Genotyping Results Research Articles

Pharmacogenetic-guided dosing for fluoropyrimidine (DPYD) and irinotecan (UGT1A1*28) chemotherapies for patients with cancer (PACIFIC-PGx): A multicenter clinical trial.

PACIFIC-PGx evaluated the feasibility of implementing pharmacogenetics (PGx) screening in Australia and the impact of DPYD/UGT1A1 genotype-guided dosing on severe fluoropyrimidine (FP) and irinotecan-related toxicities and hospitalizations, compared to historical controls. This prospective single arm trial enrolled patients starting FP/irinotecan for any cancer between 7 January 2021 and 25 February 2022 from four Australian hospitals (one metropolitan, three regional). During the accrual period, 462/487 (95%) consecutive patients screened for eligibility for DPYD and 50/109 (46%) for UGT1A1 were enrolled and genotyped (feasibility analysis), with 276/462 (60%) for DPYD and 30/50 (60%) for UGT1A1 received FP/irinotecan (safety analysis). DPYD genotyping identified 96% (n = 443/462) Wild-Type, 4% (n = 19/462) Intermediate Metabolizers (50% dose reduction), and 0% Poor Metabolizers. UGT1A1 genotyping identified 52% (n = 26/50) Wild-Type, 40% (n = 20/50) heterozygous, and 8% (n = 4/50) homozygous (30% dose reduction). Key demographics for the FP/irinotecan safety cohorts included: age range 23-89/34-74 years, male 56%/73%, Caucasian 83%/73%, lower gastrointestinal cancer 50%/57%. Genotype results were reported prior to cycle-1 (96%), average 5-7 days from sample collection. PGx-dosing for DPYD variant allele carriers reduced high-grade toxicities compared to historic controls (7% vs. 39%; OR = 0.11, 95% CI 0.01-0.97, p = 0.024). High-grade toxicities among Wild-Type were similar (14% vs. 14%; OR = 0.99, 95% CI 0.64-1.54, p = 0.490). PGx-dosing reduced FP-related hospitalizations (-22%) and deaths (-3.7%) compared to controls. There were no high-grade toxicities or hospitalizations for UGT1A1*28 homozygotes. PGx screening and prescribing were feasible in routine oncology care and improved patient outcomes. Findings may inform expanded PGx programs within cancer and other disease settings.

Clinical impact of genetic testing in a large cohort of pediatric cardiomyopathies

BackgroundThere are limited data that can explain the earlier penetrance and the different expressivity of pediatric cardiomyopathy (pCM) compared to adult-onset cardiomyopathy (aCM). In addition, the relationship between genotype and pCM results is poorly described. ObjectiveWe compared the genotypes between a cohort of aCM and a cohort of pCM to propose hypotheses on the earlier penetrance and expressivity of pCM. Finally, we report how genetic testing was used to guide genetic counseling in pCM. Methods253 pCM (<18 years old) and 1466 aCM patients were sequenced on a panel of 67 cardiomyopathy genes. Risk factors for death and heart transplantation were analyzed. ResultsIn pCM, the variant of interest (VOI) yield was 53.7 % including 24.2 % carrying two VOI. De novo variants represented 11 % of VOI in pCM and 50 % in restrictive pCM. An age at diagnosis younger than 1 year (HR = 2.07, p = 0.029), restrictive phenotype (HR = 2.87, p = 0.03) and the presence of two VOI (HR = 2.97, p = 0.001) were independent risk factors for death or heart transplantation.In comparison with aCM, pCM patients harbored more frequently two VOI (p = 0.02), or de novo variants (p = 4.10−13). In addition, the distribution of VOI was different in aCM and pCM. Genotyping of pCM improved genetic counseling in families and led to ten prenatal-diagnosis.Conclusions: Genetic testing provides clues for earlier penetrance of pCM. The presence of two VOI in children with CM is a risk factor for severe and early cardiac events.

Performance of LDL-C only compared to the Dutch Lipid Clinic Network Score for screening of familial hypercholesterolaemia: the Austrian experience and literature review.

