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Technical Briefs |
-Thalassemia Mutations
Departments of
1
Pediatrics and
2 Obstetrics & Gynecology, National University of Singapore 119074, Singapore
3 The Children’s Medical Institute and Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore 119074, Singapore
4 Departments of Pediatrics and Gynecology & Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287
5 Department of Pathology, The University of Hong Kong and Queen Mary Hospital, Hong Kong, People’s Republic of China
6 Departments of Medicine and Pathology, Boston University School of Medicine, Boston, MA 02118
aaddress correspondence to this author at: Department of Pediatrics, National University of Singapore, Level 4, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore; fax 65-6779-7486, e-mail paecs{at}nus.edu.sg
-Thalassemia is the most common globin disorder in the world, and the severe forms are especially prevalent among Southeast Asians. It is a disorder of absent or reduced production of -globin chains resulting from mutations in the -globin gene cluster on chromosome 16p13.3. Most -thalassemia mutations involve deletions of one (-) or both (- -) -globin genes, whereas point mutations within the -globin genes (T or T) are much less frequent. Nonetheless, the number of point mutations that have been described has been steadily increasing, with >40 identified to date (1)(2).
The importance of nondeletional -thalassemia mutations is underscored by the observation that patients with nondeletional hemoglobin (Hb) H disease (T/- -) are generally more severely affected and more likely to require transfusions compared with deletional Hb H disease patients (-/- -) (3). There have also been a few reports of Hb H disease caused by homozygosity or compound heterozygosity of nondeletional mutations involving the 2-globin gene (T/T) (1)(3). Additionally, nondeletional Hb H disease involving the 2 codon 30 or codon 59 mutation can cause the fatal Hb H hydrops fetalis syndrome, especially if associated with large 
--globin gene deletions (4). In certain regions of Southeast Asia, nondeletional Hb H disease can account for as many as 50% of all Hb H disease patients (3).
Several specific and reliable molecular tests have previously been developed to diagnose and screen the most common deletional (5)(6) and nondeletional (7)(8) -thalassemia mutations. We now describe a multiplex minisequencing assay to detect seven Southeast Asian nondeletional mutations: Hb Constant Spring (2 codon 142 TAA
CAA), Hb Paksé (2 codon 142 TAATAT), Hb Quong Sze (2 codon 125 CTGCCG), 2 codon 0 
1bp (-T), 2 codon 30 3bp (-GAG), Hb Suan Dok (2 codon 109 CTGCGG), and 2 codon 59 (GGCGAC).
Genomic DNA samples of various -thalassemia genotypes were used in initial assay optimization. The point mutations in these samples were previously determined by direct 2-globin gene resequencing and/or multiplex amplification refraction mutation system-PCR, whereas the deletions were determined by Southern blot or multiplex-PCR analysis. We analyzed DNA samples heterozygous for each point mutation, as well as samples compound heterozygous for a deletion and the point mutation. No patient samples carrying a Hb Suan Dok or codon 59 mutation were available. These two mutations were therefore artificially created by PCR mutagenesis and cloned into bacterial plasmids (data not shown). Plasmid DNA was mixed with an equimolar amount of either wild-type DNA (/) to mimic heterozygosity (T/) or homozygous - -SEA DNA (- -SEA/- -SEA) to mimic a nondeletional Hb H disease genotype (T/- -SEA). The mutant allele of codon 30 was also cloned from the DNA of a Cd30/ carrier and mixed with - -SEA/- -SEA genomic DNA to generate the corresponding Hb H disease genotype.
The template for the multiplex minisequencing was obtained from either of two sources. For the first, in the context of a comprehensive -thalassemia mutation screen, a seven-deletion multiplex-PCR was first performed on the DNA sample, using previously described conditions (6), with the exception that primer 2-R was changed to 2-R(g/c) (5'-AGACCAGGAAGGGCSGGTG-3', where S = dG or dC; HUMHBA4 74757457), which was used at a final concentration of 0.2 µM. In situations where only a double -globin gene deletion was detected in a Hb H disease patient or where only a single -globin gene deletion was detected in a patient suspected to be compound heterozygous for two single -globin gene mutations, an aliquot of the remaining multiplex-PCR product was used directly for nondeletional multiplex minisequencing analysis. This was possible because the seven-deletion multiplex-PCR assay amplifies the 2-globin gene fragment whenever at least one -globin gene cluster is intact, i.e., not deleted (6).
Alternatively, to screen for only point mutations, the 2-globin gene was specifically amplified using primer pair -AF (5'-CCCCAAGCATAAACCCTGGC-3'; HUMHBA4 66306649) and 2-R(g/c) to generate an amplicon of 846 bp. Each 50-µL PCR reaction contained 200µM each deoxynucleotide triphosphate, 1x Q-solution (Qiagen), 1 U of HotStarTaq DNA polymerase in supplied reaction buffer (Qiagen), 100 ng of genomic DNA, and 0.2 µM each primer. Samples were amplified in a T3 thermal cycler (Biometra) with the following cycling condition: initial denaturation at 95 °C for 15 min, followed by 35 cycles of denaturation at 98 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min.
