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Technical Briefs |
-Thalassemias
1 Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)-Casa Sollievo Sofferenza (CSS), San Giovanni Rotondo and CSS-Mendel Institute, Rome, Italy;2 Department of Experimental Medicine and Pathology, University of Rome "La Sapienza", Rome, Italy;3 Department of Biomedical Sciences, University "G. D’Annunzio", Chieti, Italy;4 Centro Microcitemia Associazione Nazionale Microcitemia Italiana-ONLUS, Rome, Italy;5 Human Genetic Laboratory, Galliera Hospital, Genoa, Italy;6 Laboratorio di Patologia Genetica IRCCS Oasi Maria Santissima, Troina, Italy;
aaddress correspondence to this author at: CSS-Mendel Institute, Viale Regina Margherita 261, 00198 Rome, Italy; fax 39-06-44160548, e-mail v.guida{at}css-mendel.it
-Thalassemias (OMIM 141850 and 141800; GenBank accession no. NT037887) are recessively inherited hemoglobin disorders caused by loss of function of either one of the two duplicated -globin genes (1 and 2), both located on chromosome 16p13.3 (1)(2). More than 95% of -thalassemia phenotypes result from meiotic unequal recombinational events between the highly homologous 1- and 2-globin loci, which lead mainly to large genomic deletions (3–100 kb), which remove one to four -globin genes, and rarely to -gene triplication or quadruplication. Although less frequent, at least 48 different nondeletional mutations (including point mutations and deletions/insertions of a few nucleotides), mostly located in the 2-globin gene, have also been reported as causative mutations of +-thalassemia (3)(4). At present, molecular identification of this type of nucleotide mutation is carried out by specific PCR amplification of the 2 or 1 gene, followed by methods that have only a limited rate of detection (60–80%) and are technically demanding, such as single-strand conformation polymorphism (SSCP) analysis and denaturing gradient gel electrophoresis (5)(6), or are costly, cumbersome, and time-consuming, such as direct sequencing and reverse dot-blot analysis (7)(8).
We have evaluated the performance of a relatively simple and semiautomated technique, denaturing HPLC (DHPLC), that separates heteroduplex and homoduplex molecules on a stationary phase under partially denaturing conditions (9)(10). We tested a TransgenomicTM Wave DHPLC-based protocol for the molecular identification of -globin gene nondeletional mutations in 50 wild-type individuals and 50 heterozygous carriers of Italian origin whose genes had previously been molecularly defined by restriction endonuclease digestion of PCR fragments and/or reverse dot-blot analysis.
Blood samples were collected from heterozygous individuals and healthy controls at the Centro Studi Microcitemie (Rome), the Galliera Hospital Genova, and the Oasi ONLUS Troina after informed consent was given. Genomic DNA was isolated from leukocytes in peripheral blood by salting out procedures (11). Purified DNA was solubilized in Tris-EDTA buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8) and stored at –20 °C. Four overlapping primers (DHPLC1 through -4), were designed to amplify the entire region of both -globin genes, including the 5' and 3' noncoding regions, at the same temperature. Because the third exon and the 3'-untranslated region of both -globin genes are considered mutational hot-spot regions, we chose two pairs of specific primers located in regions of sequence dissimilarity, thus allowing the selective amplification and analysis of 1 or 2 genes. Furthermore, because the -globin gene cluster is located within a GC-rich DNA region, which makes clean amplification quite difficult, we explored the use of different Taq DNA polymerases, also changing the composition of the Mg2+-containing buffer. After testing several commercially available DNA polymerases, including AmpliTaqTM (PE Applied Biosystems), Taq GoldTM (PE Applied Biosystems), and Optimase DNA polymerase (Transgenomic; data not shown), we used Platinum® Taq DNA Polymerase High-Fidelity (InvitrogenTM). After empirical optimization, we performed PCR amplifications in a Gene Amp PCR system 9700 (PE Applied Biosystems); the PCR mixture (total volume, 25 µL) contained 250 µM each of the deoxynucleotide triphosphates, 100 ng of template DNA, 0.5 µM each of the primers, and 1.25 U of Platinum Taq DNA Polymerase High Fidelity (Invitrogen) in 1x reaction buffer [60 mM Tris-SO4 (pH 8.9), 18 mM (NH4)2SO4, and 2.0 mM MgSO4]. To preserve the DNASep column, we replaced the Invitrogen buffer with the 1x Transgenomic Optimase buffer (2 mM MgSO4), obtaining the same results. The PCR conditions consisted of an initial denaturation step (94 °C for 3 min), followed by 30 cycles at 94 °C for 30 s, 62 °C for 30 s, and 68 °C for 45 s, with a final extension step of 7 min at 68 °C.