Familial hypercholesterolaemia (FH) is a severely underdiagnosed, inherited disease, causing dyslipidaemia and premature atherosclerotic cardiovascular disease. In order to facilitate screening in a broad clinical spectrum, we aimed to analyse the current yield of routine genetic diagnostics for FH and to evaluate the performance of the Dutch Lipid Clinic Network Score (DLCNS) compared to a single value, the off-treatment LDL-cholesterol exceeding 190 mg/dL. We investigated all patients that underwent molecular genotyping routinely performed for FH over a 4-year period in two Austrian specialist lipid clinics. Variants reported in FH-causing genes including LDLR, APOB, PCSK9, LDLRAP, and APOE were collected and classified. For clinical classification, the DLCNS was calculated retrospectively and compared to the original scores documented in patient charts. Additionally, a literature review on comparisons of DLCNS to LDL-C was performed. Of 469 patients tested, 21.3% had a disease-causing variant. A median of 3 out of 8 (excluding genotyping results and LDL-C) DLCNS criteria were unavailable. DLCNS was documented in 48% of cases, with significant discrepancies compared to retrospective scoring (P < 0.001). DLCNS did not outperform off-treatment LDL-C alone (Δ = 0.006; P = 0.660), analogously to several reports identified in the literature. A single cut-off of 190 mg/dL LDL-C compared to DLCNS ≥ 6 showed excellent sensitivity (84.9% vs. 53.8%) and acceptable specificity (39.0% vs. 84.1%). Missing criteria and severe discrepancies observed between retrospective and on-site scoring by treating physicians were highly prevalent, confirming limited utility of DLCNS in clinical routine and warranting a single off-treatment LDL-C cut-off of 190 mg/dL for enhanced index-case identification.

Experience of Genotyping of the Class 1 Major Histocompatibility Complex Genes in a Rhesus Macaques Collection

The utility of animal models in preclinical research depends on the completeness of our knowledge about these models. Nonhuman primates are the closest animal model to humans, available for preclinical evaluation of biotechnological products. The lack of immunogenetic characteristics of the primates used in a particular study significantly limits the possibilities of analyzing the results obtained. The availability of information about the haplotype of the major histocompatibility complex (MHC) increases the overall significance of the model. Genotyping of primates at the MHC locus by DNA analysis is excluded, since the representation of individual MHC proteins depends on the level of their expression in immune cells, rather than on their presence in the genome. Therefore, the genotyping of primates at the MHC locus is carried out by analyzing mRNA encoding allelic variants of proteins. This work presents the results of genotyping of a sample of rhesus macaques from the collection of the Kurchatov Complex of Medical Primatology by a next-generation sequencing technology using an IonProton sequencer.

Analysis of Tandem Repeats in Short-Read Sequencing Data: From Genotyping Known Pathogenic Repeats to Discovering Novel Expansions.

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genotyping known pathogenic tandem repeat loci Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy Support Protocol 1: Installation of tools and ExpansionHunter catalog modification Basic Protocol 2: Performing genome-wide genotyping of tandem repeats Basic Protocol 3: Discovering de novo tandem repeat expansions Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles.

Assessing the Effects of Dasatinib on Mesenchymal Stem/Stromal Cells

Abstract Introduction Progressive aging, or senescence, of mesenchymal stem/stromal cells (MSCs) is a major obstacle faced when trying to culture potent stem cells for use in therapy. Senescent cells are irreversibly nondividing cells that cease performing critical functional effects. Elimination of senescent cells using biochemical means, such as the use of senolytic drugs like dasatinib, may be useful in retaining the viable and proliferating populations of the cells. Methods An in vitro approach was used to investigate the effect of dasatinib on phenotypic, genotypic, and immunomodulatory functionality of osteogenic and adipogenic differentiated MSCs. Replicative senescence was achieved through multiple sub-culturing in vitro, then senescent and non-senescent cultures were treated with a standard dosage of dasatinib. MSCs were then differentiated into osteogenic, adipogenic or chondrogenic cultures using conditioned media to be tested for the three criteria being investigated. Results Significant changes were observed in these criteria, indicated by evidence gathered from proliferation and indoleamine 2,3 dioxygenase activity assays. Phenotypic results of dasatinib were shown to reduce the population of senescent MSCs while allowing non-senescent MSCs to continue differentiating and proliferating without interference from senescent cells. Genotypic results showed no change to upregulation in markers associated with osteogenic and adipogenic cells when exposed to dasatinib. Indoleamine Dioxygenase activity showed insignificant differences in cells exposed to dasatinib versus control groups, providing evidence against compromised cellular immune function. Conclusion This investigation provides insight into how dasatinib effects MSCs functional ability and provides a better understanding of the function of senolytic agents.

