Multiplex Allele-specific PCR Assay Research Articles

A Rapid Molecular Detection System for Sdh Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Corynespora cassiicola.

Cucumber leaf spot, caused by Corynespora cassiicola, is a serious disease of cucumbers in greenhouses. Due to the frequent application of SDHIs, resistance caused by point mutations in the SDHB/C/D gene has been reported. Different mutations lead to different resistance levels, and mutations vary over time and regions. This means that it is necessary to know the type of mutation in the field to select the appropriate SDHIs. Here, the sensitivity of mutations to SDHIs was determined, and 8 resistance patterns were obtained: pattern I (BosVHR, FluoMR, PenHR, CarR); pattern II (BosMR, FluoSS, PenS, CarS); pattern Ⅲ (BosVHR, FluoSS, PenLR, CarS); pattern Ⅳ (BosLR, FluoLR, PenS, CarR); pattern Ⅴ (BosMR, FluoLR, PenS, CarS); pattern Ⅵ (BosMR, FluoLR, PenLR, CarS); pattern Ⅶ (BosVHR, FluoHR, PenHR, CarS); and pattern Ⅷ (BosLR, FluoLR, PenLR, CarS). We successfully established 9 AS-PCR assays that can detect mutation types. The sensitivity and specificity of AS-PCR were also determined. The sensitivity results showed that most of the detection thresholds of the AS-PCR assays were 100 pg/µL, while the AS-PCR assay of the B-H278R and D-G109V mutations exhibited high sensitivity, with 10 pg/µL. To validate the use of the developed AS-PCR assay, DNA from leaves inoculated with known mutations was extracted, detected by AS-PCR and sequenced. The results showed good similarity between the two methods. Additionally, to rapidly detect mutations in the CcSdhD gene, we developed a single-tube multiplex allele-specific PCR assay. In conclusion, AS-PCR and MAS-PCR were established for mutation detection and targeted control of CLS.

Development of Multiplex Allele-specific PCR Assay for BRAFV600E Mutation Detection in Human Cancer Cells

Development of Multiplex Allele-specific PCR Assay for BRAFV600E Mutation Detection in Human Cancer Cells

Improvement of New PCR Assay Targeting Mutations for Fast Recognition of Azole Resistant Mould Fungus, Aspergillus fumigatus

Allergenic mould fungus, Aspergillus fumigatus well known for its invasive aspergillosis disease is intrinsically resistant to most of the fluconazole group of medicines. It was studied that the mutations encoding 14a-sterol demethylase has the capacity to make triazole-resistant to this clinical fungal isolates. The present study is a multiplex allele-specific PCR assay targeting mutations for rapid detection of azole-resistant A. fumigatus for suitable patient management. During the present work clinical and wild isolates of A. fumigatus were employed to evaluate MAS-PCR, which was confirmed by direct DNA sequencing of cyp5 IA98 region from selected isolates of A. fumigatus. MAS-PCR assay was successfully developed to simplify the rapid detection of L98H mutation in cyp51A gene that confers the resistance to triazoles in A. fumigatus strains. The assay would help in primary acknowledgement of triazole-resistant A. fumigatus strains for proper management of patients with invasive aspergillosis.

Ancient hybridization and mtDNA introgression behind current paternal leakage and heteroplasmy in hybrid zones

Hybridization between heterospecific individuals has been documented as playing a direct role in promoting paternal leakage and mitochondrial heteroplasmy in both natural populations and laboratory conditions, by relaxing the egg-sperm recognition mechanisms. Here, we tested the hypothesis that hybridization can lead to mtDNA heteroplasmy also indirectly via mtDNA introgression. By using a phylogenetic approach, we showed in two reproductively isolated beetle species, Ochthebius quadricollis and O. urbanelliae, that past mtDNA introgression occurred between them in sympatric populations. Then, by developing a multiplex allele-specific PCR assay, we showed the presence of heteroplasmic individuals and argue that their origin was through paternal leakage following mating between mtDNA-introgressed and pure conspecific individuals. Our results highlight that mtDNA introgression can contribute to promote paternal leakage, generating genetic novelty in a way that has been overlooked to date. Furthermore, they highlight that the frequency and distribution of mtDNA heteroplasmy can be deeply underestimated in natural populations, as i) the commonly used PCR-Sanger sequencing approach can fail to detect mitochondrial heteroplasmy, and ii) specific studies aimed at searching for it in populations where mtDNA-introgressed and pure individuals co-occur remain scarce, despite the fact that mtDNA introgression has been widely documented in several taxa and populations.

