Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions.
BACKGROUND
alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations.


METHODS
We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2).


RESULTS
Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods.


CONCLUSIONS
The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.
Puehringer
Helene
H
ViennaLab Diagnostics GmbH, Vienna, Austria. puehringer@viennalab.co.at
Najmabadi
Hossein
H
Law
Hai-Yang
HY
Krugluger
Walter
W
Viprakasit
Vip
V
Pissard
Serge
S
Baysal
Erol
E
Taher
Ali
A
Farra
Chantal
C
Al-Ali
Amein
A
Al-Ateeq
Suad
S
Oberkanins
Christian
C
D004252:DNA Mutational Analysis / Q000379:methods*; D005838:Genotype; D006801:Humans; D008403:Mass Screening / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods*; D017354:Point Mutation; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D017384:Sequence Deletion; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics
D011933:Reagent Kits, Diagnostic