Host genetic polymorphisms involved in long-term symptoms of COVID-19.

Host genetic polymorphisms are recognized as a critical determinant of diversity in clinical symptoms of Coronavirus disease 2019 (COVID-19). Accordingly, this study aimed to determine possible associations between single nucleotide polymorphisms (SNPs) in 37 candidate genetic variants and clinical consequences of COVID-19 - especially long-term symptoms, Long COVID. A total of 260 COVID-19 patients, divided into mild (n = 239) and severe (n = 21) and further categorized based on the presence of Long COVID (no, n = 211; yes, n = 49), were recruited. Genotyping of selected polymorphisms responsible for viral entry, immune response, and inflammation was performed using MassARRAY system. Out of 37 SNPs, 9 including leucine zipper transcription factor like-1 (LZTFL1) rs10490770 C allele, LZTFL1 rs11385942 dupA allele, nicotinamide adenine dinucleotide synthetase-1 (NADSYN1) rs12785878 TT genotype, plexin A-4 (PLXNA4) rs1424597 AA genotype, LZTFL1 rs17713054 A allele, interleukin-10 (IL10) rs1800896 TC genotype and C allele, angiotensin converting enzyme-2 (ACE2) rs2285666 T allele, and plasmanylethanolamine desaturase-1 (PEDS1) rs6020298 GG genotype and G allele were significantly associated with an increased risk of developing Long COVID, whereas interleukin-10 receptor subunit beta (IL10RB) rs8178562 GG genotype was significantly associated with a reduced risk of Long COVID. Kaplan-Meier curve displayed that the above gene polymorphisms were significantly associated with cumulative rate of Long COVID occurrence. Polymorphisms in LZTFL1 rs10490770, LZTFL1 rs11385942, LZTFL1 rs17713054, NADSYN1 rs12785878, PLXNA4 rs1424597, IL10 rs1800896, ACE2 rs2285666, PEDS1 rs6020298, and IL10RB rs8178562 appear to be genetic factors involved in development of Long COVID.

Association of HLA-A and HLA-B Alleles with Lamotrigine-Induced Cutaneous Adverse Drug Reactions in the Thai Population.

Background: Lamotrigine (LTG) is commonly used for treatment of epilepsy and bipolar disorder. It is one of the common cause of cutaneous adverse drug reactions (CADR). Clinical symptoms of LTG-induced CADR range from maculopapular exanthema (MPE) to severe cutaneous adverse reactions (SCAR). This study aimed to determine the association of the LTG-induced CADR with human leukocyte antigen (HLA) alleles in Thai patients. Methods: Fifteen patients with LTG-induced CADR [10 MPE; 4 Stevens-Johnson syndrome; and 1 drug reaction with eosinophilia and systemic symptoms] and 50 LTG-tolerant controls were included in the study. HLA-A and HLA-B genotyping was performed using polymerase chain reaction-sequence-specific oligonucleotides probes. Results: The proportion of HLA-A∗02:07 and HLA-B∗15:02 allele carriers were significantly higher in the LTG-induced CADR group than in the tolerant controls [odds ratio (OR): 7.83; 95% confidence interval (CI): 1.60-38.25; P = 0.013, and OR: 4.89; 95% CI: 1.28-18.67; P = 0.014]. In addition, subjects with HLA-A∗33:03, HLA-B∗15:02, and HLA-B∗44:03 were significantly higher in the LTG-induced MPE group than in the tolerant controls (OR: 8.27; 95% CI: 1.83-37.41; P = 0.005, OR: 7.33; 95% CI: 1.63-33.02; P = 0.005; and OR: 10.29; 95% CI: 1.45-72.81; P = 0.029). In contrast to the LTG-induced MPE group, there were no significant differences between HLA alleles and LTG-induced SCAR group. Conclusion:HLA-A∗02:07 and HLA-B∗15:02 were associated with LTG-induced CADR in Thai patients. We also identified an association between HLA-A∗33:03, HLA-B∗15:02, and HLA-B∗44:03 and LTG-induced MPE in this population. These results suggest that these alleles could be useful screening markers for preventing CADR before LTG treatment in Thai patients, but further replication studies with larger sample sizes are needed.

Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC-MS/MS: An application for phenotypic and genotypic correlations.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2→151.9 and the isotope 6-MMP-d3 with m/z of 170.5→152.2 were applied as an internal standard. The calibration curve covered the range of 2.5-360ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the inter-assay was less than 5.43%. This method developed for measuring TPMT by LC-MS/MS is a reliable, safe, and simple with a small volume requirement (100µl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9±12.47nmol 6-MMP/g Hb/h and 54.2nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance.

Development and Validation of Voriconazole Concentration by LC-MS-MS: Applied in Clinical Implementation.

BACKGROUND: Voriconazole (VRZ) is a triazole antifungal used for treatment of invasive fungal infection, which is a life-threatening condition. Therapeutic drug monitoring is recommended for identifying the optimal dose in patients who have hepatic/renal impairment or reduced function of the CYP2C19 metabolizing enzyme. METHODS: One hundred microliters of sample plasma was extracted by protein precipitated with 200 µl of acetonitrile containing fluconazole as internal standard (IS). After vortexing and centrifugation, supernatant was dried and reconstituted with 100 µl of mobile phase (ACN: 0.1% formic acid in 10 mM Ammonium acetate) (50:50 v/v) before injected. The column was C18, 2.7 µm, 3.0 × 50 mm at flow rate of 0.5 ml/min with retention time of 0.5 and 0.75 min for VRZ and IS, respectively. The tandem mass spectrometer was set in multiple reactions monitoring (MRM) mode with the following transition; VRZ m/z 350.10→281.10 and 307.20→220.20 (IS). RESULTS: The accuracy and precision inter- and intra-day were less than 9%, over the range 0.05-10 µg/ml. The linearity was consistent (r2 = 0.9987) and recovery was more than 85.0% for both analyses. CONCLUSION: This method is applicable for routine monitoring of patients' VRZ plasma level with fast and accurate runtime to assess CYP2C19 genotype.

Polymorphisms of the ApoE (Apolipoprotein E) gene and their influence on dyslipidemia in HIV-1-infected individuals.

The purpose of this retrospective case-control study was to investigate the frequency of Apolipoprotein E (ApoE) polymorphisms and their influence on antiretroviral therapy (ART)-induced lipodystrophy or dyslipidemia in HIV-infected Thai patients. The clinical characteristics and frequencies of ApoE genotypes were compared between the case (moderate to severe lipodystrophy, n = 67) and control (absent to mild lipodystrophy, n = 18) groups. The ApoE genotype frequencies among the 85 participants were 2.35% (n = 2) for E2/E2, 20% (n = 17) for E2/E3, 9.41% (n = 8) for E2/E4, 36.47% (n = 31) for E3/E3, 30.59% (n = 26) for E3/E4, and 1.18% (n = 1) for E4/E4. None of the ApoE genotypes showed association with ART-induced lipodystrophy. However, the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-cholesterol), and ApoB were lower in patients carrying the E2 allele but higher in E4 carriers. Interestingly, the ratios between TC and high-density lipoprotein (TC/HDL cholesterol ratio) and ApoB/ApoA-I ratio were significantly higher in the case group. Patients carrying the E2 allele displayed protective lipid profile, while those carrying E4 appeared to be at higher risk of dyslipidemia. In conclusion, ApoE polymorphisms were not associated with lipodystrophy in patients undergoing antiretroviral therapy but influenced lipid alteration.

Pharmacogenetics and clinical biomarkers for subtherapeutic plasma efavirenz concentration in HIV-1 infected Thai adults.

