α-Thalassaemia is a common hereditary disorder caused by reduced expression or complete absence of α-globin chains required for adult haemoglobin (Hb A, α2β2). The most common cause of α-thalassaemia is deletions that remove one (α+-thalassaemia) or both (α0-thalassaemia) of the duplicated α-globin (HBA) genes. Individuals missing one or two of the four HBA genes have mild microcytosis and hypochromia, but otherwise have no associated health problems. However, they are at risk for having children with more severe α-thalassemia syndromes, such as Hb H disease, Hb H hydrops fetalis syndrome, and Hb Bart’s hydrops fetalis syndrome (Higgs & Bowden, 2001).
Although α-thalassaemia is most common in regions of the world where malaria has been endemic, cases have been reported in families of northern European descent (Higgs et al, 1985; Harteveld et al, 1997; Nooitgedagt et al, 2007). More than two decades have past since α0-thalassaemia trait was first reported in multiple British families (Higgs et al, 1985). Gene mapping by Southern hybridization indicated that the deletion extends for approximately 26 kb, removes both HBA genes, and leaves the ζ-globin gene (HBZ) intact. The deletion, designated the British type of α0-thalassaemia (--BRIT), has since been reported in other individuals of British descent (Wilkinson et al, 1986; Bhavnani et al, 1989; Trent et al, 1989). A deletion with the identical gene map has been described in several African-American families, and was designated as the Black (--BLACK) deletion (Steinberg et al, 1986).
Over the past decade, we have identified 25 individuals from 18 families who have Southern hybridisation profiles similar or identical to those previously described for the British and Black types of α0-thalassaemia. Based on the published gene maps for these deletions (Higgs et al, 1985; Steinberg et al, 1986), we designed polymerase chain reaction (PCR) primers to amplify across the deletion breakpoints. Nucleotide sequence analysis of the breakpoint fragment established the deletion spans a total of 28 073 bp, extending from nucleotide positions 145 465–173 537 relative to the chromosome 16 reference sequence (GenBank Accession NC_000016) (Fig 1A). Comparison of 5′ and 3′ breakpoint sequences indicated that the deletion resulted from non-homologous recombination between two Alu elements oriented in the same direction (Fig 1B). The Alu elements share 87% sequence identity over a span of 297 bp. The HBA gene cluster has a high density of Alu elements and non-homologous recombination between such elements is a common mechanism for generating deletions within the cluster (Harteveld et al, 1997).
We developed a gap-PCR protocol for rapid diagnosis of the --BRIT deletion (Fig 1C). The PCR assay included two pairs of primers. One pair spans the --BRIT breakpoint and amplifies a 439 bp fragment that is specific for the --BRIT deletion (BRIT-F 5′-CAGG TGTC CATC ATCA GGAC TAAC-3′ and BRIT-R 5′-CCTT CACC ACCA CCTG TGTA GG-3′). The second pair serves a control and amplifies a larger fragment from the PAFAH1B1 gene on chromosome 17 (LIS1-F 5′-ATAC CATG GTTA CCCC ATTG AGC-3′ and LIS1-R 5′-TTAT GTAA TGCA CATT GCAC ATCCC-3′). The PCR was run in a total volume of 50 μl containing 10 mmol/l Tris–HCL (pH 8·3), 50 mmol/l KCl, 2·5 mmol/l MgCl2, 200 μmol/l dNTP, 750 mmol/l Betaine, 1·0% (v/v) dimethyl sulphoxide, 5 U AmpliTaq Gold polymerase, 0·33 μmol/l PAFAH1B1-F primer, 0·30 μmol/l PAFAH1B1-R primer, 0·32 μmol/l BRIT-F primer, 0·34 μmol/l BRIT-R primer, and 100–500 pg genomic DNA. The cycling conditions were as follows: initial denaturation at 94°C for 12 min followed by 38 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 20 s, and extension at 72°C for 40 s, and finishing with a final extension at 72°C for 7 min. PCR products were analysed on 7% nondenaturing polyacrylamide gels, and visualized by ethidium bromide staining and ultra violet light fluorescence.
Using the above assay, together with sequence analysis of the breakpoint fragment, we confirmed that all 25 patients have the identical deletion. The adult carriers of this deletion (--BRIT/αα) had haematological indices typical of α0-thalassaemia trait, with marked microcytosis [mean cell volume (MCV) 69·2 ± 1·5 fl, mean ± standard deviation], and hypochromia [mean cell haemoglobin (MCH) 21·9 ± 0·8 pg]. Two patients were compound heterozygotes for the −α3·7 single gene deletion and the --BRIT deletion (−α3·7/--BRIT). One was an adult female with Hb H disease (Hb 88 g/l, MVC 50·9 fl, MCH 15·4 pg), the other was identified during newborn screening for Hb H disease.
A total of 18 unrelated families were identified, 16 from Canada and 2 from the United States. It is notable that 11 of the Canadian families trace their origins to the province of Newfoundland. The population of Newfoundland and Labrador is small (c. 500 000) and genetically isolated. Approximately 98% of the population is of English or Irish descent; Protestants from the southwest of England and Roman Catholics from the south of Ireland (Rahman et al, 2003). The British α0-thalassaemia deletion was originally reported in patients from northwest England with no foreign ancestry (Wilkinson et al, 1986; Bhavnani et al, 1989). Accordingly, we propose that the Newfoundlanders identified in this report represent a founder thalassaemic population that originated in England.
Further study is needed to determine the prevalence of the α0-thalassaemia trait in Newfoundlanders. This is particularly important given that the most recent clinical practice guidelines in Canada recommend thalassaemia carrier screening if a woman and/or her partner are identified as belonging to an ethnic population whose members are at higher risk for being carriers (Langlois et al, 2008). The guidelines specifically note that Caucasians of northern European ancestry, which certainly includes Newfoundlanders, are not at increased risk.
Two of the families identified in this study are African-American, and both were identified during the course of universal newborn screening for haemoglobinopathies in California (USA). The sequence of the deletion junction was identical to that found in the Caucasian patients from Canada, providing direct evidence that the British and Black α0-thalassaemia deletions are one and the same. The existence of this deletion in the African-American population is most probably due to population admixture.