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Human b -globin gene sequence and primer locations. The mutation sites are marked in capital letters and red font . The shaded DNA sequences indicate primers. Assignments are shown in the right column and shaded in the same color as the relevant primer. Overlapping sequences are shaded in pink . The sequences for PCR amplification are shaded in cyan
Source publication
Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HB...
Contexts in source publication
Context 1
... former issue pre- sents a specific challenge because highly multiplex genotyping parameters may be difficult to optimize. For HBB, as shown in Fig.1, two sets of clustering poly- morphic sites, CD27/28 +C versus CD17 A>T, and CD43 G>T versus CD41/42 -TCTT, are in close proximity to each other and almost overlap. ...
Context 2
... efficiency of primer extension could also be caused by off-target annealing ( Su et al. 2003). This is evidenced in the case of CD41/42 -TCTT and CD43 G > T, which are only 5 bps apart, and their probing primers are partially overlapping (Fig.1). When used in multiplex primer extension reactions, the yield from these primers was low. ...
Similar publications
This report details the work performed over a period of five years in an NIJ-funded project intended primarily to develop a practical means for accessing the large reserve of genetic variation in the ~15,000 bp mtDNA coding region, as a means for augmenting the forensic discrimination provided by sequencing the hypervariable (HV) regions of the mtD...
Citations
... Routine multiplex PCR and PCR-RDB methods are low-throughput and cumbersome. Recently, MALDI-TOF-MS has been used to detect SNVs of HBA and HBB with the traditional SBE strategy: an assay for 21 HBB mutations was developed by dividing the extension probes into three reaction systems (Thongnoppakhun et al. 2009), a multiplex assay for 27 β-thalassemia mutations was performed in six reaction systems (Looi et al. 2011), and a single-tube assay for 8 β-thalassemia mutations was reported (Liao et al. 2005). These traditional SBEbased assays require multiple reaction tubes to avoid the interference of clustered SNVs, or only analyzed a limited number of independent mutations. ...
Single-nucleotide variants (SNVs) and copy number variations (CNVs) are the most common genomic variations that cause phenotypic diversity and genetic disorders. MALDI–TOF–MS is a rapid and cost-effective technique for multi-variant genotyping, but it is challenging to efficiently detect CNVs and clustered SNVs, especially to simultaneously detect CNVs and SNVs in one reaction. Herein, a novel strategy termed Target-Allele-Specific Probe Single-Base Extension (TASP-SBE) was devised to efficiently detect CNVs and clustered SNVs with MALDI–TOF–MS. By comprehensive use of traditional SBE and TASP-SBE strategies, a MALDI–TOF–MS assay was also developed to simultaneously detect 28 α-/β-thalassemia mutations in a single reaction system, including 4 α-thalassemia deletions, 3 HBA and 21 HBB SNVs. The results showed that all 28 mutations were sensitively identified, and the CNVs of HBA/HBB genes were also accurately analyzed based on the ratio of peak height (RPH) between the target allele and reference gene. The double-blind evaluation results of 989 thalassemia carrier samples showed a 100% concordance of this assay with other methods. In conclusion, a one-tube MALDI–TOF–MS assay was developed to simultaneously genotype 28 thalassemia mutations. This novel TASP-SBE was also verified a practicable strategy for the detection of CNVs and clustered SNVs, providing a feasible approach for multi-variants analysis with MALDI–TOF–MS technique.
... As a kind of high-throughput detection instrument, MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) is playing an increasingly important role in clinical chemistry [14]. Though it has been used for the detection of mutations in thalassaemia patients [15][16][17], sample pre-treatment (DNA extraction and PCR) is still timeconsuming. Direct detection of the intact haemoglobin chains in untreated blood samples is an efficient way to increase the throughput of MALDI-TOF MS. ...
Background
Thalassaemia is one of the most common inherited monogenic diseases worldwide with a heavy global health burden. Considering its high prevalence in low and middle-income countries, a cheap, accurate and high-throughput screening test of thalassaemia prior to a more expensive confirmatory diagnostic test is urgently needed.
Methods
In this study, we constructed a machine learning model based on MALDI-TOF mass spectrometry quantification of haemoglobin chains in blood, and for the first time, evaluated its diagnostic efficacy in 674 thalassaemia (including both asymptomatic carriers and symptomatic patients) and control samples collected in three hospitals. Parameters related to haemoglobin imbalance (α-globin, β-globin, γ-globin, α/β and α-β) were used for feature selection before classification model construction with 8 machine learning methods in cohort 1 and further model efficiency validation in cohort 2.
