Microsatellite Markers within —SEA Breakpoints for Prenatal Diagnosis of HbBarts Hydrops Fetalis Sherry Sze Yee Ho, 1 , Samuel S. Chong, 2,4 Evelyn S.C. Koay, 3,4 Yiong Huak Chan, 5 Ponnusamy Sukumar, 1 Lily-Lily Chiu, 4 Wen Wang, 2 Ashim Roy, 1 Mary Rauff, 1 Lin Lin Su, 1 Arijit Biswas, 1 and Mahesh Choolani 1* Background: We sought to develop a rapid prenatal diagnostic test for simultaneous detection of HbBarts hydrops fetalis and exclusion of maternal contamination. Methods: We developed a multiplex quantitative fluo- rescent PCR (QF-PCR) test that detects the presence/ absence of 2 microsatellite markers (16PTEL05/16PTEL06) located within breakpoints of the Southeast Asia ( —SEA ) deletion. HbBarts hydrops fetalis ( —SEA/—SEA ) is diagnosed by absence of both markers, and maternal contamination of fetal DNA is excluded by absence of noninherited maternal alleles. Fetal and parental DNA samples from 50 families were analyzed in a blinded clinical validation study, and QF-PCR results were compared with their respective molecular genotypes. Results: The multiplex QF-PCR results included correct diagnoses of HbBarts hydrops fetalis in 11 of the fetuses tested, correct verification as unaffected in 20 fetuses, and correct identification as either carriers (/ —SEA ) or unaffected homozygotes in 18. Misidentification as un- affected occurred for 1 carrier. Sensitivity for diagnosis of HbBarts hydrops fetalis was 100% [lower 95% confi- dence interval, 76.2%], and specificity was 100% (lower 95% confidence interval, 92.6%). None of the samples tested showed any traces of noninherited maternal al- leles; thus false-positives because of maternal contami- nation were eliminated. Conclusions: In this QF-PCR method, detection of ma- ternally and paternally inherited fetal alleles allowed diagnosis of the double-deletion syndrome, and the ability to differentiate between these alleles allowed simultaneous exclusion of maternal contamination of the fetal genetic material. This novel strategy using cell-free fetal DNA in maternal plasma could form the basis for noninvasive testing for HbBarts hydrops fetalis. © 2007 American Association for Clinical Chemistry -Thalassemia (OMIM 141800 and 141850), an inherited anemia syndrome, is the most common of the inherited hemoglobin synthesis disorders, which are the most com- mon monogenic diseases (1, 2). -Thalassemia is charac- terized by decreased or complete absence of -globin chain synthesis (3–5 ), caused by deletion of or mutation (nondeletional) in the -globin genes (1, 6). Clinically, 4 variants of the syndrome are recognizable, with increas- ing severity of the disease manifestation depending on how many normal -globin genes are present (3, 2, 1 or none) (7, 8). Retention of 3 normal -globin genes results in a silent carrier state, with minimal complications. Individuals with 2 normal -globin genes develop micro- cytosis (heterozygous -thalassemia). Those with 1 nor- mal -globin gene often have microcytosis and hemolysis (HbH disease). Loss of all 4 -globin genes, as can occur in the common Southeast Asian ( —SEA ) 6 deletion, leads to HbBarts hydrops fetalis (9, 10). Affected fetuses develop severe intrauterine anemia and become hydropic, usually in the 2nd and 3rd trimesters. They die either in utero or soon after birth. Maternal complications such as hyper- tension, preeclampsia, polyhydramnios, and severe post- partum hemorrhage can lead to fatal consequences in late gestation and at delivery (11 ). Of the many mutations that have been described, deletions at the -globin gene locus Departments of 1 Obstetrics & Gynaecology, 2 Paediatrics, and 3 Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singa- pore. 4 Molecular Diagnosis Centre, National University Hospital, Singapore. 5 Biostatistic Unit, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. * Address correspondence to this author at: Department of Obstetrics and Gynaecology, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074. Fax 65-6779-4753; e-mail obgmac@nus.edu.sg. Received June 15, 2006; accepted November 6, 2006. Previously published online at DOI: 10.1373/clinchem.2006.075085 6 Nonstandard abbreviations: —SEA , Southeast Asia; QF-PCR, quantitative fluorescent-PCR; AF, amniotic fluid; VNTR, variable number of tandem repeat; STR, short tandem repeat; PIC: polymorphism information content. Clinical Chemistry 53:2 173–179 (2007) Molecular Diagnostics and Genetics 173