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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CORRESPONDENCE 1894 THREE-BASE DELETION IN EXON 3 OF THE /3-GLOBIN GENE PRODUCED A NOVEL VARIANT (BGUNMA) W I T H A THALASSEMIA-LIKE PHENOTYPE To the Editor: Most of the molecular defects producing a @-thalassemiaphenotype are linked to a pretranslational process of @-globin chain synthesis. Only a few mutations create a @-thalassemiaphenotype, posttranslationally.’ We have now identified a novel structural mutant caused by the three-base deletion in exon 3 of the @-globin gene that creates a @-thalassemia phenotype in a Japanese individual. The patient was a 36-year-old man who presented to the hospital with a mild anemia; hemoglobin (Hb), 11.3 g/dL; hematocrit (Hct), 38.3%; mean corpuscular volume (MCV), 64.2fL; mean corpuscular hemoglobin (MCH), 19.8 pg; and mean corpuscular hemoglobin concentration (MCHC), 30.8 g/dL. His HbF and HbA2 levels were 3.4% and 4.3%. respectively. All routine examinations for liver function were within normal ranges. The serum iron and total iron binding capacity were 122 and 225 pg/dL, respectively. Blood film examination showed a slight anisopoikilocytosis and no inclusion body was observed on staining with brilliant cresyl blue. The Hb electrophoresis, the heat and isopropanol stability tests showed no abnormal Hb. The globin chain synthesis ratios were 0.12 and 0.45 for y/a and @/a,respectively. Both @-globingenes of the patient were cloned as 7.8-kb Hind111 fragments in bacteriophage Charon 28.2 The 4.9-kb EglII fragments from the recombinant phage clones were inserted into the plasmid vector, pUC 13, and the @-globingene sequences from both alleles were determined.l DNA sequence analysis demonstrated that one clone (clone B) has the normal sequence, and in another clone (clone A), a 3-bp (AGG) deletion within codons 127 and 128 in exon 3 of the @-globingene (Fig 1A). This mutation results in the deletion of two amino acid residues, glutamine and alanine at residues 127 and 128, and an insertion of a new proline at residue 127, as depicted in Fig 1A. Therefore, the @-globinchain synthesized from this mutant allele would consist of 145 instead of 146 amino acid residues. We further confirmed this mutation by dot blot hybridization of the amplified genomic DNA with specific oligonucleotide probes using procedures described previ~usly.~ Figure 1B demonstrated that the patient was heterozygous for this mutation. Haplotype analysis in the @-globin gene cluster2 demonstrated that the haplotype (+ - - - - - +) was linked to this mutant allele. Analysis of the synthetic globin chains by pulse-labeling using carboxymethyl-cellulose column chromatography failed to demonstrate abnormal protein, suggesting that this @-globinvariant is highly unstable. In the PA globin chain, codons 127 (Gln) and 128 (Ala), located within the H-helix of the globin chain, are essential for the al and 81 subunit interaction.’ Replacement of the Gln-Ala dipeptide at these positions by a Pro residue in the @-globinchain variant disrupts the H-helix of this @-globinchain and therefore interferes with the a l a 1 dimeric formation. The uncombined @-globinchain synthesized from the mutant allele would be rapidly removed by proteolysis and a @-thalassemia phenotype would ensue. This is also the case with other @-chainvariants, such as Hb Showa-Yakushiji: Hb Indianapolis,’ Hb Houston,’ and the recently described Hb Galicia,’ which are associated with a @-thalassemia phenotype. The novel @ globin chain mutant described here was named Hb Gunma. SUPAN FUCHAROEN GOONNAPA FUCHAROEN YASUYUKI FUKUMAKI Research Laboratory f o r Genetic Information Kyushu University Maidashi. Higashi-ku, Fukuoka