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Advances in biomarkers, targeted therapies, and immuno-oncology have transformed the clinical management of patients with advanced NSCLC. For oncogene-driven tumors, there are highly effective targeted therapies against EGFR, ALK, ROS1, BRAF, TRK, RET, and MET. In addition, investigational therapies for KRAS, NRG1, and HER2 have shown promising results and may become standard-of-care in the near future. In parallel, immune-checkpoint therapy has emerged as an indispensable treatment modality, especially for patients lacking actionable oncogenic drivers. While PD-L1 expression has shown modest predictive utility, biomarkers for immune-checkpoint inhibition in NSCLC have remained elusive and represent an area of active investigation. Given the growing importance of biomarkers, optimal utilization of small tissue biopsies and alternative genotyping methods using circulating cell-free DNA have become increasingly integrated into clinical practice. In this review, we will summarize the current landscape and emerging trends in precision medicine for patients with advanced NSCLC with a special focus on predictive biomarker testing.

Diffuse astrocytoma (WHO grade II) has classically been considered a slow growing tumour, typically affecting young adults, with tendency for late malignant conversion. We describe a case of early atypical malignant transformation of diffuse astrocytoma seventeen months after complete surgical removal, as an intraventricular high-grade glioma (HGG). Retrospective laboratory findings for the presence of IDH 1/2 (isocitrate dehydrogenase) mutations were negative. There is growing evidence that IDH-wildtype (wt) astrocytomas behave more aggressively, therefore identifying IDH-mutation status should be mandatory in order to determine disease prognosis and guide treatment course. info:eu-repo/semantics/publishedVersion

Strongyloidiasis represents a major medical and veterinary helminthic disease. Human infection is caused by three species; Strongyloides stercoralis, Strongyloides fuelleborni fuelleborni and Strongyloides fuelleborni kellyi, with S. stercoralis accounting for the majority of cases. Strongyloides f. fuelleborni likely represents a zoonosis acquired from non-human primates (NHPs), while no animal reservoir for S. f. kellyi infection has been found. Whether S. stercoralis represents a zoonosis acquired from dogs and cats remains unanswered. Over the past two decades various tools have been applied to genotype Strongyloides spp. The most commonly sequenced markers have been the hyper-variable regions I and IV of the 18S rRNA gene and selected portions of the cytochrome c oxidase subunit I gene. These markers have been sequenced and compared in Strongyloides from multiple hosts and geographical regions. More recently, a machine learning algorithm multi-locus sequence typing approach has been applied using these markers, while others have applied whole genome sequencing. Genotyping of Strongyloides from dogs, cats, NHPs and humans has identified that S. stercoralis likely originated in dogs and adapted to human hosts. It has also demonstrated that S. stercoralis is distinct from S. f. fuelleborni and S. f. kellyi. Two distinct genetic clades of S. stercoralis exist, one restricted to dogs and another infecting humans, NHPs, dogs and cats. Genotyping of S. f. fuelleborni has identified two separate clades, one associated with African isolates and another Indochinese peninsular clade. This review summarises the history and development of genotyping tools for Strongyloides spp. It describes the findings of major studies to date in the context of the epidemiology and evolutionary biology of these helminths, with a specific focus on human-infecting species.

Introduction Breast cancer (BC), a heterogeneous disease, features microRNA-related single nucleotide polymorphisms (miRSNPs) as underlying factors of BC development, thus providing targets for novel diagnostic and therapeutic strategies. This study investigated miRSNPs in BC susceptibility in Australian Caucasian women. Patients and Methods The study population included patients 33 to 80 years of age stratified by molecular subtypes of breast tumors (luminal A, 47.59%), stage (stage I, 36.96%), tumor-type (ductal, 44.95%), grading (intermediate, 35.52%), size (10.1-25 mm, 31.14%), and lymph node (sentinel negative, 38.93%). Sixty-five miRSNPs underwent allelic analysis in two independent case–control cohorts (GU-CCQ-BB, 377 cases and 521 controls; GRC-BC, 267 cases and 201 controls) using a MassARRAY platform. GU-CCQ-BB, GRC-BC, and the combined populations (BC-CA) (644 cases and 722 controls) underwent independent statistical analysis. Results In the GU-CCQ-BB population, miRSNPs TET2-rs7670522, ESR1-rs2046210, FGFR2-rs1219648, MIR210-rs1062099, HIF1A-rs2057482, and CASC16-rs4784227 were found to be associated with increased BC risk (P ≤ .05). Only ESR1-rs2046210 was also significantly associated (P ≤ .05) when replicated in the GRC-BC and BC-CA populations. No significant association was correlated with BC-clinical features (pathological types and ER/PR/HER2 status); however, MIR210-rs1062099 was found to be significantly associated (P ≤ .05) with age (>50 years) in the GU-CCQ-BB cohort. Conclusion This is the first study to demonstrate the association of MIR210-rs1062099 and TET2-rs7670522 with increased BC risk. Additionally, four previously reported SNPs (ESR1-rs2046210, FGFR2-rs1219648, HIF1A-rs2057482, and CASC16-rs4784227) were confirmed as BC risk variants. Replication and functional studies in larger Caucasian cohorts are necessary to elucidate the role of these miRSNPS in the development of BC.

Epidermal growth factor receptor (EGFR) genotyping, a critical examen for the treatment decisions of patients with non-small cell lung cancer (NSCLC), is commonly assayed by next-generation sequencing (NGS), but this global approach takes time. To determine whether rapid EGFR genotyping tests by the IdyllaTM system guides earlier therapy decisions, EGFR mutations were assayed by both the IdyllaTM system and NGS in 223 patients with NSCLC in a bicentric prospective study. IdyllaTM demonstrated agreement with the NGS method in 187/194 cases (96.4%) and recovered 20 of the 26 (77%) EGFR mutations detected using NGS. Regarding the seven missed EGFR mutations, five were not detected by the IdyllaTM system, one was assayed in a sample with insufficient tumoral cells, and the last was in a sample not validated by the IdyllaTM system (a bone metastasis). IdyllaTM did not detect any false positives. The average time between EGFR genotyping results from IdyllaTM and the NGS method was 9.2 ± 2.2 working days (wd) (12.6 ± 4.0 calendar days (cd)). Subsequently, based on the IdyllaTM method, the timeframe from tumor sampling to the initiation of EGFR-TKI was 7.7 ± 1.2 wd (11.4 ± 3.1 cd), while it was 20.4 ± 6.7 wd (27.5 ± 7.7 cd) with the NGS method (p &lt, 0.001). We thus demonstrated here that the IdyllaTM system contributes to improving the therapeutic care of patients with NSCLC by the early screening of EGFR mutations.

