Genotype Subgroups Research Articles

Development of rapid and efficient enterovirus typing and classification platform for real-time diagnostics

Enteroviruses are highly prevalent among children and adults worldwide. Accurate detection and genotyping of enteroviruses can be achieved using consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction (PCR)-based methods. Nevertheless, the popularized use of such methods for enterovirus detection and genotyping is limited by their relatively long processing time (>2 days), high processing cost, and the requirement for specialized and expensive instrumentation. Here, we report a relatively simple asymmetric PCR-electrochemical biosensing method that can detect enteroviruses and accurately characterize them into four distinct genotypic subgroups in less than 2 h. Specifically, genes of interest in the enteroviruses were amplified via asymmetric PCR (asy-PCR) using the designed PCR primers. The asy-PCR products were hybridized with enterovirus genotype-specific single-stranded DNA primer probes. Changes in response to electrochemical impedance signals can rapidly clarify the genotypes of enteroviruses. We observed that this method could not only effectively amplify the target gene within minimal PCR-amplified cycles of consensus-degenerate hybrid oligonucleotide (COHOP) products from the specimen but also allowed the rapid differentiation of the four subgroups of enteroviruses in 2 h. In summary, we developed an efficient and rapid electrochemical biosensing platform for enterovirus subtyping diagnostics that can be applied to other emergent infectious diseases.

Genetic Polymorphisms in Exon 5 and Intron 5 and 7 of AIRE Are Associated with Rheumatoid Arthritis Risk in a Hungarian Population.

Rheumatoid arthritis (RA) is chronically persistent synovitis and systemic inflammation. Although multiple contributors are detected, only one is pivotal in the neonatal period: the negative selection of autoimmune naïve T-cells by the autoimmune regulator (AIRE) transcriptional factor. Single-nucleotide polymorphisms (SNPs) in the DNA-binding site of AIRE may determine its function and expression. We intended to analyse site-specific allelic polymorphisms in two exon (rs878081 and rs1055311) and three intron (rs1003853, rs2075876, and rs1003854) loci with an RA risk. Our analytical case-control study analysed 270 RA patients and 322 control subjects in five different genetic models using quantitative real-time PCR (qPCR) with TaqMan® assays. Statistically significant differences were found between the odds of allelic polymorphisms in the loci of rs878081, rs1003854, and rs1003853 among the controls and RA patients, and the disease activity seemed to be significantly associated with the genotypic subgroups of rs878081 and rs1055311. Our in silico analysis supported this, suggesting that allele-specific alterations in the binding affinity of transcriptional factor families might determine RA activity. Our findings highlight the involvement of neonatal self-tolerance in RA pathogenesis, providing novel insights into disease development and paving the way for an analysis of further site-specific genetic polymorphisms in AIRE to expand the intervention time for RA.

Survival in a Real-World Cohort of Patients With Transthyretin Amyloid Cardiomyopathy Treated With Tafamidis: An Analysis From the Transthyretin Amyloidosis Outcomes Survey (THAOS)

In the pivotal Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT), tafamidis significantly reduced mortality rates, leading to its approval in many countries for the treatment of transthyretin amyloid cardiomyopathy (ATTR-CM). Real-world evidence on survival in patients with ATTR-CM following tafamidis treatment has not been extensively reported. The Transthyretin Amyloidosis Outcomes Survey (THAOS) was a longitudinal, observational, phase 4 study of patients with transthyretin amyloidosis and asymptomatic participants carrying pathogenic transthyretin variants. Patients from THAOS with a predominantly cardiac phenotype at enrollment were included, and survival was analyzed according to tafamidis treatment status (treated or untreated). Results are based on the completed THAOS dataset. In tafamidis-treated (n = 587) and tafamidis-untreated (n = 854) patients, respectively, median age at enrollment was 77.7 and 76.4 years, 91.8% and 90.0% were male, and 91.8% and 83.8% had wild-type disease. Survival rates (95% CI) at 30 and 42 months, respectively, were 84.4% (80.5-87.7) and 76.8% (70.9-81.7) in tafamidis-treated patients, and 70.0% (66.4-73.2) and 59.3% (55.2-63.0) in tafamidis-untreated patients. Survival rates in genotype subgroups (wild-type and variant) were similar to those of the overall cohort. Survival rates were better in a contemporary cohort, as reflected by a sensitivity analysis performed in patients enrolled after vs before 2019. No new safety signals were identified. In this real-world cohort of patients with ATTR-CM, survival rates were higher than in ATTR-ACT and consistent with more recent reports, suggesting early diagnosis and treatment with tafamidis has improved life expectancy in ATTR-CM. These results provide further evidence supporting tafamidis' safety and effectiveness. ClinicalTrials.gov identifier: NCT00628745.

