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

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

Prenatal diagnosis (2015-02-03)
Min Chen, Jerry K Y Chan, Sadhana Nadarajah, Arnold S C Tan, Melinda L H Chan, Joyce Mathew, Eugene E L Saw, Cheryl Lim, Wendy Wong, Felicia S H Cheah, Hai-Yang Law, Peng-Cheang Wong, Samuel S Chong
RESUMO

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. 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 α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application. 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. 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.

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Sigma-Aldrich
Formamide, ≥99.5% (GC), BioReagent, for molecular biology
Sigma-Aldrich
Formamide, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
Formamide solution, NMR reference standard, 90% in DMSO-d6 (99.9 atom % D), NMR tube size 10 mm × 8 in.
Supelco
Formamide solution, NMR reference standard, 90% in DMSO-d6 (99.9 atom % D), NMR tube size 5 mm × 8 in.