Circulating tumor DNA (ctDNA) enables real-time genomic profiling of cancer without the need for tissue biopsy. ctDNA-based technology is seeing rapid uptake in clinical practice due to the potential to inform patient management from diagnosis to advanced disease. In metastatic disease, ctDNA can identify somatic mutations, copy-number variants (CNVs), and structural rearrangements that are predictive of therapy response. However, the ctDNA fraction (ctDNA%) is unpredictable and confounds variant detection strategies, undermining confidence in liquid biopsy results. Assay design also influences which types of genomic alterations are identifiable. Here, we describe the relationships between ctDNA%, methodology, and sensitivity-specificity for major classes of genomic alterations in prostate cancer. We provide recommendations to navigate the technical complexities that constrain the detection of clinically relevant genomic alterations in ctDNA.

Purpose To develop a genotype assay to assess associations with common and rare age-related macular degeneration (AMD) risk variants, to calculate an overall genetic risk score (GRS), and to identify potential misdiagnoses with inherited macular dystrophies that mimic AMD. Design Case-control study. Participants Individuals (n = 4740) from 5 European cohorts. Methods We designed single-molecule molecular inversion probes for target selection and used next generation sequencing to sequence 87 single nucleotide polymorphisms (SNPs), coding and splice-site regions of 10 AMD-(related) genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, and SLC16A8), and 3 genes that cause inherited macular dystrophies (ABCA4, CTNNA1, and PRPH2). Genetic risk scores for common AMD risk variants were calculated based on effect size and genotype of 52 AMD-associated variants. Frequency of rare variants was compared between late AMD patients and control individuals with logistic regression analysis. Main Outcome Measures Genetic risk score, association of genetic variants with AMD, and genotype–phenotype correlations. Results We observed high concordance rates between our platform and other genotyping platforms for the 69 successfully genotyped SNPs (>96%) and for the rare variants (>99%). We observed a higher GRS for patients with late AMD compared with patients with early/intermediate AMD (P Conclusions This study reports a genotype assay for common and rare AMD genetic variants, which can identify individuals at intermediate to high genetic risk of late AMD and enables differential diagnosis of AMD-mimicking dystrophies. Our study supports sequencing of CFH, CFI, and C3 genes because they harbor rare high-risk variants. Carriers of these variants could be amendable for new treatments for AMD that currently are under development.

Knowledge of the genetic landscape of a specific population group is vital for population-specific diagnosis and treatment of familial breast cancer. Although BRCA-related diagnostic testing has long been implemented in South Africa, the genotyping approach previously failed for the SA Indian population as it was based on other SA population groups. Because this population is uniquely admixed, the lack of population-specific data resulted in the implementation of comprehensive mutation screens for BRCA1/2. A total of 223 female patients were screened for clinically actionable variants. High-resolution melting analysis (HRMA) was used to screen 88 patients for DNA alterations in the coding and splice site boundaries of BRCA1 exons 2-9, BRCA1 exons 11-23, BRCA2 exons 2-9 and BRCA2 exons 12-27. The protein truncation test (PTT) was used to screen the three larger exons (BRCA1 exon 10 and BRCA2 exons 10 and 11) for protein termination changes. Multiplex ligation-dependent probe amplification (MLPA) was used to determine the presence of larger indels and possible copy number differences. Next Generation Sequencing (NGS) was performed on the remaining 135 samples. All potential variants were confirmed by performing Sanger DNA sequencing. The search revealed 28 different pathogenic heterozygotic variants, together with nine variants of unknown significance (VUS). The results suggested that the SA Indian population represents a different genetic admixture compared to that of mainland India, as only five pathogenic variants corresponded to those reported for mainland India. Familial breast cancer testing for SA Indian patients should therefore be performed as comprehensively as possible as the pathogenic variants seem to be family- rather than population-specific. Furthermore, predictive testing of family members will contribute to relieve the financial burden on the country's healthcare system, as increased surveillance and appropriate management could prevent disease.

PURPOSE Plasma circulating tumor DNA (ctDNA) analysis is routine for genotyping of advanced non–small-cell lung cancer (NSCLC); however, early response assessment using plasma ctDNA has yet to be well characterized. MATERIALS AND METHODS Patients with advanced EGFR-mutant NSCLC across three phase I NCI osimertinib combination trials were analyzed in this study, and an institutional cohort of patients with KRAS-, EGFR-, and BRAF-mutant advanced NSCLC receiving systemic treatment was used for validation. Plasma was collected before treatment initiation and serially before each cycle of therapy, and key driver mutations in ctDNA were characterized by droplet digital polymerase chain reaction. Timing of plasma versus imaging response was compared in a separate cohort of patients with EGFR-mutant NSCLC treated with osimertinib. Across cohorts, we also studied ctDNA variability before treatment start. RESULTS In the NCI cohort, 14/16 (87.5%) patients exhibited ≥ 90% decrease in mutation abundance by the first on-treatment timepoint (20-28 days from treatment start) with minimal subsequent change. Similarly, 47/56 (83.9%) patients with any decrease in the institutional cohort demonstrated ≥ 90% decrease in mutation abundance by the first follow-up draw (7-30 days from treatment start). All 16 patients in the imaging cohort with radiographic partial response showed best plasma response within one cycle, preceding best radiographic response by a median of 24 weeks (range: 3-147 weeks). Variability in ctDNA levels before treatment start was observed. CONCLUSION Plasma ctDNA response is an early phenomenon, with the majority of change detectable within the first cycle of therapy. These kinetics may offer an opportunity for early insight into treatment effect before standard imaging timepoints.

Whole genome sequencing is instrumental for the study of genome variation in natural populations, delivering important knowledge on genomic modifications and potential targets of natural selection at the population level. Large dormant eggbanks of aquatic invertebrates such as the keystone herbivore Daphnia, a microcrustacean widespread in freshwater ecosystems, provide detailed sedimentary archives to study genomic processes over centuries. To overcome the problem of limited DNA amounts in single Daphnia dormant eggs, we developed an optimized workflow for whole genome amplification (WGA), yielding sufficient amounts of DNA for downstream whole genome sequencing of individual historical eggs, including polyploid lineages. We compare two WGA kits, applied to recently produced Daphnia magna dormant eggs from laboratory cultures, and to historical dormant eggs of Daphnia pulicaria collected from Arctic lake sediment between 10 and 300 years old. Resulting genome coverage breadth in most samples was ~70%, including those from >100-year-old isolates. Sequence read distribution was highly correlated among samples amplified with the same kit, but less correlated between kits. Despite this, a high percentage of genomic positions with single nucleotide polymorphisms in one or more samples (maximum of 74% between kits, and 97% within kits) were recovered at a depth required for genotyping. As a by-product of sequencing we obtained 100% coverage of the mitochondrial genomes even from the oldest isolates (~300 years). The mitochondrial DNA provides an additional source for evolutionary studies of these populations. We provide an optimized workflow for WGA followed by whole genome sequencing including steps to minimize exogenous DNA.