Retrospective analysis: Does 5-FU dose adjustment per DPYD mutation status affect toxicity?

11172 Background: 5-Fluorouracil (5-FU) forms a cornerstone in neoadjuvant chemotherapy (NACT) regimens for solid tumors including head and neck cancers. The DPYD gene encodes for enzyme dihydropyrimidine dehydrogenase (DPD) which catalyses an essential, rate-limiting step in the metabolism of 5-FU. Heterozygous genomic alteration of DPYD (30-35% of population) result in reduced potency of DPD enzyme, while homozygous DPYD alteration (0.1-0.2% of population) results in no functional DPD. Reduced functioning of DPD leads to slower metabolism of 5-FU causing toxicity such as bone marrow suppression, cytopenias etc. The Clinical Pharmacogenetics Implementation Consortium (CPIC) have released guidelines recommending reducing the dose of 5-FU in patients with these genomic alterations of DPYD from 25% to avoiding its use based on a calculated activity score (DPYD-AS). Methods: A phase III randomized study compared a DC vs DCF regimen as NACT in technically unresectable oral cancer was published in March 2024. We utilised data from the ‘DCF’ arm of this study. Patients underwent DPYD testing and we stratified these patients based on DPYD status. In the study, patients underwent the appropriate dose reductions per the CPIC guidelines based on DPYD-AS scores. We evaluate the frequency and degree of adverse events in these groups to determine whether this dose reduction effectively reduces toxicity from 5-FU in these patients. Adverse events were graded per CTCAE. Analysis by Fishers exact test. Results: 247 patients in the DCF arm were included. 85 (34.4%) did not undergo DPYD testing. 118 (47.8%) did not have any DPYD mutation, 44 (17.8%) had heterozygous mutation while 0 had homozygous mutation. The adverse events by DPYD genotypic subgroup are shown in the table. Conclusions: There was no statistically significant difference in adverse events between the group with no DPYD mutation who received the full dose of 5-FU and the heterozygous DPYD mutation group receiving a lower 5-FU dose based on DPYD-AS score. Thus, the dose reduction as set out by the CPIC appears to appropriate. [Table: see text]

Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.

The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Genotyping of Croatian Olive Germplasm with Consensus SSR Markers

Leaf samples of 226 cultivated olive trees were collected from traditionally managed olive orchards and genotyped with eleven consensual SSR markers. The proportion of shared allele distance was used for the estimation of distances between olive genotypes. Cluster analyses were performed using a Fitch–Margoliash least-squares algorithm. The number of different genetic subgroups of olive genotypes (K) was investigated using STRUCTURE analysis. The standardization of allele lengths was performed to enable the comparison SSR profiles of Croatian olive genotypes with olive profiles obtained with the same SSR primers in OleaDB and WOGB databases. Overall, 73 SSR profiles of known Croatian varieties and 53 profiles of unknown olive genotypes were differentiated. Synonyms were detected in 18 varieties, and we found intra-varietal differences in 15 varieties. Three genetic subgroups of olive genotypes were determined. Following allele length standardization achieved using nine referral samples, the genetic profiles of 126 cultivated olive genotypes were compared to OleaDB and WOGB databases, out of which 92 genotypes were found to be unique to Croatian olive germplasm. The results revealed the wide genetic diversity of olive germplasm beyond the known, registered varieties. The FAZ_oliveDB database containing the profiles of 126 Croatian olive genotypes was created and made available for public use.

