Barry Eng

Three New α-Thalassemia Point Mutations Ascertained Through Newborn Screening

We report three new α-thalassemia (thal) point mutations detected during newborn screening for hemoglobinopathies. The first mutation is a single nucleotide deletion (−A) that abolishes the translation initiation codon of the α2-globin gene, detected in a newborn of Hmong ethnicity who carried the Southeast Asian α0-thal deletion (αTα/– –SEA). The second mutation, a frameshift caused by a single nucleotide deletion in exon 2 of the α1-globin gene [codon 78 (−C)], was detected in a Black/Chinese newborn who also carried the Southeast Asian α0-thal deletion (ααT/– –SEA). The third mutation was a frameshift in exon 3 of the α2-globin gene, codons 113/114 (−C). This mutation was detected in a newborn who carried the 3.7 kb α+-thal deletion (αTα/–α3.7).

Characterization of a rare single alpha-globin gene deletion in a Chinese woman with Hb H disease.

A Chinese patient with Hb H (β4) disease was found to be a compound heterozygote for a 2.4 kb α+-thalassemia (thal) deletion and the common Southeast Asian α0-thal deletion. The endpoints of the 2.4 kb deletion were identified by sequence analysis of the deletion junction. The deletion removes the entire α1-globin gene and leaves the α2-globin gene intact.

Characterisation of the British α0‐thalassaemia deletion: evidence of a founder effect in Newfoundland, Canada

a-Thalassaemia is a common hereditary disorder caused by reduced expression or complete absence of a-globin chains required for adult haemoglobin (Hb A, a2b2). The most common cause of a-thalassaemia is deletions that remove one (a-thalassaemia) or both (a-thalassaemia) of the duplicated a-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 a-thalassemia syndromes, such as Hb H disease, Hb H hydrops fetalis syndrome, and Hb Bart’s hydrops fetalis syndrome (Higgs & Bowden, 2001). Although a-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 a-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 f-globin gene (HBZ) intact. The deletion, designated the British type of a-thalassaemia (–), 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 (–) 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 a-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 – deletion (Fig 1C). The PCR assay included two pairs of primers. One pair spans the – breakpoint and amplifies a 439 bp fragment that is specific for the – 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 ll containing 10 mmol/l Tris–HCL (pH 8Æ3), 50 mmol/l KCl, 2Æ5 mmol/l MgCl2, 200 lmol/l dNTP, 750 mmol/l Betaine, 1Æ0% (v/v) dimethyl sulphoxide, 5 U AmpliTaq Gold polymerase, 0Æ33 lmol/l PAFAH1B1-F primer, 0Æ30 lmol/l PAFAH1B1-R primer, 0Æ32 lmol/l BRIT-F primer, 0Æ34 lmol/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 (–/aa) had haematological indices typical of a-thalassaemia trait, with marked microcytosis [mean cell volume (MCV) 69Æ2 ± 1Æ5 fl, mean ± standard deviation], and hypochromia [mean cell haemoglobin (MCH) 21Æ9 ± 0Æ8 pg]. Two patients were compound heterozygotes for the )a single gene deletion and the – deletion ()a/–). One was an adult female with Hb H disease (Hb 88 g/l, MVC 50Æ9 fl, MCH 15Æ4 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). correspondence

Characterisation of a novel 49·3 kb Gγ(Aγδβ)0-thalassaemia deletion in seven families of Asian descent

The human b-globin gene cluster consists of five functional genes located on the short arm of chromosome 11 and arranged in the linear order e-c-c)d)b (centomere fi telomere). More than 40 different deletions of the b-globin gene cluster have been reported, the phenotype of which depends on the number of genes affected (Giardine et al, 2007). The c(cdb)-thalassaemias are due to deletions or rearrangements that abolish expression from the c, d, and b-globin genes while leaving the c-globin gene intact. Ten different c(cdb)-thalassaemia deletions and rearrangements have been reported in a wide range of ethnic groups (Higgs et al, 2001; Wood, 2001). The 5¢ deletion endpoints are tightly clustered immediately upstream and within the c-globin gene, and extend downstream of the b-globin gene for varying distances. Carriers of c(cdb)-thalassaemia typically have mild microcytosis and hypochromia, coupled with elevated levels of Hb F (a2c2). Herein, we describe a novel deletion identified in multiple unrelated individuals with c(cdb)-thalassaemia trait. Southern hybridisation using a probe specific for c-globin genes (c-IVS2) detected the following deletion-specific fragments: Bam HI (4Æ6 kb), Bgl II (8Æ2 kb), Xba I (5Æ9 kb) and Eco RV (9Æ3 kb). Based on the Southern hybridisation results, the 5¢ deletion endpoint was localised to a region immediately

