Journal of Clinical Laboratory Analysis 20:1–7 (2006)
Identification of the Four Most Common b-Globin Gene Mutations in Greek b-Thalassemic Patients and Carriers by PCR-SSCP: Advantages and Limitations of the Method Konstantinos V. Kakavas,1,2 Argiris Noulas,2 Christos Chalkias,3 Christos Hadjichristodoulou,4 Ioannis Georgiou,5 Elena Georgatsou,1 and Sophia Bonanou1 1
Department of Biochemistry, School of Medicine, Faculty of Health Sciences, University of Thessaly, Larissa, Greece 2 Department of Clinical Chemistry, School of Medical Laboratories, Faculty of Health and Care, Highest Technological Educational Institution, Larissa, Greece 3 Department of Transfusions, b-Thalassemia Unit, General Municipal Hospital, Larissa, Greece 4 Department of Hygiene-Epidemiology, School of Medicine, Faculty of Health Sciences, University of Thessaly, Larissa, Greece 5 Genetics Unit, Department of Obstetrics and Gynecology, School of Medicine, University of Ioannina, Greece
In the present study we investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) the four most common b-globin gene mutations in the Greek population: IVS-I-110, Cd39, IVS-I-1, and IVS-I-6. Using DNA from 50 b-thalassemic patients and carriers, we amplified by PCR the appropriate 238-bp region of the human bglobin gene, analyzed the reaction products by nondenaturing polyacrylamide gel electrophoresis, and visualized the bands by silver staining. Single-stranded DNA (ssDNA) fragments showed a reproducible pattern of bands that was characteristic of the mutations present. With the use of
control samples containing six of the 10 possible combinations of the four most common b-globin gene mutations, we were able to predict the mutations present in a quarter of the patients studied. Our predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. We conclude that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in patients, provided that suitable controls are available. Moreover, the method is easy to apply to the identification of mutations in carriers, which makes it particularly useful for population screening. c 2006 J. Clin. Lab. Anal. 20:1–7, 2006. Wiley-Liss, Inc.
Key words: single-strand conformational polymorphism; b-thalassemia; DNA mutational analysis; population characteristics
Abbreviations: ARMS, amplification refractory mutation system; bp, base pair; DGGE, denaturing gradient gel electrophoresis; dHPLC, denaturing high-performance liquid chromatography; dNTP, deoxynucleoside triphosphates; EDTA, ethylene diamine tetraacetic acid; IVS, intervening sequence; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; ssDNA/dsDNA, single-stranded/doublestranded DNA; SSCP, single-strand conformational polymorphism; TBE, Tris-borate-EDTA; TAE, Tris-acetate-EDTA
c
2006 Wiley-Liss, Inc.
Grant sponsor: European Union; Grant sponsor: Greek Ministry of Education (Elective Studies Program ‘‘Medical Biochemistry’’). Correspondence to: Sophia Bonanou, Department of Biochemistry, School of Medicine, Faculty of Health Sciences, University of Thessaly, Papakyriazi 22, Larissa 41222, Greece. E-mail:
[email protected] Received 22 July 2005; Accepted 16 September 2005 DOI 10.1002/jcla.20091 Published online in Wiley InterScience (www.interscience.wiley.com).
