Rapid and reliable preimplantation genetic diagnosis of common hemoglobin <scp>B</scp>art's hydrops fetalis syndrome and hemoglobin <scp>H</scp> disease determinants using an enhanced single‐tube decaplex polymerase chain reaction assay
Authors: Min Chen, Seong Feei Loh, Su Ling Yu, Suresh Nair, Heng Hao Tan, Sadhana Nadarajah, Peng‐Cheang Wong, Soon Chye Ng, Ethiraj B. Prasath, Arnold S.C. Tan, Felicia S.H. Cheah, Eugene E.L. Saw, Samuel S. Chong
Journal: American Journal of Hematology (2015)

To the Editor: Hemoglobin (Hb) Bart's hydrops fetalis syndrome and Hb H disease are the most severe forms of α-thalassemia, with estimated annual affected births of 14,000 globally 1, 2. Within Asia, both the -α3.7 and -α4.2 α+-thalassemia deletions are prevalent, as are the --SEA, --FIL, --THAI, and --SA α0-thalassemia deletions, whereas -α3.7, --MED, and -α(20.5) are common in the Mediterranean region 3. Preimplantation genetic diagnosis (PGD) for Hb Bart's hydrops fetalis syndrome can be achieved by gap polymerase chain reaction (PCR) 4, 5, which requires customized primer sets for different combinations of deletion types, or by PCR of intradeletion microsatellite markers 6. Indirect linkage-based PGD can be offered in cases where one or both members of the couple carry a nondeletional or undetermined mutation 7. We recently described a general strategy for PGD of deletional Hb Bart's hydrops fetalis syndrome through multiplex PCR of intradeletion markers 16PTEL05 and 16PTEL06, supplemented by haplotype analysis of seven flanking microsatellite markers 8. A significant deficiency of the marker panel, however, was its inability to detect the most common Hb H disease genotype (-α3.7/--). We now describe an enhanced single-tube assay that can be used for PGD of all common deletional determinants of Hb Bart's hydrops fetalis syndrome and Hb H disease. We did this by adding an amplicon from the Y1 box region between the HBA2 and HBA1 genes to the nine closely linked microsatellite markers (Fig. 1A). Multiplex PCR amplification was performed essentially as described 8, but with the addition of 0.05 µM of primers Y1-F (5′-GACCTGATGCACTCCTCAAAG-3′) and Y1-R (5′-AAGGATATGTATTAGGTGGAGGAGGT-3′). Single-tube PCR assay for PGD of Hb Bart's hydrops fetalis syndrome and Hb H disease. (A) Schematic illustration of the alpha-globin gene cluster showing relative positions of the nine microsatellite markers, Y1 box, and extents of several common deletions involving -α3.7(Z84721.1:g.34164_37967del3804), -α4.2(Z84721.1:g.30646_34903del4258), --SEA, --FIL, --THAI, --SA, --MED, and -(α)20.5 (modified from Chen et al. 8). (B) Schematic illustration of the expected outcome of PCR amplification of 16PTEL05, Y1, and 16PTEL06 from embryos that have the --SEA/--SEA Hb Bart's genotype, and the -α3.7/--SEA and -α4.2/--SEA Hb H genotypes. A check mark (√) indicates positive amplification product, whereas a cross (×) indicates absence of amplification product. (C) Electropherograms (left) and haplotype phasing (right) for the first Hb H disease PGD case. For each embryo, the electropherogram result for one of the two blastomeres tested is shown. The x-axis indicates marker/fragment size in base pairs. The y-axis indicates product yield in terms of relative fluorescent units (RFUs, ×103). Marker/fragment names are indicated below their corresponding allele peaks. Arrows indicate drop-out of one of the two expected alleles. Dotted oval indicates null amplification of Y1 fragment. Haplotyped markers are ordered from the p-arm telomere (top) to centromere (bottom) in the "pedigree." Numbers indicate amplicon sizes in base pairs. A dash (–) and plus (+) denote absence and presence, respectively, of amplification product. Paternally and maternally derived haplotypes of fetuses are shown on the left and the right, respectively. Dark-shaded haplotypes are linked to the mutation, whereas light-shaded haplotypes are linked to the normal allele. (D) Electropherograms (left) and haplotype phasing (right) for the second Hb H disease PGD case. Asterisk indicates a stutter peak. Dotted oval indicates null amplification of 16PTEL05 marker. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] With this enhanced assay, deletional Hb Bart's hydrops fetalis syndrome will now be characterized by null amplification of 16PTEL05, 16PTEL06, and Y1 products. More importantly, Hb H disease involving the common -α3.7/-- genotype can now be detected, based on null amplification of the Y1 box fragment, whereas Hb H disease involving the less common -α4.2/-- genotype is detected by null amplification of 16PTEL05 (Fig. 1B). The optimized assay was applied to six clinical PGD cases. The first case involved PGD for Hb H disease due to -α3.7/--SEA genotype. Embryo 1 displayed one allele each for 16PTEL05 and 16PTEL06 whereas Y1 was absent, indicative of affected genotype (-α3.7/--SEA) (Fig. 1C). Embryo 2 displayed two alleles each for 16PTEL05 and 16PTEL06, and positive amplification of Y1, indicating either an unaffected (αα/αα) or silent carrier (αα/-α3.7) embryo. Embryo 3 displayed one allele each for 16PTEL05 and 16PTEL06 and was positive for Y1, indicative of α-thal-1 carrier genotype (αα/--SEA). Flanking marker diplotypes were consistent with the mutation genotypes, identifying Embryo 2 to be a silent carrier (αα/-α3.7). Both carrier embryos were transferred, a singleton pregnancy ensued, and a healthy baby girl was delivered. The second PGD case involved Hb H disease due to -α4.2/--SEA genotype. Three embryos were analyzed and diagnosed as affected (-α4.2/--SEA), unaffected (αα/αα), and silent carrier (αα/-α4.2), respectively (Fig. 1D). The unaffected and silent carrier embryos were transferred without pregnancy success. The third PGD case involved a woman affected with -α3.7/--SEA type Hb H disease and her αα/--SEA carrier spouse. One embryo was diagnosed as a silent carrier (αα/-α3.7), three were α-thal-1 carriers (αα/--SEA), one had Hb H disease (-α3.7/--SEA), and three had Hb Bart's hydrops fetalis syndrome (--SEA/--SEA) (data not shown). Two carrier embryos were transferred, resulting in a biochemical pregnancy (elevated maternal human chorionic gonadotropin 2 weeks after embryo transfer), which was subsequently lost. The remaining three PGD cases involved Hb Bart's hydrops fetalis syndrome. Altogether, five embryos were affected (--SEA/--SEA), five were carriers (αα/--SEA), and two were unaffected (αα/αα). Carrier embryos were transferred in two cases, but without pregnancy success. Although allele drop-out, which is the random failure to detect either one of two expected target alleles, was observed at 1–3 marker loci in some samples, the remaining markers in the panel enabled unambiguous haplotype phasing of unaffected and mutant chromosomes to be established in all instances. This enabled unambiguous diagnoses for all embryos in all six clinical PGD cases, underlining the utility of mutation detection plus haplotype analysis in α-thalassemia PGD. This single-tube decaplex PCR assay should be applicable to PGD involving other common α-thalassemia deletional determinants and should also be useful for PGD of nondeletional disease determinants when an index affected family member is available to establish disease haplotype phase. S.S.C. conceptualized the assay design and revised the manuscript. M.C. performed the assay optimization and validation and wrote the manuscript. S.L.Y., Sa.N., S.C.N., H.H.T., Su.N., P.C.W., and S.F.L. co-ordinated patient care, and E.B.P. the IVF/ICSI and embryo transfer procedures, as well as reviewed and approved the manuscript. A.S.C.T., F.S.H.C., and E.E.L.S. performed the clinical PGD testing and revised the manuscript. Min Chen1,2 Seong Feei Loh3 Su Ling Yu4 Suresh Nair5 Heng Hao Tan6Sadhana Nadarajah6 Peng-Cheang Wong7 Soon Chye Ng8 Ethiraj B. Prasath3Arnold S.C. Tan1,9 Felicia S.H. Cheah9 Eugene E.L. Saw9 Samuel S. Chong1,9,10* 1Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore; 3Thomson Fertility Centre, Thomson Medical Centre, Singapore; 4Centre for Assisted Reproduction, Department of Obstetrics & Gynaecology, Singapore General Hospital, Singapore; 5Gynecology Consultants Clinic and Surgery, Singapore; 6KKIVF, Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore; 7Clinic for Human Reproduction, Department of Obstetrics and Gynecology, National University Hospital, Singapore; 8Sincere IVF Center, Sincere Healthcare Group, Singapore; 9Preimplantation Genetic Diagnosis Center, Khoo Teck Puat–National University Children's Medical Institute, National University Health System, Singapore; 10Department of Laboratory Medicine, National University Hospital, Singapore