Targeting virulence of resistant Escherichia coli by the FDA-approved drugs sitagliptin and nitazoxanide as an alternative antimicrobial approach.

The spread of multidrug-resistant Escherichia coli in healthcare facilities is a global challenge. Hospital-acquired infections produced by Escherichia coli include gastrointestinal, blood-borne, urinary tract, surgical sites, and neonatal infections. Therefore, novel approaches are needed to deal with this pathogen and its rising resistance. The concept of attenuating virulence factors is an alternative strategy that might lead to low levels of resistance and combat this pathogen. A sub-inhibitory concentration (¼ MIC) of sitagliptin and nitazoxanide was used for phenotypic assessments of Escherichia coli virulence factors such as biofilm production, swimming motility, serum resistance, and protease production. Moreover, qRT-PCR was used to determine the impact of sub-MIC of the tested drugs on the relative expression levels of papC, fimH, fliC, kpsMTII, ompT_m, and stcE genes encoding virulence factors in Escherichia coli. Also, an in vivo model was conducted as a confirmatory test. Phenotypically, our findings demonstrated that the tested strains showed a significant decrease in all the tested virulence factors. Moreover, the genotypic results showed a significant downregulation in the relative expression levels of all the tested genes. Besides, the examined drugs were found to be effective in protecting mice against Escherichia coli pathogenesis. Sitagliptin and nitazoxanide exhibited strong anti-virulence activities against Escherichia coli. In addition, it is recommended that they might function as adjuvant in the management of Escherichia coli infections with either conventional antimicrobial agents or alone as alternative treatment measures.

Study on the relationship between KCNQ1 gene-environment interaction and abnormal glucose metabolism in the elderly in a county of Hechi City, Guangxi.

This study aimed to understand the potassium voltage-gated channel KQT-like subfamily, member 1 gene polymorphism in a rural elderly population in a county in Guangxi and to explore the possible relationship between its gene polymorphism and blood sugar. The 6 SNP loci of blood DNA samples from 4355 individuals were typed using the imLDRTM Multiple SNP Typing Kit from Shanghai Tianhao Biotechnology Co. The data combining epidemiological information (baseline questionnaire and physical examination results) and genotyping results were statistically analyzed using GMDR0.9 software and SPSS22.0 software. A total of 4355 elderly people aged 60 years and above were surveyed in this survey, and the total abnormal rate of glucose metabolism was 16·11 % (699/4355). Among them, male:female ratio was 1:1·48; the age group of 60-69 years old accounted for the highest proportion, with 2337 people, accounting for 53·66 % (2337/4355). The results of multivariate analysis showed that usually not doing farm work (OR 1·26; 95 % CI 1·06, 1·50), TAG ≥ 1·70 mmol/l (OR 1·19; 95 % CI 1·11, 1·27), hyperuricaemia (OR 1·034; 95 % CI 1·01, 1·66) and BMI ≥ 24 kg/m2 (OR 1·06; 95 % CI 1·03, 1·09) may be risk factors for abnormal glucose metabolism. Among all participants, rs151290 locus AA genotype, A allele carriers (AA+AC) were 0.70 times more likely (0.54 to 0.91) and 0.82 times more likely (0.70 to 0.97) to develop abnormal glucose metabolism than CC genotype carriers, respectively. Carriers of the T allele at the rs2237892 locus (CT+TT) were 0.85 times more likely to have abnormal glucose metabolism than carriers of the CC genotype (0.72 to 0.99); rs2237897 locus CT gene. The possibility of abnormal glucose metabolism in the carriers of CC genotype, TT genotype and T allele (CT + TT) is 0·79 times (0·67-0·94), 0·74 times (0·55-0·99) and 0·78 times (0·66, 0·92). The results of multifactor dimensionality reduction showed that the optimal interaction model was a three-factor model consisting of farm work, TAG and rs2237897. The best model dendrogram found that the interaction between TAG and rs2237897 had the strongest effect on fasting blood glucose in the elderly in rural areas, and they were mutually antagonistic. Environment-gene interaction is an important factor affecting abnormal glucose metabolism in the elderly of a county in Hechi City, Guangxi.

