Background: A beta-hemoglobin variant (beta 126 (H4) Val-->Gly) was reported from Thailand and Naples (Southern Italy) as Hb Dhonburi (1) and Hb Neapolis (2), respectively. This abnormal hemoglobin, resulting from a valine to glycine substitution in the contact region between alpha and beta subunits, gives rise to instability at non-physiological conditions. However, it was difficult to distinguish this variant from Hb A using hemoglobin electrophoresis and cation exchange liquid chromatography. Hb Dhonburi was rarely reported, possibly due to a relatively milder phenotype in heterozygote with slightly decreased MCV. Thus several Hb Dhonburi carriers might have been under-diagnosed.
Methods: Combined molecular analyses by PCR-single strand conformation polymorphism (PCR-SSCP) and direct genomic sequencing of the beta globin genes were carried out in 2 pediatric patients with mild thalassemia intermedia. A novel amplification refractory mutation system (ARMS-PCR) was developed and performed in five individuals with microcytosis and borderline Hb A(2).
Results: Both patients were compound heterozygotes for Hb E and Hb Dhonburi. In addition, 5 Hb Dhonburi heterozygotes, including 3 identified through thalassemia carrier screening, were identified by ARMS-PCR. Linkage analysis of the affected families revealed that the haplotype of Hb Dhonburi in Thailand (VII) was different from that of Hb Neapolis (V) suggesting 2 independent mutational events.
Conclusions: The molecular strategy described provides a robust and economical measure, alternative to the whole beta globin genes sequencing, to identify rare or unknown beta globin mutations. To overcome its 'silent' nature on electrophoresis, we proposed a novel ARMS-PCR for a rapid diagnosis of Hb Dhonburi in future cases.