Download THREE-BASE DELETION IN EXON 3 OF THE /3

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.
Copyright 2011 by The American Society of Hematology; all rights reserved.