Hemoglobin, 2012; 36(6): 586–588Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630269.2012.736442

SHORT COMMUNICATION

A NOVEL β0-THALASSEMIA FRAMESHIFT MUTATION:

[HBB:c.216delT]

Christopher Konialis, Birgitta Hagnefelt, Sophia Sevastidou, Katerina Pispili,

and Constantinos Pangalos

InterGenetics, Diagnostic Genetics Centre, Athens, Greece

� A 33-year-old adult male of Greek ethnicity, with hematological indices suggesting β0-thalassemia(β0-thal) trait, was investigated for HBB gene mutations in the course of preparation for preimplanta-tion genetic diagnosis (PGD). Application of a routine diagnostic protocol, consisting of sequenceanalysis of the HBB gene, coupled to multiplex ligation-dependent probe amplification (MLPA),identified a single nucleotide deletion (–T) at codon 72 [HBB: c.216delT], leading to a novelpathogenic frameshift and protein-truncating β0-thal mutation (p.Phe72LeufsX18).

Keywords β0-Thalassemia (β0-thal), Frameshift mutation, Truncating mutation

A 33-year-old female and a 33-year-old male, both of Greek ethnicity,presented for genetic counseling to discuss the possibility of having preim-plantation genetic diagnosis (PGD) performed for their next pregnancy. Theywere both determined to be β-thalassemia (β-thal) carriers by routine hema-tological testing. The reported hematological indices of the father: RBC 6.96� 1012/L, hemoglobin (Hb) 13.9 g/dL, MCV 65.1 fL, MCH 20.0 pg, Hb A2

4.1%, Hb F 2.9%, were indicative of β0-thal trait. A pathological HBBmutation[IVS-I-1 (G>A)] was identified only in the mother, while prior genetic testinghad failed to identify a pathological HBB gene mutation in the father.

As part of our routine diagnostic protocol, a DNA sample was obtainedfrom the father and the β-globin gene was amplified by polymerase chain

Received 12 June 2012; Accepted 24 July 2012.Address correspondence to Dr. Christopher Konialis, Department of Molecular Genetics,

InterGenetics, Diagnostic Genetics Centre, Vas. Sofias 120, Athens 11526, Greece; Tel.: þ210-7705010,Fax: þ210-7705011, E-mail: genetic@ath.forthnet.gr

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reaction in two fragments, from approximately�150 bp relative to the mRNACap site to approximately þ160 bp relative to the translation stop. The result-ing fragments were subjected to fluorescent bi-directional automated DNAsequencing, using the BigDye™ v3.1 Terminator Cycle Sequencing Kit(Applied Biosystems, Foster City, CA, USA) and analysis on the ABIPRISM™ 3730�l DNA sequencer (Applied Biosystems). The sequence datafiles were subsequently exported to the Mutation Surveyor V3.97 software(SoftGenetics LLC, State College, PA, USA) for variant identification(Reference sequence: NG_000007.3.). Additionally, multiplex ligation-dependent probe amplification analysis was performed in parallel (probemixP102-B1; MRC-Holland, Amsterdam, The Netherlands) for the identificationof deletions/duplications within the β-globin gene cluster.

Two heterozygous coding sequence variants were identified: the knownbenign polymorphism c.9C>T (p.H3H) and a single nucleotide (–T) deletionat codon 72 (c.216delT), occurring at a stretch of three consecutive Ts(Figure 1). This single bp deletion is a frameshift mutation, leading to pre-mature termination of protein synthesis 18 codons downstream (p.Phe72LeufsX18) and a severely altered truncated protein, consistent with aβ0-thal phenotype. To the best of our knowledge, this is a novel β-thal muta-tion, as it has not yet been reported in the HbVar database (1) or anywhere inthe literature. The presence of the known intronic sequence polymorphisms,IVS-II-16 (C>G) (heterozygous), IVS-II-81 (C>T) (heterozygous), IVS-II-66(T>C) (heterozygous) and IVS-II-74 (G>T) (homozygous), was also detected.The parents of the proband were unavailable for testing, although it wasreported that his father presented with similar anemia (Figure 2).

It is worth noting that this novel mutation occurs at a region of exon 2 of theHBB gene spanning codons 68-76, where at least six other small insertion/deletion mutations have been reported, the majority of which, however, arepresent almost exclusively in Asian populations (1–3). In the Greek population,

FIGURE 1 Bidirectional DNA sequencing of the β-globin gene, exon 2 region carrying the c.216delTmutation. a) Top: reference sequence; b) middle: sequence trace alignment of the forward strand; c)bottom: sequence trace alignment of the reverse strand. Vertical line depicts the position of the mutation.

Novel β0-Thalassemia Frameshift Mutation 587

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except for the common codon 39 (C>T) (c.118C>T) mutation, there are lessthan four very raremutations within the entire exon 2 that have been describedto date (1,4).

The identification of this novel β0-thal mutation, through a routine com-prehensive protocol involving DNA sequencing of the entire HBB gene,demonstrates the merit of this diagnostic approach as opposed to commonlyemployed (screening) techniques, such as denaturing gradient gel electro-phoresis, which often fails to uncover single base substitutions and single bpdeletions (5). Furthermore, the novel mutation reported herein will aid themolecular diagnosis of β-thal, adding further evidence to the molecularheterogeneity of the disease.

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

REFERENCES

1. Giardine B, van Baal S, Kaimakis P, et al. HbVar database of human hemoglobin variants and thalassemiamutations: 2007 update. Hum Mutat. 2007;28(2):206 (http://globin.bx.psu.edu/hbvar).

2. Yu N, Kim HR, Cha Y J, Park EK. A novel frameshift mutation at codon 66 (HBB:c.del201A) in theβ-globin gene leads to β-thalassemia. Ann Hematol. 2011;90(2):243–244.

3. Angalena R, Aggarwal S, Phadke SR, Dalal A. Compound heterozygote condition in β thalassemia majordue to a novel single nucleotide deletion (–T) at codon 69 in association with IVS 1-5 (G>C) mutation.Int J Lab Hematol. 2012;34(4):e7–e9.

4. Boussiou M, Karababa P, Sinopoulou K, Tsaftaridis P, Plata, E, Loutradi-Anagnostou A. The molecularheterogeneity of β-thalassemia in Greece. Blood Cells Mol Dis. 2008;40(3):317–319.

5. Meldrum, CJ, McPhillips, M, Crooks R, et al. A comparison between denaturing gradient gel electro-phoresis and denaturing high performance liquid chromatography in detecting mutations in genesassociated with hereditary non-polyposis colorectal cancer (HNPCC) and the identification of 9 newmutations previously unidentified by DGGE. Hered Cancer Clin Pract. 2003;1:39–48.

FIGURE 2 Family tree depicting the proband (arrow), who is a carrier of the c.216delT mutation. Hisfather’s carrier status was reported but not confirmed by genetic testing.

588 C. Konialis et al.

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