Accurate genotyping is important to improve the treatment of hepatitis C virus (HCV) infection. We herein report a 44-year-old Japanese man with hemophilia A and coinfection of HCV and human immunodeficiency virus (HIV) who was diagnosed with HCV genotype 4 by direct sequencing. Two genotyping tests based on the nested polymerase chain reaction method that we used misdiagnosed his genotype as 2b and 1b. Although several HCV genotyping tests are available in Japan, it is important to recognize that some cannot detect genotype 4. Care should be taken when genotyping HCV patients who have received non-heated coagulation factor preparations or were infected abroad.

Methylation of host-cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high-risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host-cell DNA methylation markers for triage in an hrHPV-positive cohort derived from primary HPV-based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation-specific PCR (qMSP) in clinician-collected cervical samples (n = 715) from hrHPV-positive women (age 29-60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end-point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single-marker and bi-marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV-positive clinician-collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi-marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3-85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1-77.9%). In conclusion, our study shows that the individual host-cell DNA methylation classifiers and the bi-marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV-positive women invited for routine screening.

Objective Low levels of neutrophils can be an intrinsic condition, with no clinical consequences or immunity impairment. This condition is the benign constitutional neutropenia (BCN), defined as an absolute neutrophils count (ANC) ≤ 2000 cells/mm. Diagnosis of BCN is of exclusion where patients are submitted to blood tests and possibly to invasive diagnostic search until secondary causes of neutropenia are ruled out. The natural history of the disease suggests benign evolution and Brazilian study showed an overall frequency of 2.59%. The main mechanisms include reduced neutrophil production, increased marginalization, extravasation to the tissues and immune destruction. Genetic studies showed strong association between the single nucleotide variant rs2814778 located on chromosome 1q23.2 in the promoter region of the atypical chemokine receptor 1 (Duffy blood group system) gene (ACKR1, also termed DARC) and BCN. The aim of this study is to evaluate FY phenotypes and genotypes including the analysis of the rs2814778 SNP in Brazilian patients with BCN in order to determine an effective diagnostic tool, allowing reassurance of the patient and cost reduction in their care. Methods Case control study, with 94 individuals (18 patients and 76 controls). Phenotyping was performed by gel test and genotyping was performed by PCR-RFLP. Results White blood cell (WBC) and absolute neutrophils (AN) counts showed lower levels in patients compared to controls. In the patient group 83.3% were genotyped as FY*B/FY*B. The SNP rs2814778 (-67T > C) was identified in 77.8% of the patients genotyped as FY*B-67C/FY*B-67C. In the control group, 72.7% were homozygous for the wild type and 23.3% were heterozygous. Conclusion This study reinforces that FY phenotyping and genotyping can be used to detect most people with BCN, avoiding excessive diagnostic investigation. Besides, this procedure may reduce health costs and be reproductible in clinical practice.

The objective of this study was to determine the correlation between single-nucleotide polymorphism rs4769628 in POMP gene and the development of allergic disease in children. Methods. Genotyping of single nucleotide polymorphism rs4769628 in POMP gene in children with manifestations of atopic march and apparently healthy children using real-time polymerase chain reaction. Results. 62.26 % of children with atopic diseases and 53.06 % healthy children had major allele rs4769628 in POMP gene. 33.67 and 37.76 % of children, respectively, had heterozygous AG variant. Minor GG genotype was not detected in children with atopic diseases, 9.18 % of healthy people are carriers of minor allele (р < 0.05). Conclusions. Minor G allele of rs4769628 in РОМР gene is associated with reduced risk of atopic diseases in children. Polymorphism rs4769628 in РОМР gene can be used as an important prognostic marker for the development of atopic diseases in children.

Objective: to determine the association between single-nucleotide polymorphism rs510432 in gene ATG5 and the development of atopic march in children, to find out the probability of clinical manifestation and the features of the clinical course of atopic diseases in individuals with different allelic variants of polymorphism. Methods. Genotyping assay of polymorphism rs510432 in gene ATG5 was performed in children with atopic diseases and apparently healthy children using real-time polymerase chain reaction. Results. The minor T allele of rs510432 in ATG5 gene was significantly more often found in patients with atopic diseases than in apparently healthy children (χ2 = 6.36; р < 0.05). The results of logistic regression demonstrated that the risk of atopic diseases in children with minor genotype is 2.4 times higher than in carriers of major genotype. The average values of forced expiratory volume in 1 second (FEV1) are significantly lower in asthma patients with minor genotype than in thereof with major genotype (Q = 2.71; р

The present paper discusses methodological approaches to the creation of nomenclatural standards and genetic passports for Russian cultivars, currently being developed at the N.I. Vavilov Institute of Plant Genetic Resources (VIR) in collaboration with different breeding research centers. Plant material of potato cultivars bred in the Siberian Research Institute of Plant Cultivation and Breeding was collected by the cultivar creator A.D. Safonova in the experimental field of this institute and transferred to the VIR herbarium for preparation of their nomenclatural standards. Plant shoots and tubers of potato cultivars bred in other Siberian research centers in collaboration with the A.G. Lorkh All-Russian Research Institute of Potato Farming (VNIIKH) was collected by the representative of this institute in the experimental field of VNIIKH. As a result of joint research, nomenclatural standards were accomplished for 11 cultivars, namely ‘Аntonina’, ‘Zlatka’, ‘Lina’, ‘Lûbava’, ‘Nakra’, ‘Pamâti Rogačeva’, ‘Sarovskij’, ‘Safo’, ‘Solnečnyj’, ‘Tuleevskij’, ‘Ûna’** bred in five different Siberian breeding institutes including cultivars developed in collaboration with VNIIKH. Nomenclatural standards were prepared according to the ‘International Code of Nomenclature for Cultivated Plants’. DNA samples isolated from nomenclatural standards were used for preparation of genetic passports of these 11 cultivars. These genetic passports include information of the polymorphism of eight chromosome-specific microsatellites, data on the markers of 11 R-genes conferring resistance to various harmful organisms, as well as the information about cytoplasm types. Voucher specimens of additional three Siberian cultivars ‘Kemerovčanin’, ‘Kuznečanka’, ‘Tanaj’ and five Ural cultivars ‘Аlâska’, ‘Bravo’, ‘Irbitskij’, ‘Lûks’, ‘Terra’ from the Ural Research Institute for Agriculture were also registered in the VIR herbarium collection. For these eight cultivars, the genetic passports were not issued, but the results of SSR genotyping and molecular screening of voucher specimens performed with the same set of the DNA markers are presented in this report. A similar set of DNA markers was used for genotyping cultivar accessions from the in vitro and field collections of various institutes as well as cultivar specimens from eco-geographical tests performed within the framework of the Comprehensive Research Plan of the subprogram “Development of potato breeding and seed production in the Russian Federation”. The comparison of cultivar genetic passport data with genotyping results of specimens having the same name, but obtained from different sources made it possible to verify this plant material.

