The first validated criteria for effective screening and a new simplified method for α-globin gene sequencing for diagnosis of uncommon α-globin mutations.
No well-defined phenotypes that distinguish between unknown α- and β-globin mutations have been reported to date. Direct DNA sequencing of α-globin genes can be technically challenging, as α1- and α2-globin genes are nearly indistinguishable. To detect hemoglobin variants (HbXs) on Hb analysis, the entire β- and α-globin genes were directly sequenced using a newly developed sequencing protocol for α-globin genes. An algorithm to distinguish between α- and β-HbXs was constructed and subsequently validated in the independent validation group. Distinctive characteristics that can distinguish 39 α-HbXs from 24 β-HbXs were the presence of unidentifiable variants of HbA2and/or HbX of <37% on isoelectric focusing and <31% on high-performance liquid chromatography. Another set of 67 HbXs was employed to validate our algorithm. This accurately predicted 33 α-HbXs with 100% sensitivity and 97.1% specificity. Our sequencing protocol for α-globin genes was able to identify 11 rare mutations among all exons of both α-globin genes from 72 subjects. Six of these variants were first discovered in Thais. This is the first well-characterized algorithm for distinguishing unknown Hb variants in a large cohort. Our validated criteria and DNA sequencing procedure are highly efficient for molecular characterization of rare Hb mutations.
Division of Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, 10330, Thailand. drnoppacharn@yahoo.com.
Noppacharn
http://orcid.org/0000-0003-4562-9139
N
Uaprasert
Division of Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, 10330, Thailand.
Rung
R
Settapiboon
Division of Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, 10330, Thailand.
Supaporn
S
Amornsiriwat
Division of Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, 10330, Thailand.
Pranee
P
Sutcharitchan
Division of Hematology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, 10330, Thailand.
Ponlapat
P
Rojnuckarin
D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D005260:Female; D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007525:Isoelectric Focusing; D008297:Male; D009154:Mutation; D055542:alpha-Globins / Q000235:genetics*
D055542:alpha-Globins
DNA sequencing; Screening algorithm; Validation; α-Hemoglobin variants