Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification

Abstract

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4- kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.

Phenotypic expression of Hb F in common high Hb F determinants in Thailand: roles of α‐thalassemia, 5′ δ‐globin BCL11A binding region and 3′ β‐globin enhancer

“…While the levels of Hb A 2 were within the normal range in all cases, variability in Hb F levels ranging from 12.6 to over 30% were noted. DNA analysis using gap‐PCR identified the 12.6 kb deletion δβ 0 ‐thalassemia (NG_000007.3:g.64383_76994del12,612) in 79 cases, the 79.2 kb deletion HPFH‐6 (NG_000007.3:g.45595_124872del79278) in 65 cases, and the Indian deletion‐inversion G γ( A γδβ) 0 ‐thalassemia (NG_000007.3:g.48400_49245del846;49246_64567inv; 64568_72051del7484) in 15 cases. Further screening for other high Hb F determinants found in the Asian population revealed a Chinese G γ( A γδβ) 0 ‐thalassemia (NG_000007.3:g.48795_127698del78904) with 78.9 kb deletion in the remaining case.…”

“…Genomic DNA was prepared from peripheral blood leukocytes using a standard method. Screening for DNA deletions causing δβ 0 ‐thalassemia and HPFH found in Thailand and other Asian countries was carried out using multiplex PCR as previously described . Identifications of α o ‐thalassemia (SEA and THAI‐deletions), α + ‐thalassemia (3.7 and 4.2 kb deletions), Hb Constant Spring and Hb Pakse' mutations are routinely performed in our laboratory using PCR methodologies described elsewhere .…”

Thalassemia intermedia phenotype resulting from rare combination of c.46delT [Codon15 (‐T)] mutation of beta globin gene and HPFH3

“…To detect the mutation causing elevated Hb F, primers flanking the break points of ψβ , δ , β gene were used (HPFH‐type 3 deletion) . HPFH‐type 3 deletion involves the deletion of 48.5 kb DNA including codon 15 of the β ‐globin gene.…”

Detection of unknown deletions in β‐globin gene cluster using relative quantitative PCR methods

“…Almost 200 mutations including at least 60 different deletions have been characterized in β‐globin gene cluster so far (1, 4). A considerable number of deletions with variable extent and position such as hereditary persistence of fetal hemoglobin (HPFH) types 1, 2, 3, Spanish (δβ), Sicilian (δβ), Chinese G γ( A γδβ), Turkish inversion‐deletion (δβ), Asian Indian inversion–deletion G γ( A γδβ) and hemoglobin (Hb) Lepore have been detected that all involve the β‐globin gene (5).…”

“…Moreover, its accuracy critically depends on the quality of the hybridization probes and needs large amounts of DNA. FISH analysis as a qualitative approach is time consuming with cell culturing to generate metaphase chromosome spreads and has a low resolution (5, 8). The strategy for identifying the deletions in β‐globin gene cluster resulting in the δβ‐thalassemia, Hb Lepore and the HPFH deletion mutations is based on Gap PCR.…”