M. Murat Çelik

Ramazan Güneşaçar

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343 Letter to the Editor  First report from Turkey of a rare frameshift mutation [codons 9/10 (+T)] in the beta-globin gene Türkiye’de beta globin geninde nadir olarak gözlenen ilk çerçeve kayması mutasyonu [codons 9/10 (+T)] raporuRamazan Güneşaçar1, M. Murat Çelik21Department of Medical Biology and Genetics, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey2Department of Internal Medicine, Faculty of Medicine, Mustafa Kemal University, Hatay, TurkeyAddress for Correspondence: Assoc. Prof. Ramazan Güneşaçar, Department of Medical Biology and Genetics, Faculty of Medicine, Kahramanmaraş Sütçü İmam University, Kahramanmaraş, Turkey Phone: +90 506 307 92 90 E-mail: rgunesacar@hotmail.comdoi:10.5152/tjh.2011.95To the Editor,Beta-thalassemia  (β-thal)  is  one  of  the  most common autosomal recessive single gene disorders worldwide [1]. At least 200 different mutations in the β-globin gene that result in the β-thal minor and major phenotypes have been described [2,3]. The incidence of β-thal is high in Mediterranean regions, Iran, India, The Arabian Peninsula, Southeast Asia, and  Turkey  [4].  β-thal  is  characterized  by  point mutations, small deletions, or insertions that result in a decrease in or lack of expression of the β-globin chain, and each ethnic group or population has its own set of common mutations. In Turkey β-thal is common and 12 mutations accounted for 83.3% of 1500 unrelated cases with homozygous β-thal [5]; the remaining mutations were rare or newly identi-fied. To date, at least 39 nucleotide insertions in 3 exons of the β-globin gene that result in a modified C-terminal sequence of the β-globin protein have been  reported  (http://globin.bx.psu.edu/cgi-bin/hbvar/query_vars3).  Codon  9/10  (+T)  insertion mutation was first described in a Greek family by Wave et al. in 1994 [6], followed by the report of an Iranian patient of Kurdish origin by Rahimi et al [7]. Herein we present a 30-year-old Arab male with the β-thal trait living in Hatay, Turkey that had β-globin gene codon 9/10 (+T) frameshift mutation, which was noted during premarital genetic screening. To the  best  of  our  knowledge  this  is  the  first  case reported from Turkey and only the third case world-wide. In our laboratory where molecular testing for the premarital screening of thalassemia mutations are routinely performed, we encountered a case of a 30 year-old  male  meeting  the  diagnostic  criteria  of β-thal  trait.  After  written  informed  consent,  the patient accepted to undergo mutation analysis and laboratory tests.Genetic analysis showed a frameshift mutation-an insertion of T between codons 9 and 10 in the first exon of the β-globin gene. Hematological data TJH-ARALIK-2011-4-crossref.indd   95 02.12.2011   10:14were obtained via an automated cell counter and routine  methodology.  Red  cell  lysates  were  ana-lyzed using high-performance liquid chromatogra-phy (HPLC). Hemoglobin species assay via HPLC showed the following: Hb A: 86.8%; Hb A2: 4.7%; Hb F: 1.0%. Complete blood count findings were as fol-lows: Hb: 12.3 g/dL; Hct: 39.0%; MCV: 62.2 fL (low), MCH:  19.6  pg  (low);  MCHC:  31.5  g/dL  (low)-red blood cell count, white blood cell count, and plate-let  count  were  as  follows;  5.2  million/mm3, 6200/mm3,  255000/mm3,  respectively.  Iron  status, serum  ferritin,  and  other  hematological  indices were normal. DNA was isolated from whole blood samples  in  EDTA  using  a  commercially  available DNA  extraction  kit  (RTA  Lab,  Ltd.,  Sti,  Turkey). Regions of the β-globin gene were sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA., USA), according to the manufacturer's instruc-tions,  and  the  sequence  reaction  was  analyzed using an ABIPRISM 310 genetic analyzer (Applied Biosystems, Foster City, California, USA). The muta-tion was confirmed via sequencing of the anti-sense DNA strand, which was performed twice (Figure 1). Genetic defects that result in β-thal are primarily point mutations, rather than gene deletions or inser-tions. The mutations result in defects in transcrip-tion, RNA splicing and modification, translation via frameshifts,  and  nonsense  codons  that  produce highly unstable β-globin, which cannot be utilized. Codon 9/10 (+T) is a rare mutation in the β-globin gene and was first described in a Greek family with β-thal, and then in an Iranian of Kurdish origin. The present  case  had  the  β-thal  trait,  is  the  first  case reported  from  Turkey,  and  is  only  the  third  case worldwide. The insertion of a thymine nucleotide between codons 9 and 10 in exon 1 in the β-globin gene causes a reading frameshift from this point, leading to an in-frame stop codon at codon 22. The insertion of a T nucleotide in exon 1 alters the pro-tein structure due to a reading frameshift and the formation of a stop codon in position 22. The result-ing  truncated  protein  is  completely  inactivated because of the premature termination of translation at  codon  22,  instead  of  at  codon  147.  