The aim of this study was to assess the influence of host genetic variations and clinical factors in relation to efavirenz level in HIV-1 infected Thai adults. A total of 100 HIV-infected subjects treated with efavirenz/lamivudine/tenofivir were prospectively enrolled. The panel of CYP2A6, CYP2B6 and CYP3A4/5 polymorphisms was genotyped. At steady state, plasma efavirenz concentrations were measured using high performance liquid chromatography. The relationship between host genetic and clinical factors in terms of efavirenz pharmacokinetics in HIV-1 infected Thai adults was analyzed. The minor allele frequency for CYP2A6 -48T>G, CYP2B6 g.18492T>C, CYP3A4*1B c.-392A>G, CYP3A4*18 c.878T>C and CYP3A5*3 c.6986A>G was 0.14, 0.27, 0.01, 0.03 and 0.38, respectively. Univariant and multivariant analysis indicated associations for CYP2B6 g.18492T>C (p < 0.001 and p = 0.001), aspartate aminotransferase (AST; p = 0.001 and p = 0.006) and blood urea nitrogen (BUN; p = 0.011 and p = 0.016) with plasma efavirenz concentration. However, CYP2A6 -48T>G, CYP3A4*1B c.-392A>G, CYP3A4*18 c.878T>C and CYP3A5*3 c.6986A>G had no significant impact on plasma efavirenz concentration in HIV-1 infected Thai adults. The CYP2B6 g.18492T>C polymorphism, AST and BUN were significantly associated with low efavirenz concentrations. The results from this study can be used to improve the prediction of efavirenz plasma concentration and to optimize its dose in antiretroviral therapy.

HLA-B allele and haplotype diversity among Thai patients identified by PCR-SSOP: evidence for high risk of drug-induced hypersensitivity.

BACKGROUND: There are 3 classes of HLA molecules; HLA class I, II and III, of which different classes have different functions. HLA-B gene which belongs to HLA class I play an important role predicting drug hypersensitivity. MATERIALS AND METHODS: Nine hundred and eighty-six Thai subjects who registered at a pharmacogenomics laboratory were determined for HLA-B genotype using a two-stage sequence-specific oligonucleotide probe system (PCR-SSOP). RESULTS: In this study, HLA-B alleles did not deviate from Hardy-Weinberg equilibrium (P > 0.05). The most common HLA-B alleles observed in this population were HLA-B (*) 46:01 (11.51%), HLA-B (*) 58:01 (8.62%), HLA-B (*) 40:01 (8.22%), HLA-B (*) 15:02 (8.16%) and HLA-B (*) 13:01 (6.95%). This finding revealed that HLA-B allele frequency in the Thai population was consistent with the Chinese population (p > 0.05), however, differed from the Malaysian population (p < 0.05). The top five HLA-B genotypes were HLA-B (*) 40:01/46:01 (2.13%), HLA-B (*) 46:01/46:01 (2.03%), HLA-B (*) 40:01/58:01 (2.03%), HLA-B (*) 46:01/58:01 (1.93%) and HLA-B (*) 15:02/46:01 (1.83%). This study found that 15.92% of Thai subjects carry HLA-B (*) 15:02, which has been associated with carbamazepine-induced severe cutaneous adverse drug reactions (SCARs). Moreover, 16.33% of Thai subjects carry the HLA-B (*) 58:01 allele, which has been associated with allopurinol-induced SCARs. CONCLUSION: This study demonstrates a high diversity of HLA-B polymorphisms in this Thai population. The high frequency of HLA-B pharmacogenomic markers in the population emphasizes the importance of such screening to predict/avoid drug hypersensitivity.

CYP2C19 polymorphisms in the Thai population and the clinical response to clopidogrel in patients with atherothrombotic-risk factors.

Genetic variation in the cytochrome P450 2C19 (CYP2C19) gene has been documented gradually as the determinant conversion and variability in the antiplatelet effect of clopidogrel. The aims of this study were to determine the prevalence of clinically relevant allele variants (CYP2C19*2, CYP2C19*3, and CYP2C19*17) in a Thai study population, and finally determine whether the allele distributes and predicts metabolic phenotypes in clopidogrel treated patients. A total of 1,051 Thai patients participated in this study. Genotypes for CYP2C19 polymorphisms were detected by the microarray-based technique. Furthermore, results of genotyping and platelet aggregation in 96 cardiovascular disease patients on 75 mg clopidogrel maintenance daily dose therapy also were analyzed. Among 1,051 samples, the allele frequencies of CYP2C19 *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *1/*17 were found in 428 (40.72%), 369 (35.10%), 72 (6.85%), 77 (7.32%), 59 (5.61%), and 45 (4.30%) of the patients, respectively. Homozygous CYP2C19 *3/*3 was found in one patient (0.10%). Therefore, 40.72% of the patients were predicted as extensive metabolizers, 41.95% as intermediate metabolizers, 13.03% as poor metabolizers, and 4.30% as ultra-rapid metabolizers. Among 96 patients, the frequency of poor metabolizers was significantly higher in the clopidogrel non-responder group than in the responder group (36.0% and 15.5%, respectively, P = 0.03). CYP2C19*1/*17 was observed in responders (n = 2; 2.8%). As a result, CYP2C19 variants were associated with clopidogrel non-responders. However, there is a need for further elucidation of the clinical importance and use of this finding to make firm and cost-effective recommendations for drug treatment in the future.