YUTAKA NAKAYAMA Akita City Hospital Akita YUKIO HATTORI KIYOMI YAMAMOTO YUZO OHBA Department of Clinical Pathology Yamaguchi University Medical School Ube, Japan REFElRENCES 1. Kazazian HH Jr, Boehm CD: Molecular basis and prenatal diagnosis of @-thalassemia.Blood 72:1107, 1988 2. Orkin SH, Kazazian HH Jr, Antonarakis SE, Goff SC, Boehm CD, Sexton JP, Waber PG, Giardina PJV: Linkage of @-thalassemia mutations and @-globingene polymorphisms with DNA polymorphisms in human 0-globin gene cluster. Nature 296:627, 1982 3. Hattori M, Sakaki Y: Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152:232, 1986 4. Fucharoen SP, Katsube T, Fucharoen G, Sawada H, Oishi H, Katsuno M, Nishimura J, Motomura S, Miura Y, Fukumaki Y: Molecular heterogeneity of @-thalassaemiain the Japanese: Identification of two novel mutations. Br J Haematol74:101, 1990 5. Bunn HG, Forget B: Hemoglobin structure, in: Hemoglobin: Molecular Genetic and Clinical Aspects. Philadelphia, PA, Saunders, 1986, p 13 6. Kobayashi Y, Fukumaki Y, Komatsu N, Ohba Y, Miyaji T, Miura Y: A novel structural mutant. Showa-Yakushiji (0110 Leu-Pro) causing a @-thalassemiaphenotype. Blood 70:1688,1987 7. Adams JG, Steinberg MH, Boxer LA, Baehner RL, Forget BG, Tsistrakis GA: The structure of hemoglobin Indianapolis (8112(G14) Arginine): An unstable variant detectable only by isotopic labeling. J Biol Chem 254:3479, 1979 8. Kazazian HH Jr, Dowling CE, Hurwitzs RL, Coleman M, A d a m JG: Thalassemia mutations in exon 3 of the @-globingene From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CORRESPONDENCE 1895 A (cloneA) Mutant 124 Pro 125 Pro [E\: (cloneB) Normal d) -4 G A T C G A T C A-T - 124 T-A 126 127 Val 125 126 C-G G-C Pro 127 C-G 128 Ala 128 A-T 129 /;I:[ C-G Tyr 129 T-A ' N G-C P normal probe m u t a n t probe Fig. 1. (A) D N A sequeneing gel representing sequences in the vicinity of codons 127 and 128 where three bases, AGO (TCC of the antisense strand), are deleted from the mutant allele of the petient. Ladders represent the nucleotide sequence of the antisense strand (anti). Clone A and clone 8 derived from themutant and normal alleles. respectively. The nucleotide sequence of the sense strand (sense), their corresponding amino acids. and codon numbers are also shown. Deletion of AGG in codons 127 and 128 shown in a shaded box leads t o the elimination of Gln-Ala dipeptide and insertion of new Pro at codon 127. (8) Dot blot analysis of the amplified DNA mmples using allele specific oligonucleotide probes for the AGG deletions in codons 127 and 128. The sequence of the oligonucleotide for normal probe is 5'-ACCAGTGCAGGCTGCCTAT-3' and i s 5'-ACCAGTGCCTGCCTATCAG-3' for its mutant counterpart. N and P indicate the normal individual and the patient, respectively. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1896 often cause a dominant form of thalassemia and show no predilection for malaria in endemic regions of the world. Am J Hum Genet 45:A242, 1989 (abstr) 9. Wilson JB, Webber BB, Hu H, Kutlar F, Codrington JF, CORRESPONDENCE Prchal JT, Hall KM, de Pablos JM, Roriguez I, Huisman THJ: Hemoglobin Birmingham and Hemoglobin Galicia: Two unstable B chain variants characterized by small deletions and insertion. Blood 75:1883, 1990 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1990 76: 1894-1896 Three-base deletion in exon 3 of the beta-globin gene produced a novel variant (beta gunma) with a thalassemia-like phenotype [letter] S Fucharoen, G Fucharoen, Y Fukumaki, Y Nakayama, Y Hattori, K Yamamoto and Y Ohba Updated information and services can be found at: http://www.bloodjournal.org/content/76/9/1894.citation.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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