Each amplicon was heated at 95 °C for 5 min and cooled slowly over 30 min at room temperature to promote heteroduplex formation. Between 3 and 5 µL of crude sample was then loaded on a DNASep column (Transgenomic) and subjected to DHPLC analysis in a WAVE DHPLC instrument (Transgenomic) as described previously (12). The reversed-phase gradient was formed by mixing buffer A (0.1 mmol/L triethylammonium acetate, pH 7.0) and buffer B (0.1 mol/L triethylammonium acetate, pH 7.0, containing 250 mL/L acetonitrile). PCR products were then separated (flow rate, 1.5 mL/min) through a 5% linear acetonitrile gradient. Oven temperature for optimal heteroduplex separation under partial DNA denaturation was determined by varying the oven temperature by ±2 °C around the melting temperature suggested by the Transgenomic software 4.1.40 (Table 1
).
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Each abnormal elution profile was sequenced in both directions by use of a 310 Genetic Analyzer (PE Applied Biosystems) according to PRISM Dye Terminator and Dye Primer Cycle Sequencing chemistries.
Using this protocol we screened 12 different nondeletional mutations, including 11 point mutations, 1 five-nucleotide deletion, and 1 point mutation associated with the –3.7 rearranged 2/1 gene (Table 1
). In addition, we used 50 Italian wild-type individuals as negative controls. All 12 different mutations analyzed showed distinct and recognizable elution peaks. A comparison of elution peaks from wild-type individuals and mutant heterozygous individuals for each DHPLC fragment is shown in Fig. 1
. The chromatographic profiles for all individuals with an identical nondeletional mutation (i.e., Hb Hasharon, Hb Icaria, Hb Quong-Sze, HphI, NcoI, and NcoI) were identical (data not shown), demonstrating the reproducibility of the method. One the most frequent Mediterranean nondeletional mutations, a single-nucleotide substitution at the start codon that abolishes a NcoI restriction site and is detected in both -globin genes, was analyzed by DHPLC. Although both variants (NcoI and NcoI) were located within the same DHPLC2 fragment, we obtained different elution profiles at the same melting temperature (64.8 °C), which allowed us to distinguish which of the two genes was mutated. Moreover, the presence or absence of a common neutral polymorphism (2 + 861G>A), in conjunction with the TSaudi mutation on the same allele, changed the number and/or the shape of elution peaks compared with fragments containing a single mutation or polymorphism (compare the peaks in panels a, b, and c in Fig. 1
).
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Two different melting temperatures (63.8 and 64.8 °C) were sufficient to rapidly detect all of the most common Mediterranean mutations, including NcoI, NcoI, HphI, Hb Hasharon, Hb J-Sardegna, and Hb Icaria. For this reason, we suggest that the initial screening of any new Italian -thalassemia patient should be performed on fragments DHPLC1, DHPLC2, and DHPLC42, which contain the vast majority of -globin nondeletional mutations found in the Italian population. Overall, the DHPLC-based protocol produced neither false-negative nor false-positive results among 200 chromosomes. In contrast to traditional molecular techniques for the detection of unknown nondeletional -globin mutations (such as denaturing gradient gel electrophoresis, SSCP analysis, and direct sequencing), our DHPLC-based protocol was not affected by the extensive sequence homology and high GC content of the -globin loci (5)(6). Furthermore, compared with the above-cited methods, DHPLC is more rapid and has lower costs because, once preliminary work has been carried out to establish optimal PCR conditions, no special manipulations or implementations are required after amplification. Our data are concordant with a previous study of mutational screening carried out on the Notch 3 gene, which contains an extremely high GC content, that showed a higher sensitivity of DHPLC compared with SSCP and heteroduplex analysis (13).
Different from other equally simple strategies, such as reverse dot-blot hybridization, DHPLC-based screening is not limited to the identification of previously reported mutations but also allows the molecular characterization of unknown point mutations and small deletions/insertions.
In conclusion, we show that DHPLC is a reliable, sensitive, and specific screening method to rapidly detect the most common nucleotide mutations described in the Italian population. Many individuals with different or identical mutations can be simultaneously screened by this method. The analysis time of 2.5 min/sample offers the possibility of semiautomated analysis of 96 samples in <12 h. As such, this semiautomated method, coupled with the low costs of analysis per sample, would be suitable for rapid, large-scale mutational screening of frequent and rare mutations. In fact, although mutations affecting each globin gene are usually population specific, the widespread movement of people among communities favors a method that can identify a heterogeneous spectrum of both common and rarer -globin variants. In addition, molecular analyses are essential for the accurate diagnosis and subsequent risk ascertainment of double-heterozygous carriers for - and ß-globin alleles. In fact, in individuals who are heterozygous for ß-thalassemia, concurrent -thalassemia can mask the classic hematologic manifestations and make the ß-thalassemia carrier state less apparent, leading to errors in genetic counseling (3)(14).
Acknowledgments
This work was supported by a grant from the Italian Ministry of Health. We give special thanks to Arnaldo Morena and Roberto Cespa for their helpful information technology support.
References
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-globin gene disorders by a simple PCR methodology [Erratum published in: Haematologica 1996;81:XVI]. Haematologica 1996;81:387-396.-thalassemia point mutations by reverse dot blot analysis. Am J Hematol 2003;74:191-195.[CrossRef][ISI][Medline]
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- and ß-thalassaemia alleles: a pitfall of thalassaemia screening. Community Genet 2003;6:14-21.[CrossRef][Medline]
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