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INTRODUCTION b-Thalassemia is one of the most common single gene disorders and is characterized by the absence (b0 thal) or reduced (b1 thal) production of b-globin chains (1). More than 200 mutations are known to result in this autosomal-recessive disorder (2,3), which presents with varying severity and is particularly common in Mediterranean countries. Given the heterogeneity of the genetic defects and the seriousness of the resulting b-thalassemia syndromes, great efforts are being made to identify mutations and detect carriers. The limited number of mutations prevalent in each ethnic group makes it easier to perform such screening. In Greece, where 7.5% of the population are carriers (4), four mutations have been reported to account for 79.9% (4) or 86.8% (5) of the total mutations encountered in patients with b-thalassemia major. These mutations, their individual frequency of occurrence, and the resulting phenotype are as follows: IVS-I-110 (C-to-A): 43.2% (b1); codon 39 (Cto-T): 20.9% (b0); IVS-I-1 (G-to-A): 13.9% (b0); and IVS-I-6 (T-to-C): 8.8% (b1) (5). Similar findings were recently reported in a large regional study of 1,081 Greek b-thalassemia carriers (6). The most commonly used procedures to identify known b-thalassemia mutations in PCR-amplified DNA are the reverse dot blot analysis with allelespecific oligonucleotide probes (7), and the amplification refractory mutation system (ARMS) (8). For the detection of unknown mutations, the indirect methods of denaturing gradient gel electrophoresis (DGGE) (9) or single-strand conformation polymorphism (SSCP) analysis (10) have been employed, followed by direct sequencing to determine the mutation. The advantages and disadvantages of each of these methods are discussed on the European Molecular Genetics Quality Network website (11). Recently, systems based on a microelectronic chip (12) or denaturing high-performance liquid chromatography (dHPLC) (13) have been applied for large-scale, fast, and reliable mutational screening of known b-globin alleles. For routine mutation identification in the clinical setting, a simple, sensitive, inexpensive, nonradioactive, reliable, and preferably automated system is required. In this study we investigated whether PCR-SSCP, a method that satisfies most of these criteria and has been used extensively for mutation detection (10,14–16), could also be employed for the presumptive identification of known mutations, e.g., the four most common b-globin gene mutations present in Greek patients and carriers. In a system optimized for SSCP electrophoretic analysis, we demonstrated that the denatured PCR J. Clin. Lab. Anal. DOI 10.1002/jcla
reaction products of DNA samples from b-thalassemic patients, during polyacrylamide gel electrophoresis under nondenaturing conditions, showed a distinct and reproducible pattern of bands. This was due to the unique three-dimensional conformations of the dissociated DNA strands, as determined by their primary sequence. Similarly unique patterns were obtained in carriers of each of the four most common b-globin gene mutations, which makes this method useful for population screening. MATERIALS AND METHODS All chemicals used in this study were of ANALAR grade and were obtained from Sigma (Germany). Taq DNA Polymerase, dNTPs, and MgCl2 were obtained from Gibco BRL (UK). The novel b-globin primers were from Minotech (Crete, Greece). Blood samples were obtained from 40 b-thalassemic patients who were receiving transfusion therapy in the local Municipal Hospital of Larissa, three healthy control individuals, six control homozygous or compound heterozygous thalassemic individuals in whom the mutations had been determined directly by DNA sequencing, and four control heterozygous individuals, each of whom carried one of the four most common mutations under investigation. We extracted genomic DNA from the peripheral leukocytes of whole blood using the method of Miller et al. (17). To amplify the appropriate region of the b-globin gene containing the mutations of interest, we designed the following novel 20mer primers, which result in the amplification of a 238-bp fragment: forward primer (50 Gl): 50 -tg aac gtg gat gaa gtt ggt-30 ; and reverse primer (30 Gl): 30 -aac agc atc agg agt gga ca-50 . Each reaction mixture contained approximately 0.4 mg of genomic DNA, 150 mM of each primer, 50 mM KCl, 20 mM tris-HCl pH 8.4, 1.5 mM MgCl2, 200 mM of a mixture of the four dNTPs, and 2 U of Taq DNA polymerase. After the reaction mixture was heated at 941C for 5 min it was subjected to 31 cycles of heating for 30 sec at 941C, 30 sec at 521C, and 1 min at 651C in an automatic thermal cycler (Eppendorf Master cycler gradient 5331 vs. 1.2). Before they were used for SSCP, the PCR products were purified by passage through a Qiagen column (QIAquick multiwell PCR purification products) and were checked on 2% agarose gels in TAE buffer, pH 8 (18), containing ethidium bromide, for purity and correct size. Slab gels containing 5% polyacrylamide-5% bisacrylamide in Tris/phosphate/EDTA buffer, pH 6.8, were prepared in a vertical electrophoresis apparatus (180 200 5 mm) and were equilibrated at 41C. To 2 mL of PCR product (50–100 ng DNA´), 8 mL of 0.1%