Molecular Characterization of Class II Newcastle Disease Virus from Field Outbreaks in Mizoram, India

Background: Newcastle disease (ND) is a highly contagious and economically important disease affecting poultry of all ages. The causative agent, class II NDV strains are frequently virulent and are classified into at least 21 distinct genotypes with several sub-genotypes. The circulating strains of NDV from mainland India are identified as genotype XIII, whereas emergence of a new genotype XXII has been reported recently from North East Region of India. Methods: A total of 25 poultry farms comprising a total population of 2165 birds were studied for field outbreaks of ND. Detailed post-mortem examination was conducted on a total of 121 dead birds and field outbreaks were confirmed by the detection of the F gene in tissue samples by reverse transcription PCR. Deduced amino acid sequence analysis of fusion proteins and phylogenetic analysis based on complete F gene were carried out to understand the molecular epidemiology of the circulating NDV strains. Result: This study has confirmed severe NDV outbreaks in the vaccinated flock, from Mizoram state of India. The three isolates from the outbreaks revealed the presence of the multi-basic amino acid residues at the fusion protein cleavage site (112RRQKRF117) identified as characteristic of velogenic strain. The phylogenetic analysis based on the complete F gene has characterized the isolates belonging to newly identified genotype XXII and subgroup XXII.2. The evolutionary evidence of this new genotype of NDV in the unique ecosystem of NER, India, warrants detailed studies for better understanding of the variant.

Optimal folic acid dosage in lowering homocysteine: Precision Folic Acid Trial to lower homocysteine (PFAT-Hcy)

BackgroundWhile folic acid (FA) is widely used to treat elevated total homocysteine (tHcy), promoting vascular health by reducing vascular oxidative stress and modulating endothelial nitric oxide synthase, the optimal daily dose and individual variation by MTHFR C677T genotypes have not been well studied. Therefore, this study aimed to explore the efficacy of eight different FA dosages on tHcy lowering in the overall sample and by MTHFR C677T genotypes.MethodsThis multicentered, randomized, double-blind, controlled clinical trial included 2697 eligible hypertensive adults with elevated tHcy (≥ 10 mmol/L) and without history of stroke and cardiovascular disease. Participants were randomized into eight dose groups of FA combined with 10 mg enalapril maleate, taken daily for 8 weeks of treatment.ResultsThe intent to treat analysis included 2163 participants. In the overall sample, increasing FA dosage led to steady tHcy reduction within the FA dosing range of 0–1.2 mg. However, a plateau in tHcy lowering was observed in FA dose range of 1.2–1.6 mg, indicating a ceiling effect. In contrast, FA doses were positively and linearly associated with serum folate levels without signs of plateau. Among MTHFR genotype subgroups, participants with the TT genotype showed greater efficacy of FA in tHcy lowering.ConclusionsThis randomized trial lent further support to the efficacy of FA in lowering tHcy; more importantly, it provided critically needed evidence to inform optimal FA dosage. We found that the efficacy of FA in lowering tHcy reaches a plateau if the daily dosage exceeds 1.2 mg, and only has a small gain by increasing the dosage from 0.8 to 1.2 mg.ClinicalTrials.gov IdentifierNCT03472508 (Registration Date: March 21, 2018).

Higher end-of-treatment HBsAg levels is associated with later onset but not severe relapse in HBeAg-negative chronic hepatitis B patients stopping antivirals.