Genetic characterization of Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease in Lithuania.

Neisseria meningitidis is a gram-negative bacterium responsible for life-threatening invasive infections known as invasive meningococcal disease and is associated with high fatality rates and serious lifelong disabilities among survivors. This study aimed to characterize N. meningitidis isolates cultured from blood and cerebrospinal fluid collected between 2009 and 2021 in Lithuania, assess their genomic relationships with European strains, and evaluate the possibility of using a cost-effective method for strain characterization, thus improving the national molecular surveillance of invasive meningococcal disease. In total, 321 N. meningitidis isolates were collected and analyzed using multilocus restriction typing (MLRT). Amplification of the penA gene and restriction fragment length polymorphism analysis were performed to identify the modified penA genes. Based on the MLRT genotyping results, we selected 10 strains for additional analysis using whole-genome sequencing. The sequenced genomes were incorporated into a dataset of publicly available N. meningitidis genomes to evaluate genomic diversity and establish phylogenetic relationships within the Lithuanian and European circulating strains. We identified 83 different strains using MLRT genotyping. Genomic diversity of N. meningitidis genomes analysed revealed 21 different sequence types (STs) circulating in Lithuania. Among these, ST34 was the most prevalent. Notably, three isolates displayed unique combinations of seven housekeeping genes and were identified as novel STs: ST16969, ST16901, and ST16959. The analyzed strains were found to possess virulence factors not commonly found in N. meningitidis. Six distinct penA profiles were identified, each with different frequencies. In the present study, we also identified N. meningitidis strains with new penA, NEIS0123, NEIS1320, NEIS1525, NEIS1600, and NEIS1753 loci variants. In our study, using the cgMLST scheme, Minimum Spanning Tree (MST) analysis did not identify significant geographic relationships between Lithuanian N.meningitidis isolates and strains from Europe. Discussion: To our knowledge, this is the first study to employ whole genome sequencing (WGS) method for a comprehensive genetic characterization of invasive N. meningitidis isolates from Lithuania. This approach provides a more detailed and precise analysis of genomic relationships and diversity compared to prior studies relying on traditional molecular typing methods and antigen analysis.

Genotyping Apolipoprotein E (APOE) Isoforms with Sequence-Specific-Primer (SSP)-PCR in Early‑Onset Alzheimer’s Disease Patients: A Rapid and Revised Methodology

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive damage to brain cells, leading to impairment in cognitive functions. The Apolipoprotein E (APOE) variants play a significant role in the genetic basis of AD, especially late-onset AD (LOAD), and increase the disease risk at an earlier age. Although controversial, some studies reveal the association between APOE genotype and early-onset AD (EOAD) regardless of family history. Therefore, diagnostic laboratories widely perform routine tests to determine the APOE genotype. In the present study, we implemented a revised methodology for the Sequence-Specific-Primer-PCR (SSP-PCR) test for rapid APOE genotyping in 67 EOAD patients. Then, the findings were validated using automatic sequencing with newly designed primers for the related region of the APOE. We state clearly that the applicability of the SSP-PCR method was improved when the primer concentrations of control genes were increased 2-fold, as we reported. All data obtained from SSP-PCR were consistent with Sanger sequencing confirmations. Based on the genotyping results, the four different APOE genotypes were detected: E2/E4, E3/E3, E3/E4, and E4/E4. The frequencies were 1.5% (n=1) for E2/E4, 76.1% (n=51) for E3/E3, 16.4% (n=11) for E3/E4, and 6% (n=4) for E4/E4. In the study group, 23.9% (n=16) of the patients had homozygous or heterozygous APOE E4. However, we detected no significant association between the clinical features and the APOE genotype. As a result, this method is reliable, cost-effective, and rapid for performing genotyping analysis of the APOE for routine tests and research studies with larger EOAD cohorts.