Paternal leakage and mtDNA heteroplasmy in Rhipicephalus spp. ticks

Paternal leakage of mitochondrial DNA (mtDNA) and heteroplasmy have been recently described in several animal species. In arthropods, by searching in the Scopus database, we found only 23 documented cases of paternal leakage. Therefore, although arthropods represent a large fraction of animal biodiversity, this phenomenon has been investigated only in a paucity of species in this phylum, thus preventing a reliable estimate of its frequency. Here, we investigated the occurrence of paternal leakage and mtDNA heteroplasmy in ticks belonging to one of the most significant tick species complexes, the so-called Rhipicephalus sanguineussensu lato. By developing a multiplex allele-specific PCR assay targeting a fragment of the 12S rRNA ribosomal region of the mtDNA, we showed the occurrence of paternal leakage and mtDNA heteroplasmy in R. sanguineuss.l. ticks originated from experimental crosses, as well as in individuals collected from the field. Our results add a new evidence of paternal leakage in arthropods and document for the first time this phenomenon in ticks. Furthermore, they suggest the importance of using allele-specific assays when searching for paternal leakage and/or heteroplasmy, as standard sequencing methods may fail to detect the rare mtDNA molecules.

Abstract 643: An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of KRAS/BRAF/NRAS mutations in colorectal cancer

Abstract RAS and BRAF gene mutation assessment is essential for therapy decision in metastatic colorectal cancer patients with anti-EGFR monoclonal antibody treatment. However the currently approved assays in clinic is mainly focusing on tissue samples with detecting sensitivity lower than 1% in 10ng genomic DNA background. The emerging analysis of ctDNA offers advantages in clinical diagnosis as a feasible method, which requires much more sensitive methodology due to low abundance in plasma samples. Herein, we developed a multiplex allele-specific real-time (ARMS) PCR assay for detection fourteen mutations of KRAS/BRAF/NRAS in an 8-tube PCR strip. The reagent and format of mutation detection were particularly optimized to reach high specificity and sensitivity. Performance evaluation showed that the assay is remarkably sensitive with lower detection limit of 0.1% in a background of 10ng wild-type genomic DNA. A total of 136 CRC FFPE samples were screened by using the assay. Sixty-two samples were positive for KRAS/BRAF/NRAS mutations, and fifty-eight were confirmed by the Sanger sequencing. The four discrepant samples were tested further by ddPCR and the results revealed that the allelic frequencies in these samples were as low as 0.1% to 5%. The evaluation of ctDNA detection is being carried in metastatic colorectal cancer patients. The multiplex allele-specific real-time PCR assay provides a rapid, reliable and low-cost diagnostic tool for accurate detection of KRAS/BRAF/NRAS mutations for clinical application. Citation Format: Jianxin Zhai, Youmin Wu, Xuan Luo, Xinsheng Li, De-Hua Yu. An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of KRAS/BRAF/NRAS mutations in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 643.

Applied multiplex allele specific PCR to detect second-line drug resistance among multidrug-resistant tuberculosis in China

Rapid detection of resistance to the second-line drugs is essential for early initiation of appropriate anti-tubercular treatment regimen among multi-drug tuberculosis (MDR-TB). In this study, we applied a multiplex allele-specific PCR (MAS-PCR) to identify the mutations on codons 90 and 94 of gyrA and nucleotide 1401 of rrs for detecting ofloxacin (OFX) and kanamycin (KAN) resistance in 139 MDR-TB isolates from China. Using the traditional phenotypic method as the reference, MAS-PCR detected resistance to OFX and KAN with sensitivities of 67.3% and 76.5%, respectively, and specificities of 100.0%. Therefore, MAS-PCR assays can be used for rapid detection of second-line drug resistance among MDR-TB in China, enabling early administration of appropriate treatment regimens to the affected MDR-TB patients.