Quantitative hepatitis B surface antigen (qHBsAg) level at end-of-treatment (EOT) predict clinical relapse (CR) after nucleos(t)ide analogues (Nuc) in chronic hepatitis B(CHB) patients. It is unclear if higher EOT qHBsAg leads to earlier onset or more severe off-Nuc CR. This large cohort study investigates the association between EOT qHBsAg and CR onset/severity. This study enrolled HBeAg-negative CHB patients who had achieved undetectable HBV DNA for over 1 year after receiving Nuc therapy before discontinuation. The EOT qHBsAg level was categorised into three groups: <100, 100-999, ≥1000 IU/mL. The study assessed the predictability of qHBsAg levels for CR, and analysed and compared the incidence, time to onset and severity of CR among these three groups. Patients with higher EOT qHBsAg showed a higher incidence of CR (≥1000, 100-999, <100 IU/mL: 73%, 65%, and 38%, p < 0.01) but a later onset of CR (median time to CR: 35, 33 and 27 weeks, p < 0.01). The predictabilities of EOT qHBsAg for CR were greater in patients aged <50-year-old or with genotype C than in those aged ≥50-year-old or with genotype B. There's no correlation between EOT qHBsAg level and ALT folds at CR (Pearson correlation coefficient: r = -0.03, p = 0.35). EOT qHBsAg was neither a predictor for severe hepatitis flare nor a predictor for hepatic decompensation. Predictability using EOT qHBsAg levels for CR differed in subgroups of age and genotypes. Higher EOT qHBsAg levels correlate with higher incidence but later onset of CR. No correlation between EOT qHBsAg and relapse severity was observed.

Genomic Epidemiology of African Swine Fever Virus Identified in Domestic Pig Farms in South Korea during 2019–2021

African swine fever (ASF), a contagious viral disease, poses a significant threat to the global swine industry. In South Korea, ASF outbreaks have occurred since 2019, highlighting the need for a comprehensive understanding of the epidemiology and genetic characterization of the circulating African swine fever viruses (ASFVs). We obtained 21 ASFV isolates from domestic pig farms and analyzed their whole-genome sequences using the Illumina MiniSeq. Phylogenetic analysis was conducted using the maximum likelihood and time-scaled approaches to determine the genetic relationships and evolutionary dynamics of the Korean ASFV isolates. Comparative analysis of the 21 ASFV genomes with the reference strain Georgia 2007/1 revealed that while Korean isolates shared 11 mutations, they also had 22 discrete mutations, including single nucleotide polymorphisms and insertion/deletion polymorphisms (Indels). Phylogenetic analysis indicated that all Korean isolates were within the Asian subgroup of ASFV genotype II but were further divided into at least three distinct subclusters. Spatiotemporal analysis indicated multiple introductions of ASFVs into South Korea, crossing the national border with North Korea. In addition, we observed putative self-recombination between MGF 505-9R and MGF 505-10R genes in the ASFV/Korea/Pig/Inje2/2021 strain. Our findings provide insights into the genetic variations and evolution of ASFVs on South Korean pig farms from 2019 to 2021, uncovering multiple introductions of ASFVs across the national border, and highlighting the need for enhanced disease control strategies.

Efficacy of Immediate Antihypertensive Treatment in Patients With Acute Ischemic Stroke With Different Blood Pressure Genetic Variants.

