SHORT COMMUNICATION

Co-existence of the Codon 16 (–C) (b0) andCodon 10 (C!A) (bþ) Mutations on the Same

b-Globin Gene

Roshan B. Colah,* Anita Nadkarni, Aruna Pawar, Ajit Gorakshakar,

Supriya Phanasgaonkar, and Dipika Mohanty

Institute of Immunohaematology, Indian Council of Medical Research (ICMR),

King Edward Memorial Hospital, Mumbai, India

The spectrum of b-thalassemia (thal) mutations has been defined in many population

groups worldwide. An interesting observation was reported in a recent study, where it was

shown that the codon 10 (C!A) mutation, reported by us earlier as a novel bþ mutation in

an Indian b-thal heterozygote, was in fact a rare polymorphism that was present on the

same b-globin gene as the frameshift codon 16 (–C) mutation. It was suggested that the

codon 16 (–C) b0-thal mutation had arisen on an ancestral allele carrying the codon 10

polymorphism (1). A similar association has also been reported in an Afghan family where

a compound homozygosity for both these rare mutations was present (2). We had detected

the codon 10 (C!A) mutation by denaturing gradient gel electrophoresis (DGGE)

analysis, followed by manual DNA sequencing using the Sequenase Version 2 kit [United

States Biochemicals (USB), Cleveland, OH, USA] in a b-thal heterozygote (3). The codon

10 (C!A) mutation could be clearly seen in the mutant sample. However, a signal for the

presence of a C at codon 16 was also seen, albeit weakly. Furthermore, it is generally

observed that the reading frame of the sequence gets distorted after a frameshift mutation

and it is difficult to read the subsequent sequence due to overlapping of the bands, but in

our case this was not apparent after codon 16. In view of this, we considered codon 16 (–C)

*Correspondence: Dr. Roshan B. Colah, Assistant Director, Institute of Immunohaematology,

Indian Council of Medical Research (ICMR), 13th Floor, King Edward Memorial Hospital, Parel,

Mumbai-400 012, India; Fax: þ91-22-4138521; E-mail: mohanty@bom5.vsnl.net.in.

HEMOGLOBIN

Vol. 27, No. 2, pp. 133–135, 2003

DOI: 10.1081=HEM-120021549 0363-0269 (Print); 1532-432X (Online)

Copyright # 2003 by Marcel Dekker, Inc. www.dekker.com

133

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to be absent in this case. We had hypothesized that the codon 10 (C!A) mutation created

an alternate splice site, resulting in a bþ-thal phenotype.

In this report we re-examined our b-thal heterozygotes showing the codon 16 (–C)

mutation. The presence of both the codon 16 (–C) and codon 10 (C!A) mutations in cis

in two cases was confirmed by cloning the 875 bp DNA fragment of the mutant clone into

the pGET-M vector (Promega, Madison, WI, USA), followed by automated DNA

sequencing on the ABI PRISMTM 310 Sequencer (Applied BioSystems, Foster City,

CA, USA) (Fig. 1). A further six cases with the codon 16 (–C) mutation were studied by

PstI enzyme digestion, as the codon 10 (C!A) mutation creates a PstI site. All of them

showed the presence of the PstI polymorphism, while it was absent in normal individuals.

The codon 10 polymorphism was absent in 123 normal individuals and 120 b-thal

heterozygotes with other mutations. Thus, we confirm the association of both these

mutations in Indian b-thal heterozygotes.

There are several earlier reports on the presence of the codon 16 (–C) mutation from

different population groups, but none had noted the association with the codon 10

polymorphism (4). Perhaps specific mutation detection methods such as amplification

refractory mutation system (ARMS) or dot-blotting were used, and the codon 10

polymorphism was thus missed. It would be important to retrospectively study all such

cases along with a large number of normal individuals from different population groups to

ascertain this association.

REFERENCES

1. Old JM, Khan SN, Verma I, Fucharoen S, Kleanthous M, Ioannou P, Kotea N, Fisher C,

Riazuddin S, Saxena R, Winichagoon P, Kyriacou K, Al-Quobaili F, Khan B. A multi-

center study in order to further define the molecular basis of b-thalassemia in Thailand,

Pakistan, Sri Lanka, Mauritius, Syria, and India and to develop a simple molecular

diagnostic strategy by amplification refractory mutation system-polymerase chain

reaction. Hemoglobin 2001; 25(4):397–407.

2. Krugluger W, Hopmeier P. Identification of a compound b-thalassemia homozygosity

[codon 10 (GCC!GCA) and codon 16 (�C)] in an Afghan family. Hemoglobin 2002;

26(3):317–320.

Figure 1. Automated DNA sequencing (ABI PRISMTM 310; Applied BioSystems) of a WEB COLORmutant

clone showing the codon 16 (–C) mutation along with the codon 10 (C!A) polymorphism.

134 Colah et al.

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onal

use

onl

y.

3. Pawar AR, Colah RB, Mohanty D. A novel bþ thalassemia mutation (codon 10

GCC!GCA) and a rare transcriptional mutation (�28 A!G) in Indians. Blood 1997;

89:3888–3889.

4. Huisman THJ, Carver MFH, Efremov GD. A Syllabus of Human Hemoglobin Variants.

2nd ed., Augusta: The Sickle Cell Anemia Foundation, 1998 (http://globin.

cse.psu.edu).

Received December 23, 2002

Accepted January 20, 2003

Codon 16 (–C) and Codon 10 (C!A) in cis 135

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