α-Thalassemia Caused by Two Novel Splice Mutations of the α2-Globin Gene: IVS-I-1 (G>A and G>T)

We report the identification of two different mutations involving the first nucleotide of intron 1 of the α2-globin gene: IVS-I-1 G→A and G→T. The available data indicated that both mutations reduce the efficiency of proper mRNA splicing, resulting in α+-thalassemia (α+-thal).

Three New β-Globin Gene Promoter Mutations Identified Through Newborn Screening

We report three new β-globin gene promoter mutations identified in newborns with hemoglobin (Hb) profiles consistent with Hb S/β+-thalassemia (thal) (Hbs FSA). All three mutations are in close proximity to the conserved ATAA sequence located at positions −31 to −28 relative to the mRNA Cap site. Two cases involved single base substitutions at positions −25 (G→C) and −32 (C→T). The remaining case involved the deletion of two bases (−AA) at positions −27 and −26.

β+-Thalassemia Trait Due to a Novel Mutation in the β-Globin Gene Promoter: −26 (A>C) [HBB c.−76A>C]

We report the case of a woman with β+-thalassemia (β+-thal) trait, in which there were two sequence variants within the β-globin gene promoter: −54 (G>A) [HBB c.−104G>A] and −26 (A>C) [HBB c.−76A>C]. Data from other patients indicate that the −54 substitution is a non pathogenic sequence variant. Therefore, the β-thal phenotype is most likely due to the −26 mutation that is adjacent to the conserved ATAA box.

High Hb A2 β-Thalassemia Due to a 468 bp Deletion in a Patient with Hb S/β-Thalassemia

We describe a case of Hb S/β-thalassemia (thal) involving a 468 bp deletion that removes the β-globin gene promoter but leaves the coding regions intact. This is the second report of this deletion, and our family study establishes that this deletion causes β0-thal with unusually high levels of Hb A2 and Hb F. As with other genotypes involving deletions of the 5′ region of the β-globin gene, our patient had a mild form of Hb S/β-thal.

NOVEL β-THALASSEMIA MUTATION IN A β-THALASSEMIA INTERMEDIA PATIENT [POLY A (AATAAA →GATAAA)]

8.0 g=dL, PCV 0.261 L=L, MCV 69.0 fL, MCH 21.0 pg, MCHC 30.5 g=dL, RDW 26.3%, platelet count 764 10 9 =L, Hb A 72.4%, Hb A2 4.6%, Hb F 23.0%, serum ferritin 1600mg=L, bilirubin 31mmol=L. The blood film showed microcytosis, hypochromia, marked anisopoikilocytosis, ovalocytes, target cells, acanthocytes,

Second report of Hb Toulon [alpha77(EF6)Pro-->His] in a Canadian family of Italian descent.

More than 700 different human hemoglobin (Hb) variants have been described to date, with approximately 200 resulting from mutations of the a-globin genes (1). Although the majority of the a chain Hb variants have little or no clinical significance, some are associated with altered stability or function. In this report, we describe a 26-year-old man of Italian descent. who was originally referred for thalassemia (thal) screening because his wife had P-thal intermedia. Although there was no indication for a P-thal trait (Hb 14.6 g/dL, MCV 82.8 fL), isoelectrofocusing (IEF) revealed the presence of a Hb variant that migrated in the position of Hb F. The Hb variant could not be detected by starch gel electrophoresis or acid agarose electrophoresis. An a chain variant was indicated since the unknown Hb variant quantified at 25.3% and was associated with a split Hb A2. Functional testing indicated that the Hb variant had normal stability with heat and isopropanol, and normal oxygen affinity. Based on the IEF profile and