It remains unclear whether blood pressure (BP) genetic variants could modify the efficacy of immediate antihypertensive treatment after acute ischemic stroke. We conducted a secondary analysis of the CATIS (China Antihypertensive Trial in Acute Ischemic Stroke) to investigate the effect of early antihypertensive treatment on clinical outcomes among patients with acute ischemic stroke according to 5 BP-associated genetic variants. The CATIS randomized 4071 patients with acute ischemic stroke with elevated systolic BP to receive antihypertensive treatment or discontinue all antihypertensive agents during hospitalization. Randomization was conducted centrally and was stratified by participating hospitals and use of antihypertensive medications. Five BP-associated single nucleotide polymorphisms (rs16849225, rs17030613, rs1173766, rs6825911, and rs35444 in FIGN-GRB14, ST7L-CAPZA1, NPR3, ENPEP, and near TBX3, respectively) were genotyped among 2590 patients. The primary outcome was a combination of death and major disability at 14 days or hospital discharge. A weighted BP genetic risk score was constructed by the 5 single nucleotide polymorphisms. At 14 days or hospital discharge, the primary outcome was not significantly different between antihypertensive treatment and control groups based on genotype subgroups for all 5 single nucleotide polymorphisms (all P>0.05 for interaction). In addition, the BP genetic risk score did not modify the effect of antihypertensive treatment. The odds ratios (95% CIs) for the primary outcome were 0.95 (0.71-1.26), 1.08 (0.80-1.44), and 0.91 (0.69-1.22) in patients with low, intermediate, and high BP genetic risk score, respectively (P=0.88 for interaction). Early antihypertensive treatment had a neutral effect on clinical outcomes among patients with acute ischemic stroke according to 5 BP-associated genetic variants. URL: https://www.clinicaltrials.gov; Unique identifier: NCT01840072.

Rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and real-time PCR.

Analyzing all biological evidence at a crime scene presents serious time, budget, and labor constraints. Therefore, selecting valid evidence is crucial for efficient screening. The ABO blood group is a marker that can serve as valid evidence for identifying investigative leads in criminal case. Conventional identification of ABO blood groups using serological methods has only been for blood and is difficult to apply to other body fluids. ABO genotyping was conducted by analyzing single nucleotide polymorphisms (SNP) representative of each blood group. However, this method is time-consuming, expensive, and requires sophisticated instruments. In this study, we developed rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and multiplex real-time polymerase chain reaction (PCR). Three SNP sites in the ABO gene (nt 261, 526, and 803) were selected to classify the ABO genotypes. For the specificity test, we performed sequencing of 60 saliva samples to confirm that the genotyping. We conducted experiments to apply ABO genotyping using two amplification methods to mock forensic sample using cotton swab and filter paper. As a result, using LAMP, we successfully identified six ABO genotypes within 30min at a constant temperature (65 ℃). Moreover, by using multiple real-time PCR, it was possible to detect not only the major group but also the subgroup of the ABO genotype (ex. cis-AB). The amplification results using the new methods were in concordance with the sequencing results. Therefore, these ABO genotyping methods are expected to select valid evidence successfully and efficiently at the crime scene.

Effects of CYP11B2 gene polymorphisms on adrenocorticotropic hormone and angiotensin II in type 2 diabetes patients with hypertension

The aim of this study is to investigate the effects of CYP11B2 gene polymorphisms with the adrenocorticotropic hormone (ACTH) and angiotensin II (Ang II) in Chinese patients with type 2 diabetes (T2DM) and hypertension (T2MH). A total of 457 T2DM patients were genotyped for the polymorphism of rs3802228 and rs1799998 using SNP-scan high-throughput technology. The levels of adrenocorticotropic hormone in peripheral blood and related biochemical indices including aldosterone, renin, and Angiotensin II were measured and analyzed in relation to CYP11B2 polymorphisms in T2DM patients. As we found that Compared with T2DM patients, ACTH levels were higher in the rs3802228 AA/AG and rs1799998 AA/AG genotype T2MH subgroups (P < 0.001, P = 0.001). However, the difference in ACTH levels between two identical genotypes of T2MH was not statistically significant compared with T2DM patients (P > 0.05), whereas both AA and AG genotype subgroups had different AngII levels (P < 0.001, P = 0.004), which were higher than those of T2DM patients, but with a decrease in ACTH levels compared with patients with a single AA or AG genotype. Logistic regression analyses indicated that there was no significant relationship between genotype and allele polymorphisms with T2MH progression after adjustment for confounders. As a conclusion, CYP11B2 gene polymorphism was not associated with susceptibility to hypertension in Chinese T2DM patients, but its association with ACTH and Ang II levels may be a risk factor for susceptibility to hypertension in Chinese T2DM patients.