Background Variation in mitochondrial DNA (mtDNA) identified by genotyping microarrays or by sequencing only the hypervariable regions of the genome may be insufficient to reliably assign mitochondrial genomes to phylogenetic lineages or haplogroups. This lack of resolution can limit functional and clinical interpretation of a substantial body of existing mtDNA data. To address this limitation, we developed and evaluated a large, curated reference alignment of complete mtDNA sequences as part of a pipeline for imputing missing mtDNA single nucleotide variants (mtSNVs). We call our reference alignment and pipeline MitoImpute. Results We aligned the sequences of 36,960 complete human mitochondrial genomes downloaded from GenBank, filtered and controlled for quality. These sequences were reformatted for use in imputation software, IMPUTE2. We assessed the imputation accuracy of MitoImpute by measuring haplogroup and genotype concordance in data from the 1000 Genomes Project and the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The mean improvement of haplogroup assignment in the 1000 Genomes samples was 42.7% (Matthew’s correlation coefficient = 0.64). In the ADNI cohort, we imputed missing single nucleotide variants. Conclusion These results show that our reference alignment and panel can be used to impute missing mtSNVs in existing data obtained from using microarrays, thereby broadening the scope of functional and clinical investigation of mtDNA. This improvement may be particularly useful in studies where participants have been recruited over time and mtDNA data obtained using different methods, enabling better integration of early data collected using less accurate methods with more recent sequence data.

Genome sequence-driven molecular typing tools have the potential to uncover the population biology and genetic diversity of rapidly evolving plant pathogens like Magnaporthe oryzae. Here, we report a new molecular typing technique -a digitally portable tool for population genetic analysis of M. oryzae to decipher the genetic diversity. Our genotyping tool exploiting allelic variations in housekeeping and virulence genes coupled with pathotyping revealed a prevalence of genetically homogenous populations within a single-field and plant niches such as leaf and panicle. The M. oryzae inciting leaf-blast and panicle-blast were confirmed to be genetically identical with no or minor nucleotide polymorphism in 17 genomic loci analyzed. Genetic loci such as Mlc1, Mpg1, Mps1, Slp1, Cal, Ef-Tu, Pfk, and Pgk were highly polymorphic as indicated by the haplotype-diversity, the number of polymorphic sites, and the number of mutations. The genetically homogenous single field population showed high virulence variability or diversity on monogenic rice differentials. The study indicated that the genetic similarity displayed by the isolates collected from a particular geographical location had no consequence on their virulence pattern on rice differentials carrying single/multiple resistance genes. The data on virulence diversity showed by the identical Sequence Types (STs) is indicative of no congruence between polymorphic virulence genes-based pathotyping and conserved housekeeping genes-based genotyping.

Investigations in recent decades have exploited tissue DNA genotyping as a powerful ancillary tool for the precision diagnosis and subclassification of gestational trophoblastic disease. As lesions of gestational origin, the inherited paternal genome, with or without copy number alterations, is the fundamental molecular basis for the diagnostic applications of DNA genotyping. Genotyping is now considered the gold standard in the confirmation and subtyping of sporadic hydatidiform moles. Although a precise diagnosis of partial mole requires DNA genotyping, prognostic stratification according to distinct genetic zygosity in complete moles has recently gained significant clinical relevance for patient care. Beyond hydatidiform moles, DNA genotyping has fundamental applications in the diagnosis or prognostic assessment of gestational trophoblastic tumors, in particular gestational choriocarcinoma. DNA genotyping provides a decisive tool in the separation of gestational trophoblastic neoplasia from non-gestational counterparts/mimics of either germ cell or somatic origin. The FIGO/WHO prognostic scoring scheme requires ascertaining the precise index gestational event and the time interval between the tumor and index gestation, where DNA genotyping can provide highly relevant information. With rapid acquisition of molecular diagnostic capabilities in the clinical practice, DNA genotyping has become closely integrated into the routine diagnostic workup of various forms of gestational trophoblastic disease.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing, and to determine a minimal set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations on a minimal panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories in designing assays for PGx testing. When developing these recommendations, the Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations with regard to PGx testing. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document is focused on clinical CYP2D6 PGx testing that may be applied to all cytochrome P450 2D6–metabolized medications. These recommendations are not meant to be interpreted as prescriptive but to provide a reference guide for clinical laboratories that may be either implementing PGx testing or reviewing and updating their existing platform.

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus causing coronavirus disease 2019 (COVID-19), has been expanding globally since late 2019. SARS-CoV-2, an RNA virus, has a genome sequence that can easily undergo mutation. Several mutated SARS-CoV-2 strains, including those with higher infectivity than others, have been reported. To reduce SARS-CoV-2 transmission, it is crucial to trace its infection sources. Here, we developed a simple, easy-to-use genotyping method to identify SARS-CoV-2 variants using a high-resolution melting (HRM) analysis. Methods We investigated five mutation sites, A23403G, G25563T, G26144T, T28144C, and G28882A, which are known strain determinants according to GISAID clades (L, S, V, G, GH, and GR). Results We first employed synthetic DNA fragments containing the five characteristic sites for HRM analysis. All sequences clearly differentiated wild-type from mutant viruses. We then confirmed that RNA fragments were suitable for HRM analysis following reverse transcription. Human saliva did not negatively affect the HRM analysis, which supports the absence of a matrix effect. Conclusions Our results indicate that this HRM-based genotyping method can identify SARS-CoV-2 variants. This novel assay platform potentially paves the way for accurate and rapid identification of SARS-CoV-2 infection sources.

Given that binding and internalization of bacteria to host cells promotes infections and invasion, we aimed at characterizing how various S. aureus isolates adhere to and are internalized by different white blood cells. In particular, the role of genetic determinants on the association kinetics should be unveiled. A flow cytometric (FACS) whole blood assay with fluorescently labelled isolates was applied to 56 clinical S. aureus isolates. This phenotypic data was then linked to previously obtained genotyping data (334 genes) with the help of a redescription mining algorithm. Professional phagocytes showed a time-dependent increase of bacterial adhesion and internalization. Isolates showing higher affinity to granulocytes were associated with lower binding to monocytes. In contrast binding activity between S. aureus and lymphocytes could be subdivided into two phases. Preliminary binding (phase 1) was highest directly after co-incubation and was followed by S. aureus detachment or by sustained binding of a small lymphocyte subset (phase 2). Strain-dependent low granulocyte binding was observed for clonal complex 5 (CC5) isolates (MRSA), as compared to CC30 and CC45 (MSSA). S. aureus isolates associated with low granulocyte phagocytosis were characterized by the presence (cap8, can) and the absence (sasG, lukD, isdA, splA, setC) of specific hybridization signals.