Compiling molecular evidence from a tetraploid rose genome into a near-saturated map for the identification of pigment-related genes

With their high economic value and cultural significance, modern roses are one of the most important ornamental plants. Because of their complicated genetic background and tetraploid nature, the creation of high-density genetic maps of roses has been a challenge that has slowed the pace of molecular breeding for modern roses. The current construction of tetraploid genetic maps based on existing diploid rose genomes could lead to inaccurate marker information and genotyping results. Therefore, we generated the first high-quality tetraploid genome of Rosa chinensis ‘Yunzheng Xiawei.’ Utilizing Illumina, PacBio, and Hi-C sequencing technologies, we assembled a genome of 858.59 Mb with 14 pseudo-chromosomes. Mode of inheritance analysis using PolyOrigin indicated that modern roses show both quadrivalent and bivalent pairing. Based on this reference genome, high-density genetic maps were constructed using MSTmap with nearly saturated markers. Quantitative trait locus (QTL) analysis was conducted using WinQTLCart and R/qtl for flavonoids and carotenoids, and 11 QTL clusters were identified. By combining the genome annotation, phylogenetic analyses, and gene expression analyses, we were able to identify several key genes related to flavonoid and carotenoid biosynthesis. This study provides the basis for further genetic analyses of highly heterozygous tetraploid roses and could facilitate the progress of marker-assisted selection in modern roses.

Frequency of rs35803318 single nucleotide polymorphism of ACE2 gene among the Kazakhs

In the article the results of genotyping of DNA samples obtained from the study participants on the single nucleotidepolymorphism (SNP) rs35803318 (C/T) of the ACE2 gene were presented. Genotyping was carriedout by real-time polymerase chain reaction (PCR) using the technique Amplification of the Refractory MutationSystem (ARMS). The frequencies of rs35803318 (C/T) genotypes and alleles in 96 representatives of theKazakh ethnic group living in the Karaganda region were analyzed. The ACE2 gene (angiotensin-convertingenzyme 2) was extensively investigated due to its role in susceptibility to infection by the SARS-CoV-2 viruscausing COVID-19. The ACE2 gene encodes a protein that serves as a receptor for the virus, and its variationscan affect a person's susceptibility to infection. The ACE2 protein plays a role in the regulation of angiotensinand the effect on blood pressure. Therefore, there is a link between ACE2 gene polymorphisms, includingrs35803318, and the development of cardiovascular diseases. According to the results of our study,the CT genotype (63.5 %) is the most common among Kazakhs, the CC genotype was 20.8 % and theTT genotype was 15.6 %. The distribution of polymorphism alleles is as follows: allele C — 52.6 %, allele A— 47.4 %.

Missense mutations in the CITED2 gene may contribute to congenital heart disease

BackgroundCongenital heart disease (CHD) is a lifelong abnormality present from birth. Multiple studies have shown that mutations in genes involved in heart development could cause congenital heart disease. The CITED2 gene works as a transcription factor in the hypoxic pathway for the development of the heart. Therefore, five CHD types, ventricular septal defect, atrial septal defect, atrioventricular septal defect, tetralogy of fallot, and patent ductus arteriosus, were evaluated by conducting a targeted single nucleotide polymorphism (SNP) analysis of the CITED2 gene variant rs375393125 (T > C). This study aimed to identify the association of CITED2 gene mutations in CHD patients.MethodsThree hundred fifty samples, 250 from patients and 100 from controls, were collected for this genetic analysis. Allele-specific PCR and gel electrophoresis were used to identify the target missense mutations. The genotypic results of the CHDs were further validated through Sanger sequencing.ResultsThe frequency of the homozygous mutant (CC) in CHD patients was 48.4%, and of the heterozygous mutant (TC) genotype was 11.4%; these percentages are higher than controls (1%). The control samples had only one heterozygous TC and no homozygous CC genotype. The chi-square value was obtained at 103.9 with a probability of 0.05, more significant than the significance value of 21.03. The odds ratio was 43.7, which is > 1. The calculated value of ANOVA was 11.6, which was more significant than the F critical value of 3.7. As a result of sequencing, the mutant sample of each selected CHD type was found heterozygous or homozygous, and the results were like those obtained through conventional PCR.ConclusionThe samples of CHD patients showed mutations. Therefore, the CITED2 gene SNP might be associated with CHD.

