Clinical Chemistry 49: 777-781, 2003;
10.1373/49.5.777
(Clinical Chemistry. 2003;49:777-781.)
© 2003 American Association for Clinical Chemistry, Inc.
Rapid Detection of ß-Globin Gene Mutations and Polymorphisms by Temporal Temperature Gradient Gel Electrophoresis
Ramachandran V. Shaji1,a,
Eunice Sindhuvi Edison1,
Balasubramanian Poonkuzhali1,
Alok Srivastava1 and
Mammen Chandy1
1 Department of Haematology, Christian Medical College, Vellore 632004, India.
aAuthor for correspondence. E-mail rvshaji{at}cmcvellore.ac.in.
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Abstract
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Background: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations.
Methods: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 ß-thalassemia genes from homozygous ß-thalassemia patients and unrelated individuals with heterozygous ß-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE.
Results: We found common mutations in 753 ß-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the ß-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning ß-globin gene were suspected.
Conclusions: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.
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Introduction
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ß-Thalassemias are inherited hemoglobin disorders characterized by decreased or absent synthesis of ß-globin chains. Individuals heterozygous for ß-thalassemia (carriers) have hypochromia, microcytosis, increased hemoglobin A2, and an unbalanced 
/ß-globin chain synthesis ratio. The hemoglobin concentration varies from mild anemia to normal. Homozygotes or compound heterozygotes have intermediate (thalassemia intermedia) to severe (thalassemia major) phenotypes. Hypertransfusion and administration of iron-chelating agents can ensure a normal life, but this treatment must be life-long, and most families in the developing world cannot afford it. Bone marrow transplantation can be curative, but only one-third of patients will have an HLA-matching donor.
Characterization of ß-thalassemia defects in populations worldwide has revealed marked molecular heterogeneity. Approximately 200 mutations that interfere with ß-globin gene transcription, RNA processing, and translation have been described in patients with ß-thalassemia (1), but each population has a limited number of frequent mutations. Delineation of the mutations in ß-thalassemia has greatly improved preventive medical services, such as genetic counseling and prenatal diagnosis, and shed light on understanding the clinical and hematologic variations of this disorder.
Screening of the common mutations in populations can be easily achieved with methods involving allele-specific probes (2)(3) or allele-specific primers (4)(5). However, for centers that perform molecular diagnosis of ß-thalassemia in a multiethnic population or in an ethnic group with a high frequency of rare alleles, a comprehensive mutation screening strategy that detects the common as well as rare mutations is essential. Several methods that are faster and more economical than total gene DNA sequencing have been described for scanning mutations in any gene. Single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis (DGGE),
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enzymatic/chemical cleavage analysis, and conformation-sensitive gel electrophoresis are the major detection methods used for this purpose. For the analysis of mutations and polymorphisms in the ß-globin gene, a comprehensive screening method has been described that uses DGGE (6).
Recently, certain modifications have been applied to DGGE to make this method easier to perform and more reproducible in routine molecular diagnosis. One of these methods is temporal temperature gel electrophoresis (TTGE), which uses a temporal temperature gradient instead of the chemical gradient used in DGGE. TTGE has been successfully used for detection of mutations in cystic fibrosis (7) and mitochondrial genes(8).
We have developed a TTGE method to detect mutations and polymorphisms in the ß-globin gene based on the melting profiles described previously by Ghanem et al. (6) for DGGE of the ß-globin gene.
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Materials and Methods
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DNA samples were obtained from 368 patients with homozygous ß-thalassemia and 153 individuals with heterozygous ß-thalassemia. Among the 889 ß-thalassemia genes analyzed, 786 were from an Indian population, 89 were from a Maldivian population, and 14 from an Omani population.
We examined previously reported common mutations in Indian populations, i.e., Codon 15 (G
A), intervening sequence (intron)-I-1 (GT) [IVS-I-1 (GT)], IVS-I-5 (GC), Codon 8/9 (+G), and Codon 41/42 (-TCTT); two rare mutations, Codon 30 (GC) and IVS-I-1 (GA); and the mutations producing the hemoglobin variants ßE [Codon 26 (GAGAAG)] and ßs [Codon 6 (GAGGTG)] by reverse dot-blot (RDB) analysis (3). The 619-bp deletion was detected with use by PCR with primers flanking the breakpoints.