Aim: The study investigated the genetic polymorphism of the kisspeptin (KiSS1) gene and its relationship with litter size in Cyprus and Iraqi black goats. Materials and Methods: Blood samples (n=124) were collected from the two goat breeds reared at the Agricultural Research-Ruminant Research Station Breeding Station, Baghdad, Iraq. Genomic DNA was isolated using a DNA extraction kit. Polymerase chain reaction (PCR) was used to amplify the KiSS1 gene. All PCR products were sequenced and samples were used for further analysis using NCBI-Blast online on the exon 1 (595 bp) region of the KiSS1 gene. Results: The results of this study revealed a significantly (PC was identified as a single nucleotide polymorphism (SNP) associated with litter size. Furthermore, the average alleles in KiSS1 gene of both types of goats 0.567 and 0.3715 GG, were recorded. The genotyping at locus g893C>G was demonstrating domination of fecundity quality litter size, Both genotypes SNP of GC were classified at this marked region of KiSS1 gene. Conclusion: The study concluded that the role of the KiSS1 gene in fecundity, revealing the status of this gene as an indicator in the assisted of caprine breeding selection.

Proteus mirabilis is abundant in soil and water. Although this bacterium is part of the normal human intestinal flora, it can cause serious infections in humans, including complicated urinary tract infections. This pathogen is also commonly associated with multidrug resistance. In the present study, analysis of 1,093 samples from foods of animal origin and animal intestinal samples recovered 232 P. mirabilis isolates identified by PCR assay. Of these 232 isolates, 72 produced β-lactamase (determined by both phenotypic and genotypic methods), with the highest prevalence in poultry cloacal swabs (11.82%) followed by mutton (9.18%), khoa (6.32%), pork (5.63%), pig rectal swabs (5.52%), beef (5.45%), and chicken (5.13%) but none from sheep rectal swabs and bovine rectal swabs. Among β-lactamase genes, blaTEM was the predominant gene detected (59 isolates) followed by blaOXA (11 isolates), blaSHV (5 isolates), blaFOX (5 isolates), blaCIT (4 isolates), blaCTX-M1 and blaCTX-M9 (2 isolates each) and blaCTX-M2, blaDHA, and blaEBC (1 isolate each). None of the isolates carried blaACC, blaMOX, or carbapenemase genes (blaVIM, blaIMP, blaKPC, and blaNDM-1). Dendrogram analysis of enterobacterial repetitive intergenic consensus sequences and repetitive extragenic palindromic sequences obtained with PCR analysis of β-lactamase-producing isolates revealed 63 isolates, but 9 isolates did not yield bands. The analysis revealed that 6.58% of the samples had β-lactamase-producing P. mirabilis isolates that may affect food safety and contaminate the environment. Further genotyping revealed the genetic relationships between isolates of different origin. These findings emphasize the need for careful use of antibiotics to control the spread of β-lactamase-producing bacteria. HIGHLIGHTS

Pharmacogenetic testing is increasingly available from clinical and research laboratories. However, only a limited number of quality control and other reference materials are currently available for many of the variants that are tested. The Association for Molecular Pathology Pharmacogenetic Work Group has published a series of papers recommending alleles for inclusion in clinical testing. Several of the alleles were not considered for tier 1 because of a lack of reference materials. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention–based Genetic Testing Reference Material (GeT-RM) program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 18 DNA samples derived from Coriell cell lines. DNA samples were distributed to five volunteer testing laboratories for genotyping using three commercially available and laboratory developed tests. Several tier 2 variants, including CYP2C9∗13, CYP2C19∗35, the CYP2C cluster variant (rs12777823), two variants in VKORC1 (rs61742245 and rs72547529) related to warfarin resistance, and two variants in GGCX (rs12714145 and rs11676382) related to clotting factor activation, were identified among these samples. These publicly available materials complement the pharmacogenetic reference materials previously characterized by the GeT-RM program and will support the quality assurance and quality control programs of clinical laboratories that perform pharmacogenetic testing.

Objective Self-sampling for human papillomavirus (HPV) testing is an effective option to increase the cervical screening coverage. How to best triage HPV-positive self-samples remains controversial. Here, we evaluated the performance of a novel p16INK4a immunocytology approach (p16) and HPV genotyping in triaging HPV-positive self-samples. Methods A cohort of 73699 women were screened via SeqHPV assay on self-samples. HPV-positive women who met any sequential positive result of HPV16/18 or VIA or p16 were referred for colposcopy. A triage strategy was considered favorable if the NPV for CIN3+ was ≥98%, combined with an improvement of sensitivity and specificity in comparison to the comparator, being the ‘ASC-US+’ triage and the guideline strategy (HPV16/18+ or ASC-US+). Results A total of 3510 HPV-positive women were included, 422 (12.0%) CIN2+ and 247 (7.0%) CIN3+ were identified. The positivity of p16 and ASC-US+ were 36.3% and 22.2%, respectively. p16 was more sensitive and less specific than ASC-US+ (P Conclusions Our findings indicate the promising utility of p16 immunocytology via adjusting the staining score or serving as an ancillary tool to liquid-based cytology or combining with genotyping for the triage of HPV-positive self-samples, which promotes the precise screening of cervical cancer.

Vitiligo is a rare skin condition caused by an immune reaction. Vitiligo can occur anywhere on the body. This proposed explanation of vitiligo makes it clear that vitiligo is not linked to any other autoimmune diseases. The polymorphisms of some genes present in the immune system play a major function in susceptibility of vitiligo. Meta-analysis studies have shown that the Angiotensin converting enzyme (ACE) gene insertion and deletion polymorphism is closely associated with vitiligo in many ethnicities. The connection between ACE gene and vitiligo is connected through the auto immune diseases and there are no genetic polymorphism studies have been carried out with ACE gene with vitiligo in the Saudi population. Previous studies show that vitiligo patients are more likely to also have an autoimmune disorder. The current study aims to investigate the I/D polymorphism in the ACE gene with diagnosed patients with vitiligo subjects. This is a case-control study carried out in the Saudi population with 100 vitiligo cases and 100 healthy controls. Genotyping was performed through polymerase chain reaction followed by 3% agarose gel electrophoresis. Genotype and allele frequencies were carried out with genetic mode of inheritances. Statistical analysis was performed considering p 0.05) when compared between the vitiligo cases and controls. The correlation between age and genotyping was performed with Anova analysis and current study results confirmed the substantial link between 11 and 20 years (p = 0.01) and 31–40 years (p = 0.04) with the defined age groups. In conclusion, in Saudi populations, the ACE gene I/D polymorphism was identified as being correlated with vitiligo. This is the first study in Saudi Arabia to report the risk factors of vitiligo with the ACE gene polymorphism.

