system (Transgenomic Inc, USA) to detect the variants of beta globins’ gene mutation of Thalassemia. In order to generate the hetro& homoduplexes, we mixed an equal amount of HRM Real time PCR product of wild type and patients. Results: the sensivity, specifity of this method was 40. 62 and 61.12; also the PPV and NPV was 65% and 36.67% respectively. Summary/Conclusion: the specifity and NPV of DHPLC method is higher than specifity and PPV.