Abstract 5703: An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of ESR1 mutations in metastatic breast cancer

Abstract Activating mutations of estrogen receptor α (ESR1) are attributed to acquired resistance to estrogen-deprivation therapy in patients with metastatic breast cancer (MBC). Thus, detection of these mutations is clinically critical for monitoring early progression and effective therapeutic options in treatment of BC patients under hormonal therapy. However, current investigations for detection ESR1 mutations mainly focus on digital PCR technology due to low abundance of the cfDNA in plasma samples, which is not a currently feasible method for clinical diagnosis. Herein, we developed a multiplex allele-specific real-time (ARMS) PCR assay for detection of ESR1 four mutations (Y537S, Y537C, Y537N, and D538G) in a single tube reaction. In this real-time PCR system, the wild-type ESR1 DNA is completely blocked by a modified probe with a higher annealing temperature, and the mutant gene is selectively amplified by the modified ARMS primers. The assay was found to be highly specific and sensitive. With this assay, as low as 0.1% mutant DNA template in the background of a total of 10ng wild-type genomic DNA could be detected. By using this assay, we analyzed the ESR1 mutations in 50 Chinese MBC FFPE and plasma samples. The ESR1 mutations were detected in 3 FFPE and 6 plasma samples, which were confirmed by pyrophosphate sequencing. The multiplex allele-specific real-time PCR assay provides a rapid and reliable diagnostic tool for accurate detection of ESR1 mutations with potential for clinical application. Citation Format: Jiasu Liu, Yaohuai Wang, Shengrong Sun, Dehua Derek Yu. An ultra-sensitive multiplex allele-specific real-time PCR (Udx-PCR) assay for detection of ESR1 mutations in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5703. doi:10.1158/1538-7445.AM2017-5703

Molecular detection of drug resistance to ofloxacin and kanamycin in Mycobacterium tuberculosis by using multiplex allele-specific PCR

Drug resistance in tuberculosis (TB) is the biggest global health challenge as it hinders the tuberculosis control program and makes the disease more worsen. Molecular methods interrupt the spread of drug resistance by facilitating the appropriate anti- tuberculosis therapy at correct time through rapid diagnosis of multi drug resistant (MDR) and extensively drug resistant tuberculosis (XDR-TB). In this study we standardized and evaluated the diagnostic utility of multiplex allele specific PCR (MAS-PCR) targeting gyrA D94G and rrs A1401G mutations for detection of resistance against two key drugs (ofloxacin and kanamycin) of second line anti tuberculosis treatment. MAS-PCR assays targeting gyrA D94G and rrs A1401G for ofloxacin (OFL) and kanamycin (KAN) resistance respectively were carried out on 150 multidrug resistant isolates of Mycobacterium tuberculosis. The results were compared with phenotypic drug susceptibility test against ofloxacin and kanamycin by using proportion method on MGIT 960. Of 150 MDR isolates 50 were resistant to both ofloxacin and kanamycin, 36 were resistant to ofloxacin only, 8 were resistant to kanamycin only and 56 were susceptible to both the drugs. MAS-PCR correctly identified gyrA D94G and rrs A1401G mutations in phenotypically resistant isolates with a specificity of 100%. The sensitivity of MAS-PCR was 88.66%, 93.55% and 86% for OFL, KAN and XDR-TB respectively. There was no mutation detected at gyrA D94G region of 12.86% (11 of 86) OFL resistant isolates while 6.89% (4 of 58) of KAN resistant isolates did not carry rrs A1401G substitution. MAS-PCR proves to be a rapid tool for detection of drug resistance which could also be used as an initial marker for screening of XDR-TB.

Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays

ObjectivesNeither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. MethodsWe designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. ResultsThe technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. ConclusionsWe propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.

Evaluation of efficiency of nested multiplex allele-specific PCR assay for detection of multidrug resistant tuberculosis directly from sputum samples.