BACKGROUND: Non-Hodgkin’s lymphoma (NHL) is the most common hematological malignancy in the world. Many etiologic factors have been implicated in the risk of developing NHL, including genetic susceptibility and obesity. Single-nucleotide polymorphisms (SNPs) in Ghrelin (GHRL), an anti-inflammatory hormone, and tumor necrosis factor α (TNF-α), an inflammatory cytokine, have been independently associated with the risk for obesity and NHL. OBJECTIVE: To investigate the association between SNPs in GHRL and TNF-α and the risk for NHL and obesity in Kuwaitis. METHODS: We recruited 154 Kuwaiti NHL patients and 217 controls. Genotyping was performed for rs1629816 (GHRL promoter region), rs35684 (GHRL 3’ untranslated region), and rs1800629 (TNF-α promoter region). Logistic regression analysis was performed to assess the association of the investigated SNPs with NHL and the relationship between the selected SNPs with BMI in each group separately. RESULTS: We show that rs1629816 GG was associated with an increased risk for NHL in our sample (p= 0.0003, OR 1.82; CI: 1.31–2.54). None of the investigated SNPs were associated with obesity, nor was obesity found to be associated with the risk for NHL. CONCLUSIONS: Our study demonstrates an association between rs1629816, a SNP in the GHRL regulatory region, and NHL in Kuwaitis.

In developing countries, the acute gastroenteritis outbreaks submitted for viral testing are limited due to deficient surveillance programs. The aim of this study was to analyze a passive surveillance strategy for monitoring the molecular epidemiology of norovirus (NV) and counterbalance the genetic diversity data gap. Laboratory-confirmed rotavirus negative sporadic stool samples (N = 523) collected between 2010 and 2017 from children were selected from our archival collection and were tested for NV and sequencing was performed on the positive samples. Passive surveillance information was compared with the genetic diversity data that was available from local norovirus-confirmed gastroenteritis outbreaks. Each year, norovirus detection in the sporadic samples ranged from 12 to 29%. GI and GII norovirus were detected in 7 (1.3%) and 101 (19.3%) of the specimens, respectively. Four GI and six GII capsid genotypes were identified. Six out of 9 strains detected in the NV outbreaks panel were also identified in the set of sporadic samples either coincidently in the same year, the previous or the later year. Also, this set of samples depicted even better the circulating epidemic strain. Thus, implementing norovirus testing and genotyping in stool samples collected with other purposes represent a suitable strategy for providing genetic diversity information.

Background Giardia duodenalis is a zoonotic protozoan parasite causing diarrhea through waterborne or fecal-oral infection. The cysts can live in the drinking water and cause pandemic diseases. In Taiwan, very little information is available regarding the epidemiology of G. duodenalis in domestic animals. Methods Fecal samples were collected from cattle (n = 156) and pigs (n = 141) in Hualien country, eastern Taiwan. Detection and genotyping were done by microscopy examination of fecal samples and amplification of the β-giardin gene using nested PCR. Results The prevalence of G. duodenalis infection was 19.87% for cattle (31/156) and 4.26% for pigs (6/141). Using nested PCR, 30 infected samples found in cattle belonged to Assemblage E, and one sample belonged to Assemblage D. For pigs, four samples belonged to Assemblage E, one belonged to Assemblage D, and another one belonged to Assemblage A. In addition, these results showed that G. duodenalis Assemblage A was detected in pigs and may cause zoonotic transmission. Conclusion This is the first epidemiological investigation of G. duodenalis infection in animals in Hualien, Taiwan. These results could provide epidemiological information for disease control and public health protection.

Numerous canine papillomaviruses (CPVs) have been identified (CPV1–23). CPV1, 2, and 6 have been associated with inverted papillomas (IPs). We retrieved 19 IPs from 3 histopathology archives, and evaluated and scored koilocytes, inclusion bodies, giant keratohyalin granules, cytoplasmic pallor, ballooning degeneration, and parakeratosis. IHC targeting major capsid proteins of PV was performed, and CPV genotyping was achieved by PCR testing. Tissue localization of CPV DNA and RNA was studied by chromogenic and RNAscope in situ hybridization (DNA-CISH, RNA-ISH, respectively). IPs were localized to the limbs (50%), trunk (30%), and head (20%), mainly as single nodules (16 of 19). In 15 of 19 cases, immunopositivity was detected within the nuclei in corneal and subcorneal epidermal layers. PCR revealed CPV1 in 11 IPs and CPV2 DNA in 3 IPs. Overall, 14 of 17 cases were positive by both DNA-CISH and RNA-ISH, in accord with PCR results. A histologic score >5 was always obtained in cases in which the viral etiology was demonstrated by IHC, DNA-CISH, and RNA-ISH. IHC and molecular approaches were useful to ascertain the viral etiology of IPs. Although IHC is the first choice for diagnostic purposes, ISH testing allows identification of PV type and the infection phase. RNA-ISH seems a promising tool to deepen our understanding of the pathogenesis of different PV types in animal species.

BackgroundThe association of the Val66Met (rs6265) polymorphism in the brain derived neurotrophic factor (BDNF) gene with bipolar disorder (BD) and response to lithium treatment has been suggested, though inconsistently. The considerable diversity of allele frequency across different populations contributes to this. There is no data from South Asia till date. Hence, we examined the association of this polymorphism in BD cases from India, and its correlation with lithium treatment response.MethodsBD patients (N=301) were recruited from the clinical services of National Institute of Mental Health and Neurosciences (NIMHANS), India. Lithium treatment response for 190 BD subjects was assessed using Alda scale by NIMH life charts. Patients with total score ≥ 7 were defined as lithium responders (N=115) and patients with score ResultsGenotype and allele frequency of BDNF Val66Met SNP was significantly different (χ2 = 7.78, p=0.02) in cases compared to controls, and the Val(G) allele was more frequent (χ2 = 7.08, p=0.008) in BD patients. Although there was trend towards significance for greater occurrence of Val(G) allele among lithium non-responders, this difference did not persist in an analysis of extreme response groups.ConclusionsThe Val(G) allele of BDNF Val66Met polymorphism is associated with risk of BD in this sample, but is not related to response to lithium.