Results
The logistic regression model with 5 haemoglobin peak features achieved good classification performance in validation cohort 2 (AUC 0.99, 95% CI 0.98–1, sensitivity 98.7%, specificity 95.5%). Furthermore, the logistic regression model with 6 haemoglobin peak features was also constructed to specifically identify β-thalassaemia (AUC 0.94, 95% CI 0.91–0.97, sensitivity 96.5%, specificity 87.8% in validation cohort 2).
Conclusions
For the first time, we constructed an inexpensive, accurate and high-throughput classification model based on MALDI-TOF mass spectrometry quantification of haemoglobin chains and demonstrated its great potential in rapid screening of thalassaemia in large populations.
Key messages
Thalassaemia is one of the most common inherited monogenic diseases worldwide with a heavy global health burden.
We constructed a machine learning model based on MALDI-TOF mass spectrometry quantification of haemoglobin chains to screen for thalassaemia.
... Various methods for screening of the known causative mutations of β-thalassemia have been reported, including amplification refractory mutation system (ARMS) (3), allele-specific oligonuleotide (ASO) blot (4), reverse dot blot (RDB) (5), microarrays (6), fully denaturing HPLC with primer extension (7), ligase chain reaction (LCR) (8), pyrophosphorolysis-activated polymerization (PAP) technology (9), microtiter well-based chemiluminometric genotyping assay (10), and high-throughput systems based on matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (11). However, none appears ideal for rapid screening of a large number of samples. ...
Backgrounds:
β-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the β-globin gene. In Tunisia, β-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole β-globin gene (HBB) is screened for mutations covering about 90% of the spectrum.
Methods:
We have performed the validation of a DHPLC assay for direct genotyping of 11 known β-thalassemia mutations in the Tunisian population.
Results:
DHPLC assay was established based on the analysis of 62 archival β-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study.
Conclusion:
Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known β-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals.
... Because of the minimal DNA amount requirements and short amplicon length, even formalin-fixed and paraffin-embedded tissue or ancient DNA samples can be processed. Successful applications of this system in the fields of forensics, oncology, pharmacogenetics, and haematology, including βthalassemia genotyping have been previously reported [38,[45][46][47][48]. Additionally, MALDI-TOF MS has been successfully employed in genotyping foetal, paternally inherited SNPs [49]. ...
... When the primers are well designed to achieve a good separation of the primer and the extension products in a mass spectrum, the genotyping assays can be combined to perform up to 15-fold multiplex SNP analysis [62,63]. The single base primer extension assay can be applied to diagnosis and screening of hereditary diseases such as cystic fibrosis and betathalassemia [64,65]. In fact, any diseases/pathological conditions that are associated with mutations in a specific gene or a specific set of genes can be easily identified and quantified by this method. ...
The concept of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was first reported in 1985. Since then, MALDI MS technologies have been evolving, and successfully used in genome, proteome, metabolome, and clinical diagnostic research. These technologies are high-throughput and sensitive. Emerging evidence has shown that they are not only useful in qualitative and quantitative analyses of proteins, but also of other types of biomolecules, such as DNA, glycans, and metabolites. Recently, parallel fragmentation monitoring (PFM), which is a method comparable to selected reaction monitoring, has been reported. This highlights the potentials of MALDI-TOF/TOF tandem MS in quantification of metabolites. Here we critically review the applications of the major MALDI MS technologies, including MALDI-TOF MS, MALDI-TOF/TOF MS, SALDI-TOF MS, MALDI-QqQ MS, and SELDI-TOF MS, to the discovery and quantification of disease biomarkers in biological specimens, especially those in plasma/serum specimens. Using SELDI-TOF MS as an example, the presence of systemic bias in biomarker discovery studies employing MALDI-TOF MS and its possible solutions are also discussed in this chapter. The concepts of MALDI, SALDI, SELDI, and PFM are complementary to each other. Theoretically, all these technologies can be combined, leading to the next generation of the MALDI MS technologies. Real applications of MALDI MS technologies in clinical diagnostics should be forthcoming.
... Due to the short amplicon length and minimal DNA amount requirements, even formalin-fixed and paraffin-embedded tissue derived or ancient DNA samples can be processed. The system has been used successfully for instance in forensics, oncology, pharmacogenetics, and hematology, including βthalassemia genotyping [22][23][24][25][26][27][28][29][30]. Non-Mendelian mixtures of DNAs, eg, fetal DNA in maternal plasma, or somatically mutated DNA in cancerous tissue are technically challenging to detect due to the preponderance of maternal or wild-type sequences, respectively. ...