For TTGE, the ß-globin gene was amplified as seven fragments (A, B, C, D, E, F, and G) using the primers described for DGGE by Ghanem et al. (6) (Fig. 1
). Fragments A–F contained the coding and noncoding sequences of the ß-globin gene, and fragment G was designed to detect three ß-globin gene polymorphisms in IVS-II [nucleotide (nt) 16, C/G; nt 74, G/T; and nt 81, C/T]. Fragments B, C, and D each contained polymorphic sites: Codon 2, C/T; IVS-II, nt 16, C/G; and IVS-II, nt 666, T/C, respectively. One of the primers used for amplification of these fragments, except for fragment F, had a GC clamp.
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Figure 1. Fragments analyzed by TTGE (bottom) and the locations of the polymorphisms within the ß-globin gene (top).
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PCR products were analyzed in 16 x 20 cm (0.75 mm thickness) 6% polyacrylamide (acrylamide:bis = 37.5:1) gels prepared in 1.5x Tris acetate-EDTA buffer (1x: 40 mmol/L Tris acetate, 1 mmol/L EDTA, pH 8.0) containing 6 mol/L urea. We mixed 5 µL of PCR product with 5 µL of 2x gel loading dye (700 mL/L glycerol, 1 g/L bromphenol blue, 1 mL/L xylene cyanol) and loaded the mixture on the gel. The electrophoresis was carried out at 130 V at constant temperature increments of 2 °C/h on the D CodeTM mutation detection system (Bio-Rad Laboratories), which has an automatic thermal regulator that increases temperature gradually over the length of electrophoresis. The temperature range for TTGE for each PCR fragment was determined empirically with the aid of computer simulation using WinMeltTM (Bio-Rad Laboratories) and reducing the upper and lower temperatures by 12 °C because each mole of urea lowers the melting temperature by 2 °C (9). The actual temperature range for the best resolution of hetero- and homoduplexes was determined experimentally by performing the electrophoresis at temperatures within ± 1 to ± 3 °C of the calculated temperatures. The temperature ranges for the different PCR fragments are shown in Table 1
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In every TTGE gel, PCR products from healthy controls were loaded to compare the mobility shift in the test samples. Because fragments B, C, and D each contained one polymorphic site (Fig. 1
), DNA samples heterozygous for these polymorphisms were used as controls, whereas for other fragments (A, E, and F) any control from a healthy individual was used.
The DNA samples that showed abnormal TTGE patterns were sequenced with the Big Dye terminator cycle sequencing reagent set (Applied Biosystems) and analyzed on an ABI 310 genetic analyzer (Applied Biosystems) according to the manufacturer’s protocols. The sequencing data were analyzed using Sequencing Analysis, Ver. 3.0, software and compared with the GenBank sequence to identify the mutations.
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Results
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Common point mutations detected by RDB analysis and the 619-bp deletion were present in 85% (753 of 889) of the ß-thalassemia genes analyzed in our laboratory. TTGE analysis of the other 136 genes detected mutations in different fragments of the ß-globin gene in 134 genes. Examples of the patterns produced by various mutations in the heterozygous states are presented in Fig. 2
. The fragments that showed patterns different from those of healthy controls in TTGE were subjected to DNA sequencing for characterization of mutations. To validate the efficiency of TTGE to detect all possible point mutations in the ß-globin gene, samples with the nine mutations detected by RDB were also analyzed. TTGE detected these mutations as well, confirming the high sensitivity of this method for detection of all the alleles. The mutations that were detected by TTGE are listed in Table 2
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Figure 2. Examples of patterns of different mutations analyzed by TTGE.
The controls used for fragments B, C, and D show heteroduplexes and homoduplexes because they were heterozygous for the polymorphisms in these fragments.
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The neutral sequence polymorphisms in the ß-globin gene that define the four frameworks 1, 2, 3, and 3a were also identified successfully by TTGE in DNA samples with known frameworks previously analyzed by DGGE in our laboratory. The gel for fragment G in Fig. 2
shows the TTGE patterns for individuals with different combinations of these frameworks. The mutations in the fragments containing polymorphisms (B, C, and D in Fig. 2
) could be easily identified by comparison with the controls heterozygous for the polymorphisms.
For the two ß-thalassemia genes in which mutations could not be detected, we suspected large deletions spanning ß-globin. TTGE analysis in the patients and the parents in these two families showed that the patients were homozygous for a mutation in a fragment and that this mutation was present in the heterozygous state in only one of the parents. This discrepancy in the analysis of point mutations occurs when large deletions are present in compound heterozygotes with point mutations.