Mutation Identification by PCR-SSCP
SDS/10 mM EDTA was added, and the mixture was heated to 651C for 5 min. Then 15 mL of a solution containing 98% deionized formamide, 15 mM NaOH, 0.25% bromophenol blue, and 0.25% xylene cyanol were added and the mixture was heated further at 941C for 3 min in a final formamide concentration of 65%. At the end of this dissociation treatment, the reaction mixture was rapidly cooled on ice, loaded onto the precooled polyacrylamide gel, and electrophoresed at 10 W constant power (16 V, 60 mA) for 3 hr at 41 C. At the end of the electrophoretic separation the gel was carefully removed and bands were visualized by staining with silver nitrate, according to the method of Merril (19). The relevant part of the stained gel was photographed in white light with a digital camera (UVTEC CV-415.LS). The DNA samples were amplified by ARMS-PCR to determine the presence of the four most common b-globin gene mutations (8), according to previously described methods (6,20). RESULTS The DNA prepared from the patient and control samples under study was pure and of high Mr greater than 23000. A 238-bp fragment of the b-globin gene, spanning the end of the first exon, the first intron, and the beginning of the second exon (in which the four most common mutations of the Greek population are found), was amplified by PCR with the use of the novel 20mer forward and reverse primers, as shown in Fig. 1. Agarose gel electrophoresis analysis of the PCR reaction products obtained from control and thalassemic DNA samples showed a clear band corresponding to the expected 238-bp fragment (results not shown). Before we applied SSCP electrophoresis for our analysis, we proceeded to empirically determine the conditions that produced the best quality of separation of single-stranded DNA (ssDNA) molecules, as judged from the appearance and number of bands of the PCR products. Therefore, we varied the amount of DNA loaded, percentage of formamide used for denaturation, glycerol effect, methods of staining (ethidium bromide and silver nitrate), time and temperature of the electrophoretic run, power applied, and other matrixes
Fig. 1. Diagrammatic presentation of the 238-bp fragment of the b-globin gene amplified by PCR. The position of the four mutations under study is indicated: 1) IVS-I-1, 2) IVS-I-6, 3) IVS-I-110, and 4) cd39.
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as Mutation Detection Enhancement gel solution of Hydrolink (MDE). Such methods for optimizing the method of SSCP have been used in various studies in the literature, e.g., to detect mutations in the p53 gene (21). We also examined the reproducibility of the separation patterns obtained. We found that we obtained the best separation of ssDNA molecules by carefully following the conditions described in Materials and Methods. Having optimized the system, we then proceeded to perform an electrophoretic analysis of control heterozygous and b-thalassemic patient samples containing known mutations. The SSCP electrophoretic analysis of control carrier samples, containing each of the four b-globin mutations of interest, is shown in Fig. 2. One can observe that whereas the pattern of separation of the ssDNA molecules of a normal individual consists of two main bands (lane 5), the pattern given by each carrier contains either three (normal/IVS-I-110, lane 4) or four (normal/ IVS-I-1, normal/IVS-I-6, normal/codon 39, lanes 1–3, respectively) discrete bands. Moreover, the presence of each mutation in the carriers gives rise to a different and distinct pattern, which is mainly due to the slowermigrating bands, since the two bands of highest mobility are common to all four heterozygous samples examined.
Fig. 2. SSCP electrophoretic analysis of b-thalassemic heterozygous control samples. The PCR products were analyzed as described in Materials and Methods. The samples employed had the following genotypes: Lane 1: IVS-I-1/Normal. Lane 2: IVS-I-6/Normal. Lane 3: Codon 39/Normal. Lane 4: IVS-I-110/Normal. Lane 5: Normal control. The box shows the bands due to ssDNA.