Although matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) has previously been reported for high throughput blood group genotyping, those reports are limited to only a few blood group systems. This review describes the development of a large cooperative Swiss-German project, aiming to employ MALDI-TOF MS for the molecular detection of the blood groups Rh, Kell, Kidd, Duffy, MNSs, a comprehensive collection of low incidence antigens, as well as the platelet and granulocyte antigens HPA and HNA, representing a total of 101 blood group antigens, encoded by 170 alleles, respectively. Recent reports describe MALDI-TOF MS as a technology with short time-to-resolution, ability for high throughput, and cost-efficiency when used in genetic analysis, including forensics, pharmacogenetics, oncology and hematology. Furthermore, Kell and RhD genotyping have been performed on fetal DNA from maternal plasma with excellent results. In summary, this article introduces a new technological approach for high throughput blood group genotyping by means of MALDI-TOF MS. Although all data presented are preliminary, the observed success rates, data quality and concordance with known blood group types are highly impressive, underlining the accuracy and reliability of this cost-efficient high throughput method.
... For example, allele-specific arrayed primer extension has been designed for simultaneous detection of 15 nondeletion ␣-globin gene defects and 23 -globin gene mutations commonly observed in countries in southeast Asia. 25 Multiplex minisequencing has also been developed in combination with subsequent detection using a variety of platforms including capillary electrophoresis, 26 denaturing high-performance liquid chromatography, 27,28 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 29,30 However, these high-end techniques are best suited for use in a well-equipped central laboratory because of their expense and the high level of expertise required. ...
The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis-based genotyping assay (MeltPro HBB assay) for 24 β-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of β-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 β-thalassemia mutations were accurately genotyped, and β-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple β-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost.
... Increasingly multiplex PE combined with MALDI-TOF MS has been applied to genotype mutations or SNPs in variety of genes. 19,[22][23][24]32,34,35 Solid phase capturable ddNTPs in SBE have been developed using biotinylated ddNTPs to generate 3-biotinylated extension DNA products, which then are purified by streptavidin-coated magnetic beads before analysis by MS. 27 This solid phase capturable-SBE method has been applied for simultaneous genotyping of 17 Y-chromosome SNPs, 36 detection of 30 point mutations in p53 in a single tube, 37 and for concurrent analysis of 40 SNPs of CYP2C9 and 50 SNPs of CYP2A13 genes. 38 However, the cost-effectiveness of this approach has not been evaluated. ...
A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous beta-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.
... The MDM2 SNP 309 was determined according to a MALDI-TOF method as described. 18,19 The primer sets used for PCR amplification of the MDM2 SNP 309 region are as described by Bond et al. 8 Briefly, 0.1 lg genomic DNA was amplified by PCR in a final volume of 25 lL following the protocol described by Bond et al. 8 for 30 cycles (1 min at 95°C, 1 min at 62°C, and 1 min at 72°C) using a Mastercycler Ò gradient (Eppendorf AG, Hamburg, Germany). The unincorporated dNTPs and primers were removed automatically via MAPIIA (GenePure PCR purification system, Bruker, Bremen, Germany). ...
The recently identified single nucleotide polymorphism in the MDM2 promoter (SNP 309) may contribute to the early onset of both sporadic and hereditary malignancies in patients with defective p53. We tested this hypothesis by examining the effects of combined MDM2 polymorphisms and somatic p53 mutations on 351 oral squamous cell carcinomas (OSCCs) associated with areca quid chewing. We found that the G allele of MDM2 SNP 309 was associated with early age of onset (p=0.02) and poor differentiation of OSCC tumors (p=0.01). The frequency of lymph node extracapsular spread (LNECS) was increased in individuals having both the MDM2 SNP 309 GG genotype and p53 mutation (chi(2) for trend p=0.04). MDM2 GG genotype and p53 mutations were associated with poor disease-free survival in both early and lymph node positive advanced stage OSCC patients (Hazard ratio=2.77 and 1.93, respectively). Taken together, an interaction between MDM2 SNP 309 and p53 with respect to tumor behaviors (including disease onset, tumor differentiation, LNECS and disease-free survival) was observed in sporadic Taiwanese OSCCs.
... In the Southeast Asian population, the common beta-thalassemia mutations include c.-78 A>G, c.2 T>A, c.52 A>T, c.84_85 insC, c.125_128 delTCTT, c.130 G>T, c.216_217 insA, and c.316-197 C>T [4]. A wide variety of methods for genotyping the HBB gene have been developed using different detection techniques based on different principles [5], including allele-specific oligonucleotide hybridization (ASO) [6], amplification refractory mutation system (ARMS) [7], allele-specific PCR [8], reverse dot blot [9], restriction fragment length polymorphism (RFLP) [10], single base extension (SBE) [11,12], and others1314151617. These methods, however, are limited to the study of hotspot or known mutations. ...
Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB), of which more than 200 are associated with a beta-thalassemia phenotype.
We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample.
Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.
