Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single-tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta-thalassemia.
Beta (β)-thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β-globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage-based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage-based β-thalassemia PGD or in conjunction with HBB mutation detection.
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Min
M
Chen
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.|Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore.
Arnold S C
AS
Tan
Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore.
Felicia S H
FS
Cheah
Preimplantation Genetic Diagnosis Center, Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore.
Eugene E L
EE
Saw
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.|Department of Laboratory Medicine, National University Hospital, Singapore.
Samuel S
SS
Chong
D005260:Female; D005787:Gene Frequency; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics
D055544:beta-Globins
Beta-thalassemia; Linkage analysis; Microsatellite marker; Multiplex PCR; Preimplantation genetic diagnosis (PGD)