J. Clin. Lab. Anal. DOI 10.1002/jcla
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We therefore conclude that under the conditions of our electrophoretic analysis, it is possible to determine not only whether an individual is a carrier, but also to distinguish which of the four most common b-globin gene mutations is carried by the individual. Analysis of the control b-thalassemic samples, which contained six of the 10 possible different combinations of the four mutations under investigation (IVS-I-1/IVSI-1, IVS-I-1/IVS-I-6, IVS-I-1/IVS-I-110, IVS-I-1/codon 39, IVS-I-6/IVS-I-6, and IVS-I-6/IVS-I-110) showed that each combination of mutations gave a distinct pattern of bands due to ssDNA (Fig. 3A), which could be used for mutation identification. One b-thalassemic sample (IVS-I-1/IVS-I-6, lane 8) gave two bands, but in the other five cases three bands were obtained. This separation pattern was highly reproducible and differed from that given by the normal control (lane 4). In our experiments ssDNA migrated faster than doublestranded DNA (dsDNA), as deduced from the migration pattern of denatured and nondenatured samples (lanes 4 and 5), although the dissociation obtained was rarely complete. As shown more clearly on the diagram in Fig. 3B, we observed that the band of highest mobility was present in all control thalassemic samples, and as a result was not diagnostically useful, but the slowermigrating bands differed depending on the mutations present. Analysis of seven SSCP electrophoretic separations of the same control samples showed that in each sample the difference in mobility of the two slowermigrating bands was statistically significant (Po0.001), as revealed by the nonparametric test of MannWhitney/Wilcoxon. Indeed, in most of the control samples we analyzed, the mobility of the slowestmigrating ssDNA band was sufficient to identify the combination of mutations present, and only in two cases (IVS-I-1/IVS-I-110 and IVS-I-1/IVS-I-1) did we have to take the mobility of the second slowest-migrating band into account for genotype determination (Table 1). Next we attempted to identify the mutations present in the samples of the b-thalassemic patients under study, by comparing their pattern of SSCP electrophoretic separation with that produced by the known controls. We electrophoresed in the same gel a number of unknowns and some of our control samples. By this method we were able to predict, as illustrated in Fig. 4 (compare lanes 12 and 13 to lane 1, and lanes 14 and 15 to lane 3) and in other electrophoretic analyses (not shown) that the combination of mutations present in five patient samples was IVS-I-1/codon 39, and in another five was IVS-I-6/IVS-I-110. For the remaining 30 out of 40 samples, we could not make a prediction, other than they did not contain a combination of mutations corresponding to one of the six controls we had available. J. Clin. Lab. Anal. DOI 10.1002/jcla
Fig. 3. SSCP-electrophoretic analysis of b-thalassemic homozygous or compound heterozygous control samples. The PCR products were analyzed as described in Materials and Methods. A: The thalassemic samples used in lanes 1–3 and 6–8 have the genotypes indicated underneath. Normal control samples, before and after dissociation by heating in formamide, are shown in lanes 5 and 4, respectively. The box shows the bands due to ssDNA. B: Diagrammatic representation of the pattern of bands due to ssDNA, obtained in the normal and the six b-thalassemic control samples shown in Fig. 4A. The arrow points to the fastest-migrating band, which is present in all control samples examined.
