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High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females.

ABSTRACT: BACKGROUND:Like glucose-6-phosphate dehydrogenase (G6PD) deficient hemizygous males and homozygous females, heterozygous females could also manifest hemolytic crisis, neonatal hyperbilirubinemia or kernicterus upon exposure to oxidative stress induced by certain foods such as fava beans, drugs or infections. Although hemizygous males and homozygous females are easily detected by conventional G6PD enzyme assay method, the heterozygous state could be missed by the conventional methods as the mosaic population of both normal and deficient RBCs circulates in the blood. Thus the present study aimed to apply high resolution melting (HRM) curve analysis approach to see whether HRM could be used as a supplemental approach to increase the chance of detection of G6PD heterozygosity. RESULTS:Sixty-three clinically suspected females were evaluated for G6PD status using both enzyme assay and HRM analysis. Four out of sixty-three participants came out as G6PD deficient by the enzyme assay method, whereas HRM approach could identify nine participants with G6PD variants, one homozygous and eight heterozygous. Although only three out of eight heterozygous samples had G6PD enzyme deficiency, the HRM-based heterozygous G6PD variants detection for the rest of the samples with normal G6PD enzyme activities could have significance because their newborns might fall victim to serious consequences under certain oxidative stress. CONCLUSIONS:In addition to the G6PD enzyme assay, HRM curve analysis could be useful as a supplemental approach for detection of G6PD heterozygosity.

SUBMITTER: Islam MT

PROVIDER: S-EPMC6086071 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females.

Islam Md Tarikul MT Sarker Suprovath Kumar SK Talukder Shezote S Bhuyan Golam Sarower GS Rahat Asifuzzaman A Islam Nafisa Nawal NN Mahmud Hasan H Hossain Mohammad Amir MA Muraduzzaman A K M AKM Rahman Jakia J Qadri Syeda Kashfi SK Shahidullah Mohammod M Mannan Mohammad Abdul MA Tahura Sarabon S Hussain Manzoor M Saha Narayan N Akhter Shahida S Nahar Nazmun N Begum Firoza F Shirin Tahmina T Akhteruzzaman Sharif S Qadri Syed Saleheen SS Qadri Firdausi F Mannoor Kaiissar K

BMC genetics 20180810 1

<h4>Background</h4>Like glucose-6-phosphate dehydrogenase (G6PD) deficient hemizygous males and homozygous females, heterozygous females could also manifest hemolytic crisis, neonatal hyperbilirubinemia or kernicterus upon exposure to oxidative stress induced by certain foods such as fava beans, drugs or infections. Although hemizygous males and homozygous females are easily detected by conventional G6PD enzyme assay method, the heterozygous state could be missed by the conventional methods as t ...[more]

PMID: 30097005

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Project description:Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP.Intra-assay (n=5) precision of Tms for all SNPs was ?0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/?L.The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

Project description:The emergence of Mycobacterium abscessus complex (MABC) infection is the most noteworthy health care problem. Clarithromycin (CLA) and amikacin (AMK) constitute the cornerstone of treatment for patients infected with MABC; thus, early detection of resistance to these two drugs is essential for formulating effective therapeutic regimens. In the present study, we aimed to validate the use of MeltPro MAB assay, a melting curve analysis with dually labeled probes, on a set of clinical isolates to detect CLA and AMK resistance. A total of 103 clinical MABC strains were collected in our analysis, including 76 strains of M. abscessus subsp. Abscessus (MAA) and 27 strains of M. abscessus subsp. Massiliense (MAM). In vitro susceptibility testing revealed that two isolates exhibited intrinsic CLA resistance by harboring A2270T mutation in rrl, and inducible resistance was noted in 42 isolates. Additionally, two MAA isolates with erm(41)T28 genotype were susceptible to CLA. Notably, we found three out of 44 isolates had two melting curve peaks, representing the simultaneous presence of mutant and the wild type in these specimens. In contrast, no known mutations were identified in six AMK-resistant isolates. Further analysis revealed that MeltPro yielded 100% and 96.67% sensitivity and specificity for detecting CLA resistance. In summary, this study firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates, which significantly improves the turnaround time within 2 h. Approximate two fifths of MABC isolates are resistant to CLA by 23S rRNA mutation or its methylation, emphasizing the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections. IMPORTANCE Mycobacterium abscessus complex (MABC) has attracted increasing attention due to the numerous cases of infection. This pathogen is notorious for its intrinsic drug resistance, which complicates clinical management of patients with MABC infections. Clarithromycin (CLA) and amikacin (AMK) are the cornerstone of treatment regimens for MABC. Herein, our data firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates. The high frequency of CLA-resistant MABC isolates in China emphasizes the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections.

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High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females.

Publications

High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females.

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