Serum matrix metalloproteinase-9 (MMP9) and amyloid-beta protein precursor (APP) as potential biomarkers in children with Fragile-X syndrome: A cross sectional study

IntroductionFragile-X syndrome(FXS) is a neurological disease caused by abnormal repeats in the 5′untranslated region of the FMR1 gene leading to a defective fragile-X-messenger-ribonucleoprotein-1 (FMRP). Although relatively common in children, it is usually under-diagnosed especially in developing countries where genetic screening is not routinely practiced. So far, FXS lacks a laboratory biomarker that can be used for screening, severity scoring or therapeutic monitoring of potential new treatments. Methods110 subjects were recruited; 80 male children with suspected FXS and 30 matched healthy children. We evaluated the clinical utility of serum matrix metalloproteinase-9(MMP9) and amyloid-beta protein precursor(APP) as potential biomarkers for FXS. ResultsOut of 80 suspected children, 14 had full mutation, 8 had the premutation and 58 children had normal genotypes. No statistically-significant difference was detected between children with different genotypes concerning age of onset(P = 0.658), main clinical presentation(P = 0.388), clinical severity-score(P = 0.799), patient’s disease-course(P = 0.719) and intellectual disability(P = 0.351). Both MMP9 and APP showed a statistically significant difference when comparing different genotype subgroups(P = 0.019 and < 0.001, respectively). Clinically, MMP9 levels were highest in children presenting with language defects, while APP was highest in children with neurodevelopmental delay. In receiver operating curve analysis, comparing full and premutation with the normal genotype group, MMP9 has an area-under-the-curve of 0.701(95 % CI 0.557–0.845), while APP was marginally better at 0.763(95 % CI 0.620–0.906). When combined together, elevated MMP9 or APP had excellent sensitivity > 95 % for picking-up FXS cases in the clinical setting. ConclusionsScreening for circulating biomarkers in the absence of FXS genetic diagnosis is justified. Our study is the first to evaluate both MMP9 and APP in FXS suspected children in a clinical setting and to assess their correlation with disease presentation and severity.

Lipoprotein(a) in Familial Hypercholesterolemia

BackgroundLow density lipoprotein (LDL) and Lipoprotein (Lp)(a) are proatherogenic apolipoprotein (apo) B-containing members of the non–high-density lipoprotein (non-HDL) family of particles. Elevated plasma levels of LDL cholesterol (C), non-HDL-C, and apo B are defining features of heterozygous familial hypercholesterolemia (HeFH), but reports of elevated plasma Lp(a) concentration are inconsistent. MethodsWe performed retrospective chart reviews of 256 genetically characterized patients with hypercholesterolemia and 272 control subjects from the Lipid Genetics Clinic at University Hospital in London, Ontario. We evaluated pairwise correlations between plasma levels of Lp(a) and those of LDL-C, non-HDL-C and apo B. ResultsMean Lp(a) levels were not different between individuals with hypercholesterolemia and control subjects. No correlations were found between Lp(a) and LDL-C or non–HDL-C levels in controls or patients with hypercholesterolemia; all r values < 0.079 and all P values > 0.193. Borderline weak correlations between Lp(a) and apo B were identified in patients r = 0.103; P = 0.112) and controls (r = 0.175; P = 0.005). Results were similar across genotypic subgroups. ConclusionsLp(a) levels are independent of LDL-C and non–HDL-C; in particular Lp(a) levels are not increased in patients with hypercholesterolemia and molecularly proven HeFH. Apo B was only weakly associated with Lp(a). Elevated Lp(a) does not cause FH in our clinic patients. Genetic variants causing HeFH that raise LDL-C do not affect Lp(a), confirming that these lipoproteins are metabolically distinct. Lp(a) cannot be predicted from LDL-C and must be determined separately to evaluate its amplifying effect on atherosclerotic risk in patients with hypercholesterolemia.