To verify our SSCP predictions, we used the ARMS direct method of mutation identification. We tested the 10 samples in which we had identified the mutations, and another four randomly chosen samples for which
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TABLE 1. Rf of bands used in the identification of mutations present in control homozygous b-thalassaemic samples Rf of slowest band Min 0.33 Med 0.33 Max 0.331 Min 0.39 Med 0.391 Max 0.391 Min 0.41 Med 0.411 Max 0.411 Min 0.409 Med 0.411 Max 0.411 Min 0.42 Med 0.42 Max 0.42 Min 0.45 Med 0.45 Max 0.451 Min 0.46 Med 0.461 Max 0.461
Rf of second slowest band
Mutations present IVS-I-6/IVS-I-6
IVS-I-6/IVS-I-110
Min 0.437 Med 0.438 Max 0.438 Min 0.487 Med 0.488 Max 0.488
IVS-I-1/IVS-I-110
IVS-I-1/IVS-I-1
IVS-I-1/IVS-I-6
Normal
IVS-I-1/codon 39
The migration of the slowest and second slowest bands, given by the dissociation pattern of each control sample shown in Fig. 3, is expressed as Rf (ratio of distance travelled by the band to distance of gel front). The values given are the minimum, median, and maximum of seven independent electrophoretic runs.
we could not make a prediction, together with the appropriate positive and negative controls, for the presence of the four mutations, as judged by the appearance of the appropriate PCR products. Figure 5 shows the results of such an experiment, which confirm that the samples for which we had predictions were true positives, whereas the samples we had predicted to be negative did not contain these combinations of mutations. For example, patient samples 1 and 2 of Fig. 5A, which correspond to samples 12 and 13 of Fig. 4, contain the predicted mutations IVS-I-1 and codon 39; and patient samples 1 and 2 of Fig. 5B, which correspond to samples 14 and 15 of Fig. 4, contain the predicted mutations IVS-I-6 and IVS-I-110. In both 5A and B, sample 3 corresponds to a normal individual containing no mutations, sample 4 is a positive control for each mutation examined, and sample 5 is a negative control containing no DNA. All samples contain the 861-bp fragment, which serves as an internal control to ensure that the b-globin gene has been amplified. Moreover, we see that patient samples 6–9 (Fig. 5A and B), which were chosen at random among the 30 samples for which we were unable to make a prediction by SSCP with the controls we had available, contain
Fig. 4. SSCP-electrophoretic analysis of b-thalassemic patient samples and comparison with known controls. The PCR products were analyzed as described in Materials and Methods. A: Lane 1: IVSI-1/codon 39 control; lane 2: IVS-I-1/ IVS-I-1 control; lane 3: IVS-I-6/ IVS-I-110 control; lanes 4 and 5: normal control, after and before dissociation; lanes 6–15: samples of 10 different b-thalassemic patients. Only the region containing the patterns due to ssDNA is shown. Note that in lane 5 no ssDNA is present. B: Diagrammatic representation of the pattern of bands due to ssDNA, obtained by the normal, control, and 10 b-thalassemic samples shown in A. Note that the pattern of samples 12 and 13 is the same as sample 1, and the pattern of samples 14 and 15 is the same as sample 3.
none of these combinations of mutations. For example, three samples (lanes 6, 8, and 9) contained none of the mutations under investigation, whereas one sample (lane 7) was positive for IVS-I-110 but contained none of the other three most common mutations. These results demonstrate that our SSCP analysis, based on the comparison of the pattern of bands given by ssDNA in unknown samples and known controls, can be used for mutation identification. DISCUSSION In the present study we demonstrate for the first time that one can employ the SSCP method for the identification, and not simply the detection, of the four most common b-globin gene mutations found in Greek b thalassemic patients and carriers, by comparing the patterns of their ssDNA electrophoretic separation with those of appropriate controls. We expected that each of the four b-globin gene mutations examined would give rise to a characteristic band, or number of bands, that could be used to identify that mutation in whatever combination it occurred. What we found, however, was that each genotype, and J. Clin. Lab. Anal. DOI 10.1002/jcla
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Fig. 5. ARMS-PCR confirmation of SSCP results. Patient DNA samples for which we had genotype predictions from the SSCP analysis shown in Fig. 4 were analyzed by ARMS-PCR, as described in Materials and Methods and in Results. A: Samples were analyzed for the presence of mutations IVS-I-1 (upper panel) and codon 39 (lower panel). Lanes 1 and 2: Patient samples corresponding to samples 12 and 13 in Fig. 4. Lane 3: Normal control. Lane 4: Positive control for the mutation examined. Lane 5: Negative control for the ARMS reaction. Lanes 6–9: Four of the 30 patient samples for which we could not make a prediction by SSCP. B: Samples were analyzed for the presence of mutations IVS-I-6 (upper panel) and IVS-I-110 (lower panel). Lanes 1 and 2: Patient samples corresponding to samples 14 and 15 in Fig. 4. Lanes 3, 4, and 6–9 are as described in A.
