Hemoglobin, 2013; Early Online: 1–4Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630269.2012.763171

SHORT COMMUNICATION

NORMAL Hb A2 β-THALASSEMIA TRAIT: FRAMESHIFT MUTATION

(HBB: c.187_251dup) IN CIS WITH THE Hb A2’ δ-GLOBIN GENE

MISSENSE MUTATION (HBD: c.49G>C)

John S. Waye,1,2 Barry Eng,1 Laurie Hellens,1 Betty-Ann Hohenadel,1

Lisa M. Nakamura,1 and Lynda Walker1

1Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, Hamilton, Ontario,Canada2Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

� We report the case of a father and daughter who are heterozygous for a duplication of 65 bp withinexon 2 of the β-globin gene, resulting in an altered and truncated β-globin chain that is predicted to benon functional. The β-globin gene mutation is in ciswith the commonHb A2 0 missense mutation of theδ-globin gene (HBD: c.49G>C), resulting in β-thalassemia (β-thal) trait with normal levels of Hb A2.This is the second report of this β0-thal mutation, and both families were associated with the Hb A2 0

variant and normal levels of Hb A2. Laboratories should be aware of the rare occurrence of β-thal traitwith normal levels of Hb A2.

Keywords β0-Thalassemia (β0-thal), δ Chain hemoglobin (Hb) variant

The proband is a 31-year-old African-Canadian woman referred for inves-tigation of possible α-thal trait. Her father (68 years old) was referred fromanother physician for investigation of possible α- and/or β-thal trait. Bothindividuals had microcytosis and hypchromia suggestive of thalassemia trait,with normal levels of Hb A2 and slightly lower levels of a probable δ chainhemoglobin (Hb) variant (Hb X2) (Table 1). The combined percentage ofHb A2 and Hb X2 was within the range normally observed for β-thal trait.

Received 25 October 2012; Accepted 4 November 2012.Address correspondence to Dr. John S. Waye, Department of Pathology & Molecular Medicine,

McMaster University Medical Centre, Hamilton, Ontario L8N 3Z5, Canada; Tel: þ905-521-2100, ext.76273; Fax: þ905-521-2651; E-mail: waye@hhsc.ca

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Accordingly, both patients were given a provisional diagnosis of β-thal traitcomplicated by a δ chain Hb variant.

Testing of the daughter began with sequence analysis of the β-globin geneusing polymerase chain reaction (PCR) amplification, the BigDye™ TerminatorCycle Sequencing Kit, and the ABI PRISM™ 3130 automated capillary sequencer(Applied Biosystems, Foster City, CA, USA). Sequence analysis of the entire generevealed heterozygosity for two common, non pathogenic sequence variants(HBB: c.9C>T and c.315þ16C>G). In addition, there was a frameshift mutationwithin exon 2 beginning at amino acid codon 84. Sequencing in both directionsestablished that the mutation was a duplication of 65 bp (HBB: c.187_251dup)(Figure 1). The predicted product of this mutant allele is a truncated β chainconsisting of only 109 amino acid residues, with the carboxy terminal 26 residuesbeing abnormal (HBB: p.Thr85Leufs*27). The mutant transcript most likelywould be subject to nonsense mediated mRNA decay (1). Even if translated, itwas unlikely that the abnormal, truncated β chains would be capable of formingstable Hb tetramers. Accordingly, this mutation can be regarded as a β0-thalallele.

Sequence analysis of the δ-globin gene was conducted to investigate theunderlying cause for the normal level of Hb A2 and the presence of the minorHb variant (Hb X2). The analysis demonstrated that the daughter was hetero-zygous for theHb A20 missensemutation of the δ-globin gene (HBD: c.49G>C)(data not shown). In addition, she was negative for the seven commondeletional forms of α-thal, specifically ��SEA, ��FIL, ��THAI, ��MED,�(α)20.5, �α3.7, and �α4.2.

Testing of the proband’s father yielded the identical set of results. He washeterozygous for the 65 bp duplication within the β-globin gene, heterozygousfor the A20 missense mutation of the δ-globin gene, and negative for α-thal

TABLE 1 Hematologic Results For the Studied Individuals

Parameters Daughter Father

Age (years) 31 68Hb (g/dL) 9.5 13.0MCV (fL) 61.9 64.2MCH (pg) 20.2 21.8RBC (1012/L) 4.70 5.97RDW (%) 16.6 13.6Hb H bodies Negative Not testedHb A2 (%) 2.7 2.5Hb F (%) 0.6 1.1Hb X (%) 2.2 2.2β-Globin genotype c.187_251dup (heterozygous) c.187_251dup (heterozygous)δ-Globin genotype c.49G>C (heterozygous) c.49G>C (heterozygous)α-Globin genotype Normal Normal

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deletions. The most likely explanation for both the proband and her fatherbeing heterozygous for the the β- and δ-globin gene mutations is that thesemutations are in cis.

It is noteworthy that Frischknecht and Dutly have reported a patient withvery similar hematologic and molecular genetic test results (2,3). Theirpatient was heterozygous for both the 65 bp β-globin gene duplication andthe Hb A20 δ-globin gene missense mutation, lending further support to theproposition that these mutations are in cis. The only significant differencerelated to the α-globin gene cluster, with their patient being a compoundheterozygote for a single gene deletion and a triplicated α-globin gene (�α4.2/αααanti3.7).

The Hb A20 missense mutation is one of the most common δ-globin genemutations, present in approximately 1.0-2.0% of African-Americans (4).Carriers of this mutation are normal, apart from having reduced levels ofHb A2 and the presence of the Hb A20 variant. However, the presence of δgene mutations (resulting in δ chain Hb variants or δ-thal) can complicatethe diagnosis of β-thal trait because the level of Hb A2 will be normal (5). Infact, the Hb A20 missense mutation has been reported in cis with at least oneother β-thal mutation (HBB: c.143_144insA), resulting in β-thal trait withnormal levels of Hb A2 (6-8). Similarly, we have documented a normal Hb A2

β-thal allele in East Indians consisting of the codon 15 G>A β0-thal mutation(HBB: c.47G>A) in cis with another missense mutation of the δ-globin gene(Hb A2-India, HBD: c.350G>T) (8). Accordingly, diagnostic laboratoriesshould recognize the possibility of normal Hb A2 β-thal trait, particularlywhen other causes of microcytosis and hypochromia (e.g., α-thal, irondeficiency) have been excluded (5).

(A)

(B)

FIGURE 1 Bi-directional (forward A, reverse B) nucleotide sequence analysis of exon 2 of the β-globingene showing the 65 bp duplication (HBB: c.187_251dup). The duplicated sequences are underlined.

Normal Hb A2 β0-Thalassemia 3

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Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of this article.

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