Barriers to HIV rapid start among New Jersey providers

Background Rapid Start ensures that persons with HIV initiate antiretroviral therapy in less than seven days after diagnosis. Benefits of Rapid Start include timely linkage to medical care, viral suppression in a shorter time and improved retention to medical care. Despite these benefits, there is a slow uptake of Rapid Start, in New Jersey. Objective Identify barriers to Rapid Start among New Jersey providers. Methods An electronic survey, consisting of 28 questions, with the following domains was administered to New Jersey providers, using Qualtrics: provider and practice characteristics (10), knowledge (1), barriers (8) and attitudes to diverse patient types (9). The results were analyzed using descriptive statistics due to small numbers over strata. Approval to conduct the survey was obtained from the William Paterson University Institutional Review Board. Results There were 69 responses to the survey. Providers were at least 45 years old (48%), female (44/60, 73%), nurse practitioners or physician assistants (41/59, 69%). Overall, 44/63 (70%) providers did not correctly identify that integrase inhibitors had the lowest prevalence of transmitted drug resistance. Newly diagnosed patients were referred for medical care in 37 (65%) of the medical sites. Only providers from Ryan White (federally funded clinics for persons with HIV) (64%) and non-Ryan White (73%) public sites reported co-located HIV testing sites. Seventy percent of medical sites offered same-day medical appointments. However, a lower proportion of private (62%), public Ryan White (55%), and other medical sites (36%) offered same-day appointments compared to public non-Ryan White sites (82%). Despite having staff available 40 h per week (91%), only 55% of Ryan White sites offered extended office hours in the early morning, evenings, or on Saturdays. When compared to providers in public non-Ryan White sites, a lower proportion of providers in Ryan White sites were comfortable doing Rapid Start either on the day of or within one week of diagnosis, 82% and 72%, respectively, or starting antiretroviral therapy before genotype results were available, 55% and 46%, respectively. Overall, providers were not comfortable with Rapid Start for persons engaging in condomless sex (60%). Conclusions Policy and administrative decisions are needed to eliminate barriers at the clinic level. An HIV clinical scholar program, to increase providers knowledge, may increase uptake of Rapid Start.

ОЦЕНКА КОРОВ-ПЕРВОТЁЛОК ЧЁРНО-ПЁСТРОЙ ПОРОДЫ ПО ДНК-МАРКЕРАМ, АССОЦИИРОВАННЫХ С ХОЗЯЙСТВЕННО-ПОЛЕЗНЫМИ ПРИЗНАКАМИ

At present time are widely used genetic markers as means in the selection of highly productive animals. Since the genes: CSN3 (kappa-casein), PIT-1 (pituitary-specific transcription factor), PRL (prolactin), GH (somatotropin) are polymorphic and encode various proteins, the study of their influence on the productive traits of cattle is relevant. In this regard, the goal of the work is to determine the influence of complex genotypes of the studied genes on the milk productivity of first-calf cows of the Black-and-White breed bred in the collective farm-breeding plant «Kazminsky» in the Kochubeevsky district of the Stavropol Territory. The place of research is the Department of Genetics and Biotechnology, VNIIOK, a branch of the North Caucasian Federal National Research Center. This article presents the results of DNA genotyping of allelic polymorphism for the CSN3, PIT-1, PRL, GH genes using PCR-RFLP methods in first-calf cows of the Black-and-White breed. It was established that these genes in the studied sample are polymorphic. The frequency of occurrence of the desired CSN3B allele in the studied sample was 0.23. The presence of alleles PIT-1A and PIT-1B of the pituitary-specific transcription factor gene was noted with a frequency of occurrence of 0.52 and 0.48. A feature of the PRL gene polymorphism was the fairly high frequency of occurrence of the PRLA allele (0.60). In this study sample of dairy cattle, the frequency of occurrence of the desired GHL allele of the somatotropin gene was 0.62. All variants of complex genotypes were also identified and their relationship with milk productivity in black-and-white cows was noted. The study sample was dominated by animals carrying complex desirable genotypes, consisting of four and three marker alleles of three and two genes (CSN3AB/PIT-1BB/GHLV; CSN3AB/PIT1AB/GHLL or PIT-1AB/PRLAB/GHLV; PIT-1AA/GHLL ) - 46.7 %. Milk yield per lactation between groups of black-and-white first-calf cows with different complex genotypes ranged from 5839.14 to 8611.33 kg. The mass fraction of fat in milk ranged from 3.93 to 4.04 %, and protein from 2.99 to 3.14 %.