not each mutation, gave a characteristic pattern of bands. Thus each of the four control heterozygous sample we analyzed gave a clearly distinct and individual pattern of three or four bands (Fig. 2). Similarly, the pattern obtained with the six control homozygous or compound heterozygous b-thalassemic samples we analyzed was characteristic of the alleles present, and could not be deduced from the corresponding heterozygotes (Fig. 3). Moreover, we found that the difference between the two slower-migrating ssDNA bands was statistically significant, and that these bands were easily distinguishable and could be used to identify the mutations present (Table 1). By comparing the ssDNA patterns obtained by SSCP electrophoresis of the 40 thalassemic patient samples with those of the six known controls, we were able to predict the combination of mutations present in 10 J. Clin. Lab. Anal. DOI 10.1002/jcla
samples (Fig. 4). Independent identification of the mutations present in these patient samples by the direct method of ARMS-PCR confirmed our predictions (Fig. 5). Furthermore, we were able to demonstrate that samples for which we were not able to make a prediction by SSCP were truly negative for the combination of mutations examined. However, we have not been able to demonstrate that any combination of mutations always gives a distinguishable pattern of bands (i.e., that no two different combinations produce a similar pattern). The detection of only 25% of the mutations in the thalassemic samples appears to be very low. However, although the four mutations under study collectively account for approximately 80% of the total number of mutations encountered in the Greek population, their individual frequencies differ greatly (6). We calculated that the sum of frequencies of occurrence of the six control combinations of mutations we had available accounted for approximately 33% of the total mutations present in Greek b-thalassemic patients. This figure is not very different from our experimental value of 25%. Clearly, if we had employed a greater number of controls, our predictive score would have improved. However, the purpose of this study was not to identify all mutations, but to demonstrate the principle that this can be accomplished by SSCP. The identification of known mutations with the use of appropriate controls has already been accomplished with the similarly indirect method of mutation detection known as DGGE (22). DGGE is thought to have greater sensitivity than SSCP, and has the advantage that the melting temperature of a nucleotide sequence can be theoretically predicted. In this study we show for the first time that the approach of diagnosing mutations in unknown samples by comparison with known controls can also be successfully applied to SSCP, which is an intrinsically simpler method. However, because the pattern of bands obtained depends on the precise combination of alleles, the usefulness of this method for determining the genotype of b-thalassemic individuals is obviously limited by the great number of controls required. Moreover, the ability to detect mutations by SSCP is in general lower compared to other methods (e.g., DGGE or DHPLC, which are reported to detect 495% of the mutations present) (13). The SSCP method is nevertheless much easier to employ than other methods and is especially successful for b-thalassemic carriers. Thus, with the use of only four heterozygous controls, each containing one of the four most common mutations, over 80% of the carriers present in the Greek population could be identified. This method would thus be particularly useful for an initial screening of target groups. Moreover, it could be
Mutation Identification by PCR-SSCP
successfully applied for the identification, and not simply the detection, of mutations in other genetic diseases, especially if the number of known mutations is not great.
11.
ACKNOWLEDGMENTS
12.
We thank Ms R. Katsogiannou for providing us with blood samples from the b-thalassemia patients, Dr. M. Samara and Dr. P. Kollia for providing the heterozygous and homozygous control samples, and Drs. M. Papadakis, G. Simos, and N. Vamvakopoulos for many helpful suggestions.
13.
14. 15.
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