Purpose: study of associations of live weight and genotypic traits in reindeer populations.Materials and methods. The studies were carried out in 2018-2020. on the basis of 8 agricultural enterprises in Chukotka. Tissue samples (ear pinch) of deer of different sex and age groups served as material for genetic studies. In molecular genetic studies, 1002 samples were used. Individual genotyping of animals was carried out using the ISSR-PCR method. The live weight of the reindeer was determined using the materials of the zootechnical reports of the reindeer farms. Associations of the average population indices of genetic diversity and live weight of deer were established by a calculation-statistical method of comparing the values of values. The correlation coefficient was calculated by the product method according to the Pearson formula.Results. The variability of ISSR markers in populations indicates a significant similarity between them in most of the allelic frequencies, which confirms the common origin, economic and breeding use of the Chukchi breed deer. The populations are characterized by a high degree of heterogeneity. Differences in live weight between highly productive and less productive populations on average for all sex and age groups of deer amounted to 16.8%. At the same time, the populations with a high live weight of deer significantly exceeded the populations with a lower live weight in the average number of alleles per locus by 5.0% (P Conclusion. The data obtained make it possible to state the existence of a dependence of the live weight of deer on the genotypic diversity in the populations of the Chukchi breed.

Aquatic invasive species are damaging to native ecosystems. Preventing their spread and achieving comprehensive control measures requires an understanding of the genetic structure of an invasive population. Organellar genomes (plastid and mitochondrial) are useful for population level analyses of invasive plant distributions. In this study we generate complete organellar reference genomes using PacBio sequencing, then use these reference sequences for SNP calling of high-throughput, multiplexed, Illumina based organellar sequencing of fresh and historical samples from across the native and invasive range of Nitellopsis obtusa (Desv. in Loisel.) J.Groves, an invasive macroalgae. The data generated by the analytical pipeline we develop indicate introduction to North America from Western Europe. A single nucleotide transversion in the plastid genome separates a group of five samples from Michigan and Wisconsin that either resulted from introductions of two closely related genotypes or a mutation that has arisen in the invasive range. This transversion will serve as a useful tool to understand how Nitellopsis obtusa moves across the landscape. The methods and analyses described here are broadly applicable to invasive and native plant and algae species, and allow efficient genotyping of variable quality samples, including 100-year-old herbarium specimens, to determine population structure and geographic distributions.

Genome-wide association studies (GWASs) have identified regions associated with chronic obstructive pulmonary disease (COPD). GWASs of other diseases have shown an approximately 10-fold overrepresentation of nonsynonymous variants, despite limited exonic coverage on genotyping arrays. We hypothesized that a large-scale analysis of coding variants could discover novel genetic associations with COPD, including rare variants with large effect sizes. We performed a meta-analysis of exome arrays from 218,399 controls and 33,851 moderate-to-severe COPD cases. All exome-wide significant associations were present in regions previously identified by GWAS. We did not identify any novel rare coding variants with large effect sizes. Within GWAS regions on chromosomes 5q, 6p, and 15q, four coding variants were conditionally significant ( P < 0.00015) when adjusting for lead GWAS single-nucleotide polymorphisms A common gasdermin B ( GSDMB) splice variant (rs11078928) previously associated with a decreased risk for asthma was nominally associated with a decreased risk for COPD [minor allele frequency (MAF) = 0.46, P = 1.8e-4]. Two stop variants in coiled-coil α-helical rod protein 1 ( CCHCR1), a gene involved in regulating cell proliferation, were associated with COPD (both P < 0.0001). The SERPINA1 Z allele was associated with a random-effects odds ratio of 1.43 for COPD (95% confidence interval = 1.17–1.74), though with marked heterogeneity across studies. Overall, COPD-associated exonic variants were identified in genes involved in DNA methylation, cell-matrix interactions, cell proliferation, and cell death. In conclusion, we performed the largest exome array meta-analysis of COPD to date and identified potential functional coding variants. Future studies are needed to identify rarer variants and further define the role of coding variants in COPD pathogenesis.

Genotyping of lentil plant genetic resources holds the promise to increase the identification and utilization of useful genetic diversity for crop improvement. The International Center for Agriculture Research in the Dry Areas (ICARDA) lentil reference set plus collection of 176 accessions was genotyped using genotyping-by-sequencing (GBS) and 22,555 SNPs were identified. The population structure was investigated using Bayesian analysis (STRUCTURE, k = 3) and principal component analysis. The two methods are in concordance. Genome-wide association analysis (GWAS) using the filtered SNP set and ICARDA historical phenotypic data discovered putative markers for several agronomic traits including days to first flower, seeds per pod, seed weight and days to maturity. The genetic and genomic resources developed and utilized in this study are available to the research community interested in exploring the ICARDA reference set plus collection using GWAS.

Background Inherited hypertrophic cardiomyopathy (HCM) is a common heart muscle disease that damages heart function and may cause the heart to suddenly stop beating. Genetic factors play an important role in HCM. Pedigree analysis is a good way to identify the genetic defects that cause disease. Methods An HCM pedigree was determined in Yunnan, China. Whole-exome sequencing was performed to identify the genetic variants of HCM. Another 30 HCM patients and 200 healthy controls were also used to investigate the frequency of the variants by customized TaqMan genotyping assay. Results The variant NM_000257.4:c.3134G > A (NP_000248.2:p.Arg1045His, rs397516178, c.3134G > A in short) was found to cosegregate with the clinical phenotype of HCM. Moreover, the variant was not found in the 200 control subjects. After genotyping the variant in 30 HCM patients, there was one patient who carried the variant and had a family history. Conclusions Our findings suggest that this variant may be closely related to the occurrence of the disease. According the ACMG guidelines, the c.3134G > A variant should be classified as "Likely pathogenic".

Objective Denture induced stomatitis (DIS) is often associated withCandida infection. This study aimed to identify oral Candida species from DIS patients and healthy careers and to evaluate their invasive enzyme production. Methods Candida from DIS patients (n = 38) and healthy careers (n = 23) were identified using chromogenic agar and molecular techniques. Phospholipase, esterase and hemolysin activities of C. albicans were evaluated using in vitro assays. Results Chromogenic agar revealed 29 C. albicans and 09 non-C. albicans Candida among pathogenic isolates. Commensal isolates consisted of 18 C. albicans and 05 non-C. albicans Candida. PCR-RFLP analysis identified C. albicans (76.3 %) as the commonest pathogenic species followed by C. glabrata (13.2 %), C. parapsilosis (5.3 %) and C. tropicalis (5.3 %). Within commensal isolates, C. albicans (78.3 %) was leading followed by C. parapsilosis (13 %), C. glabrata (4.3 %) and C. guilliermondii (4.3 %). Furthermore, ABC genotyping revealed that pathogenic C. albicans genotype A (69 %) was predominant followed by genotypes C (20.7 %) and B (10.3 %). Commensal C. albicans had only the genotype A. Phospholipase, esterase and hemolysin activities of pathogenic C. albicans were significantly higher (P Conclusions C. albicans is the leading Candida species isolated from DIS patients and healthy careers. C. albicans genotype A remains predominant in both pathogenic and commensal isolates while prevalence of genotype C is more than the genotype B among the pathogenic isolates. C. albicans from DIS patients has significantly higher phospholipase, esterase and haemolysin activities compared to commensals which may contribute in pathogenesis.