Excess PCR primers and unincorporated deoxynucleotide triphosphates in each PCR product were removed as described previously (9). To each tube of "purified" PCR product we added 1 µL of primer mixture (consisting of seven different mutation-specific primers), 2.5 µL of HPLC-grade water, and 2.5 µL of SNaPshotTM Multiplex ready reaction mixture (Applied Biosystems) containing AmpliTaq® DNA polymerase and fluorescently labeled dideoxynucleotide triphosphates. Each mutation-specific primer contained a 5' nonspecific polynucleotide tail that differed in length from the other primers (Table 1
) and was purified by polyacrylamide gel electrophoresis or HPLC.
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Each 10-µL multiplex minisequencing reaction mixture was subjected to 25 single-base extension cycles, followed by removal of unincorporated fluorescent dideoxynucleotide triphosphates, and multiplex minisequencing products were analyzed by automated capillary electrophoresis on an ABI PRISM® 3100 Genetic Analyzer, using GeneScanTM and GenotyperTM 3.7 application software (Applied Biosystems). The principle and protocols involved in liquid-phase multiplex minisequencing analysis have been described previously (9).
The assay results appeared as electropherograms, where the position of each peak indicated the migration of each terminator-extended primer in relation to the other primers because of their different primer lengths (Fig. 1
). The positions of the different primer peaks thus specified the mutation sites, whereas the peak color/fluorescence specified the genotype (for a color version of Fig. 1
, see the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue5/).
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In all instances, the assay could differentiate between heterozygosity for a mutation and homozygosity/hemizygosity, based on color and number of peaks (1 or 2) at each mutation site in the electropherogram. The GeneScan electropherograms displayed the expected wild-type and/or mutant peaks in the presence of the wild-type and/or mutant alleles, respectively, thus confirming the specificity of the assay (Fig. 1
, left panel). There were no detectable spurious "wild-type" peaks in the homozygous/hemizygous mutant samples, confirming the absence of dye-terminator misincorporation in this assay.
Automated allele-calling was achieved by creating a "macro" file to automatically label each peak when samples were analyzed with Genotyper 3.7 application software (Fig. 1
, right panel). We created labels to provide information on the mutation site, the nucleotide incorporated, and the wild-type or mutant status of the allele. Additionally, a tabulated report could be generated together with the Genotyper electropherogram results (data not shown). Automation of the data analysis function minimizes operator manipulations and can potentially reduce operator errors of data transcription.
Assay validation was accomplished by double-blind analysis of 45 -thalassemia patient samples and wild-type controls of known genotype: /- -SEA (n = 6), CS/- -SEA (n = 4), Ps/- -SEA (n = 4), QS/ (n = 4), CS/ (n = 3), QS/- -SEA (n = 2), Cd30/ (n = 2), /- -FIL (n = 2), Ps/ (n = 1), SD/- -SEA (n = 1), SD/ (n = 1), Cd0/- -SEA (n = 1), Cd0/ (n = 1), Cd30/- -SEA (n = 1), Cd59/ (n = 1), Cd59/- -SEA (n = 1), and / (n = 10). Samples were coded and assayed by different individuals, and results were scored independently by the person performing the assay and by a third individual. Each DNA sample was subjected to both the -thalassemia seven-deletion multiplex-PCR assay (6) and the 2-globin gene seven-mutation multiplex minisequencing assay. The genotypes scored by both individuals were completely concordant, and all 45 samples were correctly genotyped (data not shown). Because many of the genotypes were represented by only one sample, analysis of additional samples with these genotypes, as well as prospective analysis of Hb H disease samples, will be needed to verify the robustness of this assay.
This new assay complements an existing deletional multiplex-PCR assay that we developed previously to detect seven common -thalassemia deletions (6) and is intended to improve the overall mutation detection sensitivity for -thalassemia, especially for Hb H disease genotyping. Because deletions account for the vast majority of -thalassemia alleles, samples sent to the molecular diagnostic laboratory for -thalassemia genotyping should first be screened for common deletional mutations. If no deletions are found or only a single or double -globin gene deletion is identified in a patient suspected to carry additional mutations, the sample should then be further screened for the presence of nondeletional mutations. If the seven-deletion multiplex-PCR assay was used in the deletional analysis (6), the multiplex minisequencing assay can be performed directly on the PCR product of the deletional assay to screen for 2-globin gene mutations, with results obtained in less than 3.5 h. If a different deletional assay was performed or if only nondeletional mutations need to be screened, the 2-globin gene can be separately amplified for the multiplex minisequencing analysis.
The automated capillary electrophoresis and analysis allow diagnostic laboratories with moderate to high sample volumes to analyze up to 192 DNA samples in a 12-h period, requiring only a 50% effort by one technologist. This includes the 2-globin gene PCR (
3 h), PCR clean-up, multiplex minisequencing and post-minisequencing clean-up (3 h), capillary electrophoresis (25 min for 16 samples), and verification of automated genotyping results (5 min for 16 samples). Including all consumables for PCR amplification, multiplex minisequencing, and capillary electrophoresis, but excluding manpower and equipment amortization, this assay costs approximately US $2.90 per sample.
Acknowledgments
This work was supported by Grant NMRC/0365/1999 (Singapore) to S.S.C.
References
-thalassaemia deletions and -globin gene triplication by multiplex polymerase chain reactions. Br J Haematol 2000;108:295-299.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
-thalassemia. Blood 2001;98:250-251. thalassaemia. Br J Haematol 1999;104:513-515.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
-thalassemia mutations using a single-tube multiplex ARMS assay. Genet Test 2001;5:327-329.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
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