Molecular characterization and PCR diagnosis of Thailand deletion of α-globin gene cluster

Abstract

Thailand deletion of alpha-Thalassemia (thal) 1 involves the zeta2-, phi zeta1-, alpha2-, alpha1-, and theta1-globin genes. In Southeast Asians and Taiwanese, this mutation is the second most common long-segment deletion of two alpha-globin genes, after the Southeast Asian deletion. To define the Thailand deletion breakpoints, we used polymerase chain reaction (PCR) to amplify the normal-sequence DNA fragments across the breakpoints. The amplified products were sequenced directly or after cloning into pGem-3Z or pCR2.1 vectors. Comparison of the normal and mutant sequences revealed that the 5' breakpoint lies between nucleotides 1,269 and 1,290 upstream of the initiator codon adenine of the zeta2-globin gene, and the 3' breakpoint lies between nucleotides 29,387 and 29,408 downstream of it. A total of 30,677 nucleotides were deleted. Both breakpoints mentioned above lie within the Alu repetitive sequences and an extensive sequence homology is present around the two breakpoints. These findings suggest that homologous recombination is the mechanism by which the deletion occurs. Based on our data, we used three oligonucleotide primers to amplify the regions across the deletion and its corresponding normal sequence. The feasibility of PCR diagnosis was confirmed in 20 carriers with this deletion.

Determination of the breakpoint of the common α‐thalassaemia deletion in Filipinos in Hawaii

“…Apart from the requirement for an unusually high magnesium concentration in the reaction, the predicted primers worked perfectly and are therefore suitable for rapid diagnosis of DNA from subjects suspected of having the Filipino (Fil) total deletion. Ko et al (1998) have reported that the Thai deletion has a crossover at exactly the same point as we report here for the Fil deletion. Since they did not report how they characterized the Thai deletion, we can only assume they had misdiagnosed the hererozygotes used for characterization of the deletion and, in fact, were dealing with the Fil deletion.…”

Prevalence study and molecular characterization of α-thalassemia in Filipinos

“…For PCR diagnosis of FIL, we used primers T83 (5b-ggtattctcaaggtgacactgagg-3b), T91 (5b-taaaccacccaatttttaaatgggc-3b), and T82 (5b-ataacctttatctgccacatgtagc-3b). The normal alleles have a 564-bp fragment with T91 and T83 and the affected alleles have a 560-bp fragment with T91 and T82 [11,12,19]. SBH using BglII and Asp718 double digestion and z-globin gene probe hybridization differentiated among SEA, rightward (-a 3.7 ) and leftward (-a 4.2 ) single a-globin gene deletion in one blot (Fig.…”

“…According to previous reports on the ethnic Chinese living in Taiwan, 3.5% are carriers of SEA, 0.17% of FIL, 0.6% of -a 3.7 , and 0.3% of -a 4.2 [12,15,19]. Significant differences in prevalence and molecular defects of a-thal also exist between the Philippines and Melanesia and regions in Southeast Asia [5,8,25,26].…”

“…These sequence characteristics result in both homologous and illegitimate recombination, which leads to various deletions in the cluster. Illegitimate recombination results in both Southeast Asian deletion (SEA) and Filipino deletion (FIL), both removing the two a-globin genes and leading to a-thal 1 [3,11,16,19,24,29]. Homologous recombination between the flanking regions of the two a-globin genes results in rightward or leftward deletion, both removing one a-globin gene and leading to a-thal 2 [9,11].…”

Microsatellite Markers within —SEA Breakpoints for Prenatal Diagnosis of HbBarts Hydrops Fetalis

“…DNA sequence analysis of each deletion breakpoint has now enabled PCR-based testing (22)(23)(24)(25)(26). Several techniques based on PCR amplification of normal and affected chromosomes (26 -28 ) have been developed to more rapidly identify globin gene mutations.…”