Identification and characterization of delta-globin gene mutations in Greek Cypriots

$\beta$ thalassemia is prevalent in the Mediterranean populations including Cyprus where mandatory carrier screening was implemented. Coinheritance of a $\delta$-globin gene mutation can confuse the diagnosis of heterozygosity for $\beta$ thalassemia which relies on the presence of a low MCV and a high Hb A$\sb2$. To help clarify the diagnosis of $\delta$/$\beta$ thalassemia carriers, we identified five $\delta$-globin gene alleles in Greek Cypriots associated with low Hb A$\sb2$ levels. The first four alleles were: G $\to$ T, C $\to$ T and T $\to$ C at codons 27, 116 and 141, and AG $\to$ GG in the IVS-2 3$\sp\prime$-acceptor, while the last allele had four different changes: C $\to$ T at $-$199 (neutral polymorphism), C $\to$ T at codons 4 and 97, and AT deletion in IVS-2. DNA haplotype analyses of the $\beta$-like globin gene cluster suggest independent origins for all alleles. Analysis of samples with borderline or low Hb A$\sb2$ using PCR and restriction enzyme digestion showed that the codon 27 mutation is the most common allele. The codon 4, 27, and 97, IVS-2 AT deletion and IVS-2 3$\sp\prime$-acceptor changes were introduced into normal $\delta$-globin genes. Using RNase protection and RT-PCR assays, we determined that the codon 27 mutation leads to activation of a cryptic splice site while the AG $\to$ GG change abolishes the 3$\sp\prime$-acceptor of IVS-2. The codon 97 and IVS-2 AT deletion transcripts are spliced normally. The codon 4 change, found on the same allele as the latter two changes, is probably the mutation leading to decreased Hb A$\sb2$ levels. We also studied effects of the codon 116 change in a yeast expression system. Heat and mechanical stability studies indicated that the codon 116 variant is as stable as Hb A$\sb2$ suggesting that reduced levels of this variant are probably due to a defect in assembly. Further studies are needed to determine the cause of the low Hb A$\sb2$ levels in the other variants. Screening for molecular defects causing $\delta$-thalassemia phenotypes will lead to improved genetic counseling