Alpha0 thalassaemia as a result of a novel 11.1 kb deletion eliminating both of the duplicated alpha globin genes

Abstract

Aims: To characterise a novel 11.1 kb deletion that eliminated both of the duplicated alpha globin genes, giving rise to a typical alpha0 thalassaemia phenotype in four carriers from a Chinese family.

Methods: Haematological investigations were carried out on all family members. The seven common forms of alpha thalassaemia were screened for by the polymerase chain reaction (PCR) and Southern blotting was used to analyse the alpha globin gene cluster. DNA sequence analysis of the entire alpha1 and alpha1 globin gene region was carried out and reverse transcription (RT)-PCR was used to investigate the transcription levels of the alpha and beta globin genes.

Results: The breakpoints were found to lie between coordinates 31695-31724 and 42846-42867 of the alpha globin gene cluster (NG_000006), with a total of about 11,135 nucleotides deleted. These sequences are involved in (CA)n repeats, suggesting a homologous recombination event. RT-PCR analysis gave a transcription level of the alpha globin gene in heterozygotes comparable with that of SEA deletion heterozygotes, confirming no output of alpha globin from the linked pair of alpha globin genes. The heterozygosity for this novel deletion was confirmed by PCR diagnosis in all four carriers from this family.

Conclusions: This rare mutation constitutes an additional heterogeneous defect causing alpha thalassaemia in the Chinese population.

Figure 1

Family pedigree. The proband is indicated by the arrow.

Figure 2

(A) Mapping of the 5′ and 3′ breakpoints of the deletion. The scale in kilobases shown at the top is relative to the projected coordinates in the α globin gene cluster. A map of the normal chromosome with the α globin complex (ψζ, ψα2, ψα1, α2, α1, and θ1) is also shown. The probes (ζ, α, and DS-θ) used in our study are marked below the chromosome map and the 3′ breakpoint of the --^SEA deletion is indicated by the upward arrow. The extent of the novel 11.1 kb deletion is indicated by a black bar. The positions of restriction enzyme sites marked by vertical lines are driven from the sequence of GenBank files NG_000006 and AE006462, and the corresponding restriction enzymes are shown to the left of the figure. The sizes of the fragments seen after enzyme digestion are shown in the corresponding rectangles. The normal fragments and the bridging restriction fragments identified by the ζ and DS-θ probes are indicated at the right of the figure (abnormal fragments in parenthesis). (B, C) Analysis of the DNA restriction fragments using (B) the ζ probe, digested with Bgl II/Acc65I, and (C) the DS-θ probe, digested with Dra I. F, father; L, leftward deletion; M, mother; N, normal control; P, proband. Abnormal fragments (indicated by an asterisk) are produced in both groups with enzymes Bgl II/Acc65I and Dra I used for digestion. (D, E) Comparison of sequences across the deletion junction of (E) the mutant allele and of (D) the corresponding normal 5′ and 3′ DNA. The downward arrows indicate the 5′ and 3′ breakpoints, which lie between coordinates 31695–31724 and 42846–42867 of the α globin gene cluster (GenBank accession number, NG_000006). (F) An electrophoretic chromatogram of DNA sequencing of the breakpoint junction amplified from the proband.

Figure 3

Detection of the deletion by 1% agarose gel electrophoresis of amplified fragments using gap-polymerase chain reaction. M, molecular size marker of 1500 (100 ladder, highest band 1500 bp). The amplification product specific for the normal allele is seen at the 1206 bp position and the mutant allele at the 584 bp position. Lanes 1–9 contain samples from the I 1, I 2, II 1, II 2, II 3, II 4, II 5, III 2, and III 1 family members (table 1), respectively. Four members of the family, the proband (III 1), her mother (II 2), her grandmother (I 1), and her eldest aunt (II 5) were found to be heterozygous carriers of this mutant allele.