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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
Author(s) -
Chen Min,
Tan Arnold S. C.,
Cheah Felicia S. H.,
Saw Eugene E. L.,
Chong Samuel S.
Publication year - 2015
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201500146
Subject(s) - loss of heterozygosity , biology , genetics , microsatellite , genetic marker , allele , genetic linkage , microbiology and biotechnology , gene
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.