For an effective control of tuberculosis, rapid detection of multidrug resistant tuberculosis (MDR-TB) is necessary. Therefore, we developed a modified nested multiplex allele-specific polymerase chain reaction (MAS-PCR) method that enables rapid MDR-TB detection directly from sputum samples. The efficacy of this method was evaluated using 79 sputum samples collected from suspected tuberculosis patients. The performance of nested MAS-PCR method was compared with other MDR-TB detection methods like drug susceptibility testing (DST) and DNA sequencing. As rifampicin (RIF) resistance conforms to MDR-TB in greater than 90% cases, only the presence of RIF-associated mutations in rpoB gene was determined by DNA sequencing and nested MAS-PCR to detect MDR-TB. The concordance between nested MAS-PCR and DNA sequencing results was found to be 96·3%. When compared with DST, the sensitivity and specificity of nested MAS-PCR for RIF-resistance detection were determined to be 92·9 and 100% respectively. For developing- and high-TB burden countries, molecular-based tests have been recommended by the World Health Organization for rapid detection of MDR-TB. The results of this study indicate that, nested MAS-PCR assay might be a practical and relatively cost effective molecular method for rapid detection of MDR-TB from suspected sputum samples in developing countries with resource poor settings.

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

BackgroundPatients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.MethodsWe developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.ResultsOverall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.ConclusionThe MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

Automation Highlights from the Literature

Automation Highlights from the Literature

A multiplex allele-specific real-time PCR assay for screening of ESR1 mutations in metastatic breast cancer

BackgroundAcquired resistance to endocrine-based therapies occurs in virtually all estrogen receptor-α (ERα, encoded by ESR1) positive breast cancer patients. The underlying molecular mechanism is attributed to the activating mutations in ESR1. These mutations provide an exciting opportunity for the development of new antagonists that specifically inhibit the mutant proteins. Therefore, accurate detection of ESR1 mutations is of critical importance in clinical practice. Materials and methodsWe carried out a single tube, multiplex allele-specific real-time PCR assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N, and D538G). ResultsThe assay was found to be highly specific and sensitive. With this assay, as low as 1% mutant DNA template in wild type DNA could be detected. Fifteen DNA samples were prepared from archived formalin-fixed paraffin-embedded metastatic breast cancer biopsies. They were further screened with this assay, and three samples were identified as ESR1 mutant. The results were validated with pyrosequencing and complete concordance was observed between the two assays. ConclusionThe multiplex allele-specific real-time PCR assay provides a rapid and reliable diagnostic tool for accurate detection of ESR1 mutations. This procedure may be used in the clinical treatment of breast cancer.

Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR.

A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-μL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-μL human whole blood followed by amplifications of 59-bp β-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis has been implemented for Cystic Fibrosis (CF) carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD). Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM) curve analysis, allele-specific PCR (AS-PCR) and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.

KRAS screening for clinically important mutants via a multiplex allele-specific PCR assay for exons 2, 3, and 4.

e14569 Background: The selection of chemotherapies is guided by the analysis of molecular targets in solid tumors for well-known somatic mutations identified in microdissected formalin-fixed and pa...

Abstract 4217: A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC).