A  similar frameshift  mutation  in  the  β-globin  gene  [codon 9/10 (+G)] was observed in a patient of Turkish ori-gin by Aulehla-Scholz et al. [8]. Codon 9/10 (+G) and codon 9/10 (+T) mutations are similar, but dif-fer in terms of nucleotide insertion. The presented case had thymine insertion between the codon 9 and codon 10 regions, whereas the case reported by  Aulehla-Scholz  et  al.  had  guanine  insertion between codon 9 and codon 10. β-thal mutations are very heterogeneous and at least 40 different mutations are common in Turkey. This molecular heterogeneity might be due to the historical migration of individuals from non-Medi-terranean regions to Turkey. β-thal mutations are usually population specific and each ethnic group has distinct common mutations. Research on dif-ferent  populations  indicated  that  4-6  mutations account for 90%-95% of β-thal chromosomes in any given  ethnic  group.  The  prevalence  of  a  limited number  of  mutations  in  such  populations  has Güneşaçar et al.Codon 9/10(+T) mutation in the β-globin gene Turk J Hematol 2011; 28: 343-5 344Figure 1. Sequence Electropherogram Showing a Frameshift Muta-tion Due to a Thymine Insertion Between Codons 9 and 10 in Exon 1 of Beta Globin GeneThymine insertion(TCT)Codon 9151 153 155 157 159 161 163 165 167(GCC)Codon 10TJH-ARALIK-2011-4-crossref.indd   96 02.12.2011   10:14facilitated molecular testing using primer-specific amplification  or  reverse  dot  blot  hybridization, with a set of sequence-specific primers or probes for  the  frequently  occurring  β-thal  mutations.  In Turkey,  β-thal  mutations  are  very  heterogeneous and the above-mentioned techniques are not suf-ficient for detecting rare or unknown mutations; therefore,  direct  DNA  sequence  analysis  of  the β-globin gene could prove to be extremely useful for  prenatal  diagnosis  and  carrier  identification. Detection of rare β-thal mutations may also be use-ful for establishing a national mutation database and in genetic counseling. Conflict of interest statementThe authors of this paper have no conflicts of inter-est,  including  specific  financial  interests,  relation-ships, and/or affiliations relevant to the subject matter or materials included.References1.  Altay C, Basak AN. Molecular basis and prenatal diag-nosis  of  hemoglobinopathies  in  Turkey.  Int  J  Pediatr Hematol Oncol 1995;2:283-90.2.  Hardison RC, Chui DHK, Giardine B, Riemer C, Patrinos GP , Anagnou N, Miller W, Wajcman H. HbVar: a relation database of human hemoglobin variants and thalas-semia mutations at the globin gene server. Hum Mutat 2002;19:225-33. [CrossRef]3.  Patrinos GP , Giardine B, Riemer C, Miller W, Chui DHK, Anagnou DP , Wajcman H, Hardison RC. Improvement in HbVar human hemoglobin variants and thalassemia mutations for population and sequence variation stud-ies. Nucleic Acids Res 2004;32:537-41. [CrossRef]4.  Cavdar AO, Arcasoy A. The incidence of ß-thalassemia and abnormal hemoglobins in Turkey. Acta Haematol 1971;45:312-8. [CrossRef]5.  Basak AN. The molecular pathology of B-thalassemia in  Turkey:  The  Bogazici  University  experience. Hemoglobin 2007;31:233-41. [CrossRef]6.  Waye JS, Eng B, Oliveri NF, Chui DH. Identification of a novel beta O-thalassaemia mutation in a Greek family and  subsequent  prenatal  diagnosis.  Prenatal  Diag 1994;14:929-32. [CrossRef]7.  Rahimi Z, Muniz A, Parsian A. Detection of responsible mutations  for  beta  thalassemia  in  the  Kermanshah Province of Iran using PCR-based techniques. Mol Biol Rep 2010;37:149-50. [CrossRef]8.  Aulehla-Scholz C, Basaran S, Agaoglu L, Arcasoy A, Holzgreve  W,  Miny  P,  Ridolfi  F,  Horst  J.  Molecular basis  of  beta-thalassemia  in  Turkey:  detection  of rare  mutations  by  direct  sequencing.  Hum  Genet 1990;84:195-7. [CrossRef]Güneşaçar et al.Codon 9/10 (+T) mutation in the β-globin gene Turk J Hematol 2011; 28: 343-5 345TJH-ARALIK-2011-4-crossref.indd   97 02.12.2011   10:14

First report from Turkey of a rare frameshift mutation [codons 9/10 (+T)] in the beta-globin gene

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