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Indian Journal of Human Genetics, Vol. 11, No. 2, May-August, 2005, pp. 76-79 Original Communication Detection of two rare β -thalassemia mutations [-90 (C → T) and CD 26 (C → T)] among Indians Gorakshakar A, Phanasgaonkar S, Colah R, Mohanty D Institute of Immunohaematology (ICMR), Parel, Mumbai, India Code Number: hg05017 Abstract BACKGROUND : β -Thalassemia (β -thal) is present in practically every caste group in Indians. Molecular characterization of β -thal in these groups has revealed an extremely heterogeneous picture.AIM : To identify all the mutations and to detect the novel mutations using a versatile mutation detection technique. MATERIALS AND METHODS : Denaturing gradient gel electrophoresis (DGGE) has been established to scan the entire β -globin gene to localize the mutation followed by DNA sequencing for characterization. The DNA samples from two families referred to us either for prenatal diagnosis or for DNA studies were studied. RESULTS : Atypical DGGE patterns in fragments B & A indicating the presence of the mutation, have been detected in both the families. DNA sequencing revealed two rare patterns fragments with patterns in fragments β -thal mutations [CD 26 (C→T) and -90 (C→T)]. CONCLUSION : DGGE is a useful mutation detection technique to identify β -thal mutations among the heterogeneous Indian population. Keywords: Beta thalassemia; denaturing gradient gel electrophoresis; India; PCR. The β -thalassemias are a group of inherited monogenic hemoglobin disorders resulting from the reduced or absent synthesis of the globin chains. Molecular analysis of various β -thal genes have shown that there exists a wide spectrum of mutations described so far all over the world.[1] Similarly each specific ethnic group or region reveals it′s own spectrum of mutations. Today more than 200 β -thal mutations have been characterized worldwide. About 20-25% of these mutations have been detected among the extremely heterogeneous Indian population 1],[2],[3][4],[5],[6],[7],[8],[9],[10] and the number is increasing continuously. Data on spectrum of mutations in any region can serve as a valuable tool for population genetic studies, which may provide insights into past migration. It also helps to plan strategies for prenatal diagnosis. The use of PCR and denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing greatly facilitated the screening, localization, and identification of mutations.[11-13] Here we report two rare β -thal mutations among Indians identified using the above mentioned strategy. Materials and methods Case history Family NB: A Sunni Muslim farmer originating from Thane district of Maharashtra was the husband of a lady who came to us for prenatal diagnosis. Their first baby died at the age of 5 months due to some respiratory problems. Their second daughter aged 5 years was diagnosed as a β -thal major at 9 months and has been on regular blood transfusions since then. During the third pregnancy, the couple came to us at 18 weeks gestation. Prenatal diagnosis was done by cordocentesis and fetal blood analysis and the fetus was found to be homozygous. The couple decided to terminate the pregnancy. During the fourth pregnancy, the couple came for first trimester prenatal diagnosis, but one of the mutations remained uncharacterized. They were called back at 18 weeks gestation and prenatal diagnosis was again done by cordocentesis and fetal blood analysis. The fetus was found to be normal. Family BR : This Kayastha family originating from Bangladesh had come for bone marrow transplant for their β -thal major baby to India.They were referred to us for mutation analysis. Peripheral blood was collected in EDTA. Hematological data were obtained with an automated cell counter (Sysmex K-1000) The HbA2 and HbF levels were determined on the Variant Hb Testing System (Biorad Laboratories Inc., Hercules, USA). DNA was isolated from WBCs using the standard phenol-chloroform protocol. Screening for the presence of the most common Indian mutations and abnormal hemoglobins [IVSI-5 (G → C), codons 8/9 (+G), codon 15 (G → A), IVSI-1 (G → T), codons 41/42 (-TCTT), codon 30 (G → C), HbE and HbS] was carried out by Reverse Dot Blot (RDB) hybridization.[14] Screening for the 619 bp deletion was done by amplifying the DNA using a specific pair of primers covering the deletion and running the amplified sample in 2% agarose containing ethidium bromide.[11] The DNA samples from the cases where the mutation was not detected using the above methods were subjected to DGGE to localize the mutation. In this, the β -globin gene is divided into seven overlapping fragments [Figure - 1] and amplification of each fragment was accomplished by PCR using a specific pair of primers of which one of the primers has a GC clamp attached to it. [11],[12],[13] Each sample was amplified for all the fragments and run on a denaturing gradient gel along with normal controls to localize the mutations. DNA sequencing was done of the fragments showing an atypical DGGE pattern on the ABI prism 310 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the same primers as those used for DGGE but without the GC clamp. Results Table 1 shows the hematological data and DGGE analysis of these two families with rare mutations. In family NB [Figure - 2], the mother had a common Indian mutation CD 15 (G → A) identified by RDB hybridization. The father revealed a mutation in fragment B, which covers exon 1 and part of IVS 1 of the β -globin gene. Hence his DNA sample was sequenced for fragment B and the mutation was found to be codon 26 (C → T). In family BR [Figure - 3], the mother had the most common Indian mutation, IVS 1-5 (G → A) detected by RDB hybridization, while the father showed an atypical DGGE pattern in fragment A which covers two proximal CACCC boxes and the promoter region of the β -globin gene. On DNA sequencing this sample revealed the -90 (C → T) mutation. Discussion CD 26 (C → T) is an amber mutation which was first reported from Thailand.[15] This mutation occurs at the same position as that of HbE, which is one of the common abnormal hemoglobins in India. This mutation interferes in detection of HbE by restriction enzyme analysis. Hence an allele specific probe approach would be used to identify HbE in the presence of this mutation. The -90 (C →T) mutation was first described in a Portuguese individual.[16] Transcription assay performed with two proximal (ACCC box mutants viz. -87 (C →G) and -88 (C →T) produced correctly initiated and spliced β -globin mRNA but at a very low level, as compared to normal mRNA.[17],[18] Similarly, the ′(C →A) substitution at the -90 position results in a tenfold decrease in the level of transcription.′[19] Hence, it can be argued that the -90 (C →T) substitution must have functional significance in producing β -thal trait. Earlier studies on molecular characterization of β -thalassemia in the Indian population [20],[21],[22],[23],[24],[25] have shown six common β -thal mutations (Asian Indian mutations). Due to the peculiar Indian population structure, the frequency of these mutations vary considerably in different states. This data helps us to establish prenatal diagnosis facilities using tests like RDB hybridization. However, the main concern is about the remaining uncharacterized β -thal chromosomes as more and more rare and novel mutations are being reported. [26],[27],[28],[29],[30],[31] This shows that the Indian population has a wide spectrum of β -thal mutations and there is a need to establish systematic screening programmes for rare β -thal mutations. In this context, versatile mutation scanning techniques like DGGE or SSCP play an important role as they greatly facilitate the localization and subsequently identification of rare β -thal mutations. References
Copyright 2005 - Indian Journal of Human Genetics The following images related to this document are available:Photo images[hg05017f1.jpg] [hg05017f3.jpg] [hg05017f2.jpg] |
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