Abstract Somatic mutations in the PIK3CA gene encoding the p110α catalytic subunit of the PI3K protein have been linked to human cancer and have been found to occur at a high frequency in BC (25-40%). Therapeutics that target key nodes in the PI3K signaling pathway are currently being developed and stratifying patients based on their PIK3CA mutation status is becoming more important. We describe the analytical performance of the cobas® PIK3CA Mutation Test (For Research Use Only), a multiplex allele-specific PCR assay designed to detect 17 mutations in exons 1, 4, 7, 9 and 20 in the PIK3CA gene in 3 PCR wells. The test detects ∼94% of known PIK3CA mutations found in BC. The amount of DNA required for the assay (150 ng) can typically be isolated from a single 5 μm FFPET section with automated test results generated within 8 hours. The analytical sensitivity of the test, defined as ≥95% mutation detection rate, was found to be ∼5% mutant allele in BC FFPET tumor sections representing 4 prevalent PIK3CA mutations (H1047R, E545K, E542K, and Q546K). Additionally, the test was able to detect all 17 mutations at ≤ 5% mutant allele in plasmid/gDNA blends. The test was also compared to 2X bi-directional Sanger sequencing using 110 BC FFPET specimens. The PCR test detected mutations in 50 specimens that were Sanger (+) (100% positive agreement). Moreover, the test detected mutations in 4 Sanger (-) specimens and detected a second mutation in 1 Sanger (+) specimen that was not detected by Sanger. All 5 of the PCR (+)/Sanger (-) were confirmed mutant by 454 sequencing. Sanger Sequencing Mutation Detected Mutation Not Detected Total cobas® PIK3CA Mutation Test Mutation Detected 50* 4 54 Mutation Not Detected 0 56 56 Total 50 60 110 *One specimen had an additional mutation. When a panel of BC FFPET specimens was repeatedly tested over multiple days by 2 operators using 2 instruments and 2 reagent lots, a correct call was obtained in 99.5% (191/192) of tests. The results demonstrate that the cobas® PIK3CA Mutation Test is a fast, accurate and reproducible assay that is more sensitive than Sanger sequencing for the detection of PIK3CA mutations in BC tumors. Citation Format: Peter Schlag, Corey Hoeppner, Anona Bristol, Namneet Kaur, Stephen Quashnick, Ramani Ravirala, Joyce Kramer, Minet Negash, Victoria Brophy, Sung Lee, Stephen Soviero. A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2013-4217

Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the β-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in P. expansum from apple in Washington State are unknown. Determination of TBZ resistance level showed that all 102 TBZ-resistant (TBZ-R) isolates were highly resistant. Sequencing of the majority of the β-tubulin gene showed that 76 TBZ-R isolates harboured the E198V mutation and 26 harboured the F167Y mutation, and all the sensitive isolates did not possess any of the mutations, indicating that these two point mutations in the β-tubulin gene were correlated with TBZ resistance in P. expansum from apple in Washington State. There was no association between levels of TBZ resistance and types of point mutations (E198V or F167Y) in the β-tubulin gene. A multiplex allele-specific PCR assay was developed to detect these two mutations simultaneously. Microsatellite-primed PCR derived presence-absence matrix used to assess the genetic relationship among 56 isolates suggested that the resistance mutations originated several times independently and that there was no correlation between the types of point mutation and the genetic background of the isolates.

Molecular and hematological characteristics of a novel form of α-globin gene triplication: The hemoglobin St.Luke's-Thailand [α95(G2)Pro→Arg] or Hb St. Luke's [A2] HBA2

ObjectivesThis study aims to report a hitherto un-described α-globin gene triplicated allele with a novel hemoglobin (Hb) variant on α2-globin gene, Hb St.Luke's-Thailand [α95(G2)Pro-Arg]. Designs and methodsA study was done on a 1.5-year-old Thai boy who showed pallor and jaundice after birth. Hb analysis was performed by HPLC and capillary electrophoresis. cDNAs of the two α-globin genes were analyzed by sequencing and novel diagnostic methods based on PCR-RFLP and multiplex PCR assays were developed. ResultsHb analysis by both HPLC and capillary electrophoresis revealed unknown variants for Hb A (amounting to 9.0 and 9.4%) and Hb A2 (0.7 and 0.6%). cDNA analysis of α-globin genes revealed a normal α1-globin gene sequence and a CCG (Pro) to CGG (Arg) mutation at codon 95 of the α2-globin gene leading to the Hb St.Luke's-Thailand or Hb St. Luke's [A2] HBA2: c.287C>G. DNA analysis of the patient and his mother identified the in cis α-globin gene triplication. This novel orientation of α-globin gene could be confirmed by PCR-NlaIV restriction digestion or a multiplex allele specific PCR assay developed. ConclusionsA relatively lower percentage of the Hb St.Luke's-Thailand due to a HBA2 gene mutation could be explained by a linked anti-3.7 α-globin gene triplication. Clinical and hematological features, effect of α-globin gene triplication on the phenotypic expression of Hb St.Luke's-Thailand and diagnostics using combined HPLC, capillary electrophoresis and molecular techniques were presented.