Single-tube nonaplex microsatellite PCR panel for preimplantation genetic diagnosis of Hb Bart's hydrops fetalis syndrome

Objective To develop a single‐tube multi‐marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non‐deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity. Methods We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha‐globin gene cluster, and optimized a single‐tube assay combining detection of α0‐thalassemia deletions with multi‐marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application. Results Of 42 markers encompassing the α‐globin gene cluster that were identified in silico, 9 were highly polymorphic (0.68 ≤ polymorphism information content ≤ 0.92; 0.66 ≤ Ho ≤ 0.90; 10 ≤ alleles ≤ 35) and optimized to co‐amplify directly from a single cell. A validation analysis of 100 single lymphoblasts yielded 100% amplification success for all markers, and individual marker allele drop‐out (ADO) rates of 0–5%. Clinical application of the assay in PGD for Hb Bart's (2 cases/cycles) resulted in a twin pregnancy and healthy live birth of two baby girls. Conclusions This single‐tube nonaplex microsatellite PCR panel can be applied directly to PGD of most deletional Hb Bart's without the need for deletion‐specific customization, and to linkage‐based PGD of non‐deletional Hb Bart's. © 2015 John Wiley & Sons, Ltd. What's already known about this topic? The preimplantation genetic diagnosis (PGD) for deletional Hb Bart's is possible using gap‐PCR or intra‐deletion markers; whereas linkage analysis by linked markers can be used for PGD of non‐deletional Hb Bart's because of known or unknown mutations. There are currently few highly polymorphic yet closely linked markers at this locus. What does this study add? We report the identification, validation and application of a highly polymorphic nonaplex marker panel for deletional and/or non‐deletional Hb Bart's PGD.