Exploration of methods for noninvasive prenatal diagnosis of beta thalassemia from circulating cell fetal DNA in maternal plasma

Abstract

Beta (and#946;) thalassemia is the most common single gene disorder in India. Prevention through carrier screening and prenatal diagnosis is possible. Traditionally prenatal diagnosis of and#946; thalassemia is achieved by analysis of cells from chorionic villi or amniotic fluid. Apart from the pain and discomfort; these invasive procedures have the risk of miscarriage, and also require special obstetric expertise. Non-invasive prenatal diagnosis (NIPD) based on cell free foetal (cff) DNA in maternal plasma has proved to be a promising approach. Development of NIPD of and#946; thalassemia would extend prenatal diagnosis to smaller towns and villages from where peripheral blood samples can be sent to a central lab. NIPD would provide an early and safe prenatal diagnosis without putting the mother through discomfort and the risk of miscarriage of an invasive procedure. newlineThe key objective of this study is to assess the feasibility of introducing NIPD of and#946; thalassemia for routine clinical diagnosis. Major challenges in this field are- low concentration of cell free foetal (cff) DNA in maternal blood, total amount of cff DNA varies between individuals, cff DNA molecules are outnumbered by maternal cell free DNA, foetus inherits half its genome from the mother. In the present study, two techniques - formaldehyde treatment of blood samples and size fractionation of cell free DNA on agarose gel after electrophoresis (allowing selection of short DNA fragments) were assessed for their ability to enrich cff DNA. Inheritance of paternal and#946; thalassemia mutation by the foetus in couples harbouring different mutations was detected by allele specific PCR (AS PCR) using locked nucleic acid (LNA) modified primers to detect paternal mutation. LNA clamps were used to suppress wild type sequences (of the mother). After informed consent, blood samples were collected from 246 couples attending Sir Ganga Ram Hospital for invasive prenatal diagnosis of and#946; thalassemia. Cell free DNA was extracted from maternal plasma using QIAamp DSP virus kit.