Vaccine-associated measles in an immunocompromised host: Hospital infection prevention and control and public health response

Introduction: Vaccine-associated measles is generally not considered to be transmissible, as opposed to wild-type measles, which is one of the most highly contagious diseases. Data on contact and exposure management of vaccine-associated measles is limited, with varied approaches to such cases described in the literature. Methods: We report the case of a 2-year-old immunosuppressed child who developed a febrile exanthem with mild conjunctivitis 18 days after receiving the measles-mumps-rubella-varicella vaccine. Results: Given the patient's recent measles-containing vaccination while on immunosuppressive medications, consistent clinical findings and the lack of epidemiological risk factors for wild-type infection, the decision was made to treat this as a presumptive case of vaccine-associated measles virus prior to return of confirmatory genotyping results. After consultation with public health experts, contact tracing was not considered necessary. No secondary measles cases were identified, despite a large exposure potential due to lack of consistent airborne precautions during hospital admission. Discussion: This case highlights the lack of transmissibility of vaccine-associated measles in immunocompromised hosts, adding to the scant body of literature on this topic, with the potential to inform hospital infection prevention and control as well as public health management in similar situations.

Antimicrobial Susceptibility of Commensal Escherichia coli from Pig Fecal Samples and Enhanced Sensitivity for Direct Detection of the blaCTX-M Gene by Nested PCR.

The commensal Escherichia coli in the gut of pigs is a major reservoir of antimicrobial resistance and can result in possible transmission to humans through the food chain. Direct detection of E. coli from fecal samples is challenging and can be used as a bioindicator of antimicrobial resistance. This study aimed to compare the antimicrobial susceptibility profiles in commensal E. coli from antibiotic- and nonantibiotic-using pig farms and developed the direct detection of ESBL genes in pig fecal samples using nested PCR (nPCR) and multiplex PCR (mPCR) techniques. All direct genotypic results were validated with the results of PCR sequencing of isolated E. coli colonies. The ESBL-producing E. coli were found in 98.6% (145 isolates) and 96.6% (144 isolates) of antibiotic-using and nonantibiotic-using farms, respectively, predominantly CTX-M-55. The nPCR decreased the limit of detection (LOD) from sPCR about 100 times, and the lower LODs of 102, 101, and 1 CFU/mL were reached after incubating samples in an enrichment medium for 2, 4, and 8 h, respectively. The mPCR, sPCR, and nPCR techniques showed sensitivities of 30.15%, 69.85%, and 91.91%, respectively, compared to PCR sequencing. The stability and recycling of ESBL genes were independent of antibiotic usage in commensal E. coli originating in pig farms.

Development and validation of a 66K SNP array for the hard clam (Mercenaria mercenaria)

BackgroundThe hard clam (Mercenaria mercenaria), a marine bivalve distributed along the U.S. eastern seaboard, supports a significant shellfish industry. Overharvest in the 1970s and 1980s led to a reduction in landings. While the transition of industry from wild harvest to aquaculture since that time has enhanced production, it has also exacerbated challenges such as disease outbreaks. In this study, we developed and validated a 66K SNP array designed to advance genetic studies and improve breeding programs in the hard clam, focusing particularly on the development of markers that could be useful in understanding disease resistance and environmental adaptability.ResultsWhole-genome resequencing of 84 individual clam samples and 277 pooled clam libraries yielded over 305 million SNPs, which were filtered down to a set of 370,456 SNPs that were used as input for the design of a 66K SNP array. This medium-density array features 66,543 probes targeting coding and non-coding regions, including 70 mitochondrial SNPs, to capture the extensive genetic diversity within the species. The SNPs were distributed evenly throughout the clam genome, with an average interval of 25,641 bp between SNPs. The array incorporates markers for detecting the clam pathogen Mucochytrium quahogii (formerly QPX), enhancing its utility in disease management. Performance evaluation on 1,904 samples demonstrated a 72.7% pass rate with stringent quality control. Concordance testing affirmed the array's repeatability, with an average agreement of allele calls of 99.64% across multiple tissue types, highlighting its reliability. The tissue-specific analysis demonstrated that some tissue types yield better genotyping results than others. Importantly, the array, including its embedded mitochondrial markers, effectively elucidated complex genetic relationships across different clam groups, both wild populations and aquacultured stocks, showcasing its utility for detailed population genetics studies.ConclusionsThe 66K SNP array is a powerful and robust genotyping tool that offers unprecedented insights into the species’ genomic architecture and population dynamics and that can greatly facilitate hard clam selective breeding. It represents an important resource that has the potential to transform clam aquaculture, thereby promoting industry sustainability and ecological and economic resilience.