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Discussion
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We have been involved in characterization of mutations in the ß-globin gene in patients with various hemoglobinopathies and thalassemias for the past 6 years for carrier detection, prenatal diagnosis in families with affected children, and genotype-phenotype correlation in individuals homozygous for ß-thalassemia. Initial analysis of previously reported common mutations showed that these mutations were present in only 85% of the ß-thalassemia chromosomes. This discrepancy is because most of the previous studies were carried out in ß-thalassemia patients and their family members in the north and east of the country, whereas our study included patients from all regions of the country. The Maldivian population has been reported to have a spectrum of mutations similar to that in the Indian population, and the common mutations detected by RDB analysis in our study constituted all of the alleles. Although Oman has certain common Indian mutations prevalent in the population, these mutations constituted only 65% of the alleles (10). Application of an efficient mutation detection method for unknown mutations was necessary in those samples in which common mutations were not detected.
DGGE and TTGE are based on the same principle, that two double-stranded DNA fragments of the same size, but differing in sequence, melt at different points in a denaturing gradient and can be distinguished by differential migration. A uniform temperature with a linear denaturant gradient formed by urea and formamide creates the denaturing environment in DGGE. In TTGE, the denaturing environment is formed by a constant concentration of urea in the gel combined with a temporal temperature gradient, which can be obtained with a temperature regulator in the electrophoresis system that increases temperature at constant increments. Because cumbersome gel casting with chemical denaturing gradient gels is not required, TTGE is simpler, faster, and more reproducible than DGGE, making this method very good for high-throughput screening. Standardization of a TTGE protocol for a given sequence is much easier because the running conditions can be easily predicted from the melting profile of the sequence obtained by WinMelt or similar computer simulations.
As with DGGE, TTGE also has increased sensitivity for detecting heterozygotes and compound heterozygotes, and the analysis of homozygotes should be done cautiously because in certain mutations the homoduplexes may not show a difference in electrophoretic mobility. Nevertheless, artificial heterozygotes can be formed by heating and annealing PCR products from the patients and controls before analysis in TTGE.
Using TTGE and subsequent DNA sequencing, we detected new and rare mutations in an Indian population. The mutations -90 (CT), -29 (AG), Int Codon (TC), Codon 17 (AT), Codon 22/23/24 (-AAGTTGG), Codon 26 (GT), IVS-I-130 (GA), Codon 36/37 (-C), Codon 41 (-C), Codon 107/108 (+G), Codon 110 (TC), Codon 126–131 (-17bp), and poly A site (TC) have not been reported previously in an Indian population. Codon 62/64 (-7bp) (11), Codon 81/87 (-22bp) (12), and IVS-II-613 (CT) were novel mutations in the ß-globin gene.
In the proposed protocol for screening for ß-globin gene mutations, DNA extraction, PCR, and mutation detection by TTGE take only 48 h with reproducible results. The speed and reproducibility of this method make it more suitable than DGGE for prenatal diagnosis of ß-thalassemia.
The sensitivity and the effectiveness of mutation detection by TTGE have been assessed in previous studies carried out with mitochondrial DNA (8) and cystic fibrosis conductance regulator (CFTR) gene (7) mutations. TTGE detected all of the previously identified mutations in the mitochondrial DNA, corresponding to 100% sensitivity, and improved the detection rate to 97.5% for CFTR gene mutations.
The temperature control during electrophoresis was monitored occasionally by use of an external thermometer to assure the reproducibility of the temperature regulation in the equipment.
In conclusion, TTGE can be used as an efficient method for detection of mutations in the ß-globin gene. When the sensitivities and limitations of other mutation detection methods are considered, TTGE appears to be a better choice in terms of throughput, cost-effectiveness, sensitivity, and simplicity. However, because TTGE does not characterize the nucleotide change, DNA sequencing must be performed in the fragment showing abnormal electrophoretic behavior. This study shows that any existing DGGE protocol can be easily converted to TTGE. The melting maps obtained by DGGE for a fragment of the gene can be used directly to calculate the upper and lower temperatures for TTGE.
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Acknowledgments
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We gratefully acknowledge financial support from the Department of Biotechnology, Government of India (Grant BT/RO948/Med/13/034/98).
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Footnotes
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1 Nonstandard abbreviations: DGGE, denaturing gradient gel electrophoresis; TTGE, temporal temperature gradient gel electrophoresis; IVS, intervening sequence (intron); RDB, reverse dot blot; and nt, nucleotide. 
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Abstract
Introduction