B-217 The study of association of FokI Polymorphism of Vitamin D Receptor (VDR) Gene, Vitamin D and Parathyroid Hormone Levels in Stage 4–5 Chronic Kidney Disease Patients: A Cross-sectional Study

Abstract Background Chronic Kidney Disease (CKD) is an important public health problem globally and almost 35% patients of CKD have deficiency in vitamin D which progresses to 80% in stage 4–5 CKD patients. Active vitamin D binds to Vitamin D Receptor (VDR) to regulate target gene transcription. Various genetic variations (Single Nucleotide Polymorphism) such as FokI polymorphism, which is a T/C transition polymorphism (ACG to ATG) may affect the VDR gene expression and thus may play an important role in pathogenesis of CKD. The aim of the study is to study association between FokI Polymorphism of Vitamin D Receptor (VDR) Gene, serum Vitamin D and PTH levels in Stage 4–5 CKD patients. Methods A total of 150 patients, aged 25–60 years, attending the Department of Nephrology AIIMS New Delhi with chronic kidney disease (CKD) stage 4–5, were enrolled for the study. FokI polymorphism was analyzed by using polymerase chain reaction-restriction fragment length Polymorphism (PCR-RFLP) in study subjects. Following DNA isolation and PCR amplification, restriction digestion was done using FokI restriction endonuclease and visualised on 1.5 % agarose gel electrophoresis. The prevalence of different genotypes and allelic frequency distribution was compared between CKD patients and healthy controls (HC). Levels of PTH, Vitamin D were estimated by eCLIA. Results No significant differences in genotype and in allelic frequencies between CKD patients and HC were observed as shown in Table 1. No significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC were observed. Conclusion The present study reveals no association of VDR FokI polymorphism with CKD. Furthermore, no significant differences in biochemical parameters were observed in FF, Ff and ff genotypic subgroups in CKD or HC groups. Further analysis revealed no significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC. Table 1.Genotype frequency and allelic frequency of VDR FOK1 gene polymorphism in CKD patients and HC.Genotype(FOK1)Genotype frequency (%)P-valueX2 (chi2)Allelic frequencyCKD (n = 150)HC (n = 150)Total study group (n = 300)0.7700.5220CKD (n = 150)HC (n = 150)FF53.33 (n = 80)57.33 (n = 86)55.33 (n = 166)FfFfFf41.33 (n = 62)37.33 (n = 56)39.33 (n = 118)0.740.260.760.24ff5.33 (n = 8)5.33 (n = 8)5.33 (n = 16)

Evidence for solanidine as a dietary CYP2D6 biomarker: Significant correlation with risperidone metabolism.

The extensive variability in cytochrome P450 2D6 (CYP2D6) metabolism is mainly caused by genetic polymorphisms. However, there is large, unexplained variability in CYP2D6 metabolism within CYP2D6 genotype subgroups. Solanidine, a dietary compound found in potatoes, is a promising phenotype biomarker predicting individual CYP2D6 metabolism. The aim of this study was to investigate the correlation between solanidine metabolism and the CYP2D6-mediated metabolism of risperidone in patients with known CYP2D6 genotypes. The study included therapeutic drug monitoring (TDM) data from CYP2D6-genotyped patients treated with risperidone. Risperidone and 9-hydroxyrisperidone levels were determined during TDM, and reprocessing of the respective TDM full-scan high-resolution mass spectrometry files was applied for semi-quantitative measurements of solanidine and five metabolites (M402, M414, M416, M440 and M444). Spearman's tests determined the correlations between solanidine metabolic ratios (MRs) and the 9-hydroxyrisperidone-to-risperidone ratio. A total of 229 patients were included. Highly significant, positive correlationswere observed between all solanidine MRs and the 9-hydroxyrisperidone-to-risperidone ratio (ρ>0.6, P < .0001). The strongest correlation was observed for the M444-to-solanidine MR in patients with functional CYP2D6 metabolism, i.e., genotype activity scores of 1 and 1.5 (ρ 0.72-0.77, P< .0001). The present study shows strong, positive correlations between solanidine metabolism and CYP2D6-mediated risperidone metabolism. The strong correlation within patients carrying CYP2D6 genotypes encoding functional CYP2D6 metabolism suggests that solanidine metabolism may predict individual CYP2D6 metabolism, and hence potentially improve personalized dosing of drugs metabolized by CYP2D6.

Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups.

While complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood. We therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome. We show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID. In conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted.

Clinical Course and Prognosis of Tubulopathies Characterized by Metabolic Alkalosis in Children.

Bartter syndrome and Gitelman syndrome are rare inherited tubulopathies characterized by hypokalemic, hypochloremic metabolic alkalosis. This study aimed to clarify the frequency of the phenotypic and genotypic subgroups, clinical features, long-term management, and prognosis of children diagnosed with Bartter syndrome and Gitelman syndrome in this study. Twenty-seven patients with Bartter syndrome and 6 patients with Gitelman syndrome, who were followed up between 2004 and 2020 in a single center, were included in the study. The median age of diagnosis was 4 months in patients with Bartter syndrome and 174 months in patients with Gitelman syndrome. At the last follow-up, a total of 12 Bartter syndrome patients had chronic kidney disease with a mean 7.79 ± 4.73 years of age; 5 (18.5%) of these patients had chronic kidney disease stage 2, 5 (18.5%) had chronic kidney disease stage 3, and 2 (7.4%) had chronic kidney disease stage 5. Of the 5 patients with Bartter syndrome with chronic kidney disease stage 2, 2 had CLCNKB and 1 had SLC12A1 gene mutation. Also, CLCNKB mutation was detected in 2 of 5 patients with Bartter syndrome with chronic kidney disease stage 3. Finally, 2 patients with Bartter syndrome with chronic kidney disease stage 5 had BSND mutation in one and CLCNKB mutation in the other. Estimated glomerular filtration rates of all patients with Gitelman syndrome were normal at the last follow-up. There was no statistically significant association of development of chronic kidney disease with genetic mutation, nephrocalcinosis, prematurity, and hypokalemia. Patients with Bartter syndrome and Gitelman syndrome may have a different clinical course due to the underlying genetic mutation. Bartter syndrome and Gitelman syndrome require lifelong treatment, and regular follow-up is important to prevent advanced-stage chronic kidney disease.

Genotyping of Candida albicans and Comparison of its Antifungal Resistance Pattern in the South Indian Region

Recent studies have documented an increase in the incidence of antifungal resistance in newly emerging species closely related to C. albicans, and the coexistence of genotypic variants. Hence, an application of PCR-based molecular typing is crucial in identifying these fungi. Our study used molecular methods to characterize the latest genotypic subgroups of C. albicans and analysed if there was a relationship between the genotypes and the antifungal resistance pattern. The study was conducted in JSS Hospital, Mysuru, Karnataka between July 2018 and December 2020. A total of 1427 Candida species were isolated from clinical samples. Candida albicans were isolated and confirmed using Germ tube test, ID VITEK 2 and PCR (ITS primer). DNA extraction was done using the Hi-Media Yeast DNA Extraction Kit. The amplified products were analysed using Agarose gel electrophoresis (2%). Among 1427 Candida species, 282 were Candida albicans. The following resistance was exhibited to major antifungals – Caspofungin (3.5%), Amphotericin B (1.4%), flucytosine (2.8%) Fluconazole (6%) Micafungin (2.8%) Voriconazole (3.1%) and all were sensitive to miconazole. ABC genotyping showed Genotype A (450 bp) predominant (87.58%) followed by genotype B (840bp) (9.92 %) and genotype C (450bp and 840 bp) (0.2%). Genotype D and E were not observed. Our study showed the growing antifungal resistance in clinical isolates. Genotype A was predominant in South Karnataka region followed by Genotype B and C. There was no correlation between genotyping and antifungal resistance. However, a study with greater number of samples from diverse geographical locations may give more insight.