Genetic variants of IL-10 promoter influence susceptibility to HIV-1 infection and disease progression in the Polish population: IL-10 polymorphisms and HIV-1

The risk of HIV-1 infection and the rate of disease progression vary considerably among individuals and the genetic makeup of the host may be one of the possible reasons for this. We aimed to determine association of functional single nucleotide polymorphism (SNPs), −1082A/G (rs1800896), −819C/T (rs1800871), and −592C/A (rs1800872) in IL-10 gene, with the susceptibility to HIV-1 infection and clinical parameters expressed as a baseline CD4+ T cell count, CD8+ T cell count, and viral load. Therapy naïve HIV-1 infected individuals and HIV-1 seronegative controls from Poland were recruited for this study. Genotyping results revealed significantly higher frequency of −1082G/G genotype (28.1 % vs 16.1 %; p = 0.0019, OR=0.49) and −1082G allele (47.6 % vs 38.8 %; p = 0.0028, OR = 0.70) as well as lower frequency of −592 and −819 heterozygosity (45.0 % vs 34.4 %; p = 0.0266, OR = 1.47) in controls compared to seropositive subjects. High producing haplotype GCC was associated with increased risk of HIV-1 infection (p = 0.0018, OR = 1.52). Individuals possessing −592 and −819 minor allele had significantly higher CD8+ T cell count compared to the wild type allele carriers (p = 0.0303). Moreover, presence of −1082G allele was related with lower viral load as well as CD4+ and CD8+ T cells counts among patients infected with R5 HIV-1 variant. Thus, IL-10 gene promoter variants may be a risk factor for HIV-1 transmission and may modulate disease progression in the Polish population.

The significance of extensive HPV genotyping for cervical high-grade neoplasia among women with atypical glandular cells.

To examine the associated risk of cervical intraepithelial neoplasm grade 3+ (CIN3+) lesions in patients with AGC and extensive human papillomavirus (HPV) genotyping. Cases with atypical glandular cell (AGC) interpretation on a Papanicolaou (Pap) test were identified along with associated extensive HPV genotyping and histologic follow-up results. Within this cohort of 469,694 Pap tests, 0.4% were diagnosed as AGCs. In total, 1267 cases had concurrent high-risk HPV (hrHPV) genotyping, and 40.3% were hrHPV positive. The percentage of AGC cases with cervical CIN3+ on histologic follow-up was 52.2% when hrHPV was positive, whereas it was 4.9% with a negative hrHPV result. The top 5 hrHPV genotypes associated with cervical CIN3+ in this cohort were HPV16, HPV18, HPV58, HPV52, and HPV33. Indeed, 92.8% of the hrHPV-associated CIN3+ lesions identified in this cohort were positive for at least one of these HPV genotypes. The sensitivity of detecting cervical CIN3+ lesions was 85.6% with the top 5 hrHPV genotypes (HPV16/18/58/52/33) and only increased to 89.0% when the additional 12 genotypes were included. In patients with an AGC Pap, the risk of having a cervical CIN3+ lesion is greatly increased by positivity for hrHPV types 16, 18, 58, 52, and/or 33. Incorporating comprehensive HPV genotyping into AGC cytology allows for refined risk stratification and more tailored management strategies.