Small-dense LDL cholesterol/large-buoyant LDL cholesterol ratio as an excellent marker for indicating lipodystrophy in HIV-infected patients.

OBJECTIVES: To examine whether the lipid parameters are predicting factors for human immunodeficiency virus (HIV)-associated lipodystrophy. METHODS: Whole-body fat compositions of HIV-positive patients receiving stavudine-containing antiretroviral regimens (n = 79) were determined. Lipodystrophy was defined as a ratio of trunk fat mass/lower limb fat mass greater than 2.28. Blood samples were analyzed for total cholesterol (TC), triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), small-dense LDL-C (sdLDL-C), apoAI, apoB, lipoprotein(a), and CD4 cell counts. Large-buoyant LDL-C (lbLDL-C) was calculated (LDL-C minus sdLDL-C). RESULTS: Twenty-six patients were classified as having lipodystrophy. The mean values of triglycerides, HDL-C, sdLDL-C, apoB, TC/HDL-C, apolipoprotein (apo) B/apoAI, and sdLDL-C/lbLDL-C showed significant differences between patients with and without lipodystrophy (P < .02). Using logistic regression analysis, sdLDL-C/lbLDL-C was identified as a significant predictor of lipodystrophy (P < .001). At a ratio of 0.554, the odds ratio was 17.8 with a likelihood ratio of 5.5. CONCLUSIONS: The sdLDL-C/lbLDL-C ratio is an excellent marker for indicating lipodystrophy in HIV-infected patients.

High plasma efavirenz concentration and CYP2B6 polymorphisms in Thai HIV-1 infections.

  Efavirenz is mainly metabolized by cytochrome P450 2B6 (CYP2B6). This study aimed to examine the frequencies of CYP2B6 and the association between CYP2B6 polymorphisms and plasma efavirenz concentrations in an HIV-1 infected Thai population. Mid-dose plasma efavirenz concentration was determined at 12 weeks following the initiation of an antiretroviral therapy (tenofovir, lamivudine and efavirenz) in 100 Thai adults with HIV-1 infection using high-performance liquid chromatography. Candidate CYP2B6 polymorphisms (c.64C>T, c.499C>G, c.516G>T, c.785A>G, c.1375A>G, c.1459C>T) were conducted by real-time PCR-based allelic discrimination. The most frequent polymorphisms among this cohort were the CYP2B6 c.785A>G and c.516G>T, which had a frequency of 0.36 and 0.32, respectively. From the cases observed, two single nucleotide polymorphisms (SNPs) (c.516G>T and c.785A>G) were significantly associated with high efavirenz plasma levels (p < 0.05). The most frequent haplotypic combinations were *1/*6, *1/*1, *1/*2 and *6/*6 at a frequency of 42.0%, 32.0%, 8.0% and 7.0%, respectively. Increased plasma concentrations of efavirenz were present in individuals with CYP2B6 *6/*6 [7.210 mg/L; interquartile range (IQR), 5.020-9.260] when compared to those with CYP2B6*1/*1 (1.570 mg/L; IQR, 1.295-2.670), p < 0.001. In our study, the impact of SNPs which are correlated with a high level of efavirenz plasma concentrations was found. The genetic configuration of SNPs which are associated with high plasma efavirenz levels may be useful in optimizing the efavirenz dose that is used in HIV-1 infected patients.