Min Chen1, Jerry K Y Chan2, Sadhana Nadarajah2, Arnold S C Tan1,3, Melinda L H Chan2, Joyce Mathew4, Eugene E L Saw3, Cheryl Lim2, Wendy Wong2, Felicia S H Cheah3, Hai-Yang Law5,6, Peng-Cheang Wong4, Samuel S Chong1,3,7. 1. Yong Loo Lin School of Medicine, Department of Pediatrics, National University of Singapore, Singapore. 2. KKIVF Center, Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore. 3. Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore. 4. Clinic for Human Reproduction, Department of Obstetrics and Gynecology, National University Hospital, Singapore. 5. Department of Paediatric Medicine, KK Women's and Children's Hospital, Singapore. 6. Paediatrics Academic Clinical Program, Duke-NUS Graduate Medical School, Singapore. 7. Department of Laboratory Medicine, National University Hospital, Singapore.
Abstract
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 α(0) -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.
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 α(0) -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.
Authors: N Samara; S Peleg; T Frumkin; V Gold; H Amir; Einat Haikin Herzberger; A Reches; Y Kalma; Dalit Ben Yosef; F Azem; M Malcov Journal: Mol Cytogenet Date: 2022-03-21 Impact factor: 2.009
Authors: Indhu-Shree Rajan-Babu; Mulias Lian; Felicia S H Cheah; Min Chen; Arnold S C Tan; Ethiraj B Prasath; Seong Feei Loh; Samuel S Chong Journal: Expert Rev Mol Med Date: 2017-07-19 Impact factor: 5.600
Authors: Mingjue Zhao; Mulias Lian; Felicia S H Cheah; Arnold S C Tan; Anupriya Agarwal; Samuel S Chong Journal: Front Genet Date: 2019-11-06 Impact factor: 4.599