Genotyping epidermal growth factor receptor (EGFR) is an essential process to indicate lung adenocarcinoma patients for the most appropriate treatment. Liquid biopsy using circulating tumor DNA (ctDNA) potentially complements the use of tumor tissue biopsy for identifying genotype-specific mutations in cancer cells. We assessed the performance of a high-fidelity sequencing method that uses molecular barcodes called the nonoverlapping integrated read sequencing system (NOIR-SS) for detecting EGFR L858R-mutated alleles in 33 advanced or recurrent patients with L858R mutation-positive lung adenocarcinoma revealed by matched tissue biopsy. We compared NOIR-SS with site-specific droplet digital PCR (ddPCR), which was taken as the reference, in terms of sensitivity and ability to quantify L858R variant allele fractions (VAFs). NOIR-SS and ddPCR had sensitivities of 87.9% (29/33) and 78.8% (26/33) for detecting L858R alleles, respectively. The VAFs measured by each assay were strongly correlated. Notably, one specimen was positive with a VAF of 30.12% for NOIR-SS but marginally positive with that of 0.05% for ddPCR because of a previously poorly recognized mechanism: two-base substitution-induced L858R (c.2573_2574delinsGA). These results indicate that NOIR-SS is a useful method for detecting ctDNA, potentially overcoming a limitation of ddPCR which highly depends on the binding ability of primers to specific targeting sequences.

Brucella canis had not been isolated in the Netherlands until November 2016, when it was isolated from a dog imported from Romania. Including this case, 16 suspected cases were notifi ed to the authorities during the following 25 months. Of these 16 dogs, 10 were seropositive; tracking investigations found another 8 seropositive littermates. All seropositive animals were rescue dogs imported from Eastern Europe. B. canis was cultured from urine, blood, and other specimens collected from the dogs. Genotyping of isolates revealed clustering by litter and country. Isolating B. canis in urine indicates that shedding should be considered when assessing the risk for zoonotic transmission. This case series proves introduction of B. canis into a country to which it is not endemic through import of infected dogs from B. canis-endemic areas, posing a threat to the naive autochthonous dog population and humans.

Background: In recent years, the disease outbreak caused by Vibrio harveyi upset the booming development of the Chinese tongue sole (Cynoglossus semilaevis) farming industry. Genomic selection (GS) is a powerful method to improve the traits of interest, which has been proved in livestock and some fishes. Besides, the single nucleotide polymorphism (SNP) array is an efficient genotyping platform that can be used for genetic studies. To improve V. harveyi resistance in C. semilaevis, we firstly constructed a reference group of 1,572 individuals and investigated accuracies of four genomic methods (genomic best linear unbiased prediction (GBLUP), weighted GBLUP, BayesB, and BayesC) at predicting the genomic estimated breeding value (GEBV) using five-fold cross-validation and SNPs varying from 0.5 k to 500 k. Then, an SNP array was developed using the Affymetrix Axiom technology, and its accuracy in genotyping was evaluated by comparing SNPs generated by the array and by the re-sequencing technology. Finally, we selected 44 candidates as the parents of 23 families of C. semilaevis to evaluate the feasibility of the SNP array for GS.Results: all genomic methods outperformed the pedigree-based BLUP (ABLUP) when at least 50 k SNPs used for prediction, of which GBLUP resulted in better estimation than ABLUP when more than 1 k SNPs used. A 38 k SNP array, “Solechip No.1”, was developed with an average of 10.5 kb inter-spacing between two adjacent SNPs. The SNPs generated by the array and by the re-sequencing reached an average consistency of 94.8 %, of which 79.3 % of loci had a more than 90 % of the consistency. The survival rates of these 23 offspring families had a correlation of 0.706 with the family GEBVs (mid-parental GEBVs), and the average survival rate of the top five families in GEBVs (79.1 %) is higher than the bottom five families (58.1 %).Conclusion: GS is an efficient method to improve the V. harveyi resistance in C. semilaevis, and the SNP array “Solechip No.1” is a convenient and reliable tool for the Chinses tongue sole selective breeding practice.

Human leukocyte antigen (HLA) genotyping gains intensive attention due to its critical role in cancer immunotherapy. It is still a challenging issue to generate reliable HLA genotyping results through in silico tools. In addition, the survival impact of HLA alleles in tumor prognosis and immunotherapy remains controversial. In this study, the benchmarking of HLA genotyping on TCGA is performed and a ‘Gun‐Bullet’ model which helps to clarify the survival impact of HLA allele is presented. The performance of HLA class I genotyping is generally better than class II. POLYSOLVER, OptiType, and xHLA perform generally better at HLA class I calling with an accuracy of 0.954, 0.949, and 0.937, respectively. HLA‐HD obtained the highest accuracy of 0.904 on HLA class II alleles calling. Each HLA genotyping tool displayed specific error patterns. The ensemble HLA calling from the top‐3 tools is superior to any individual one. HLA alleles show distinct survival impact among cancers. Cytolytic activity (CYT) was proposed as the underlying mechanism to interpret the survival impact of HLA alleles in the ‘Gun‐Bullet’ model for fighting cancer. A strong HLA allele plus a high tumor mutation burden (TMB) could stimulate intensive immune CYT, leading to extended survival. We established an up to now most reliable TCGA HLA benchmark dataset, composing of concordance alleles generated from eight prevalently used HLA genotyping tools. Our findings indicate that reliable HLA genotyping should be performed based on concordance alleles integrating multiple tools and incorporating TMB background with HLA genotype, which helps to improve the survival prediction compared to HLA genotyping alone., Human leucocyte antigen (HLA) genotyping can provide important insights for tumor prognosis and cancer immunotherapy. We performed comprehensive HLA benchmarking in TCGA cohorts, based on allele concordance results generated from eight in silico tools. Moreover, we investigated the impact of HLA alleles on tumor survival. Our proposed ‘Gun‐Bullet’ model considers immune cytotoxicity to interpret the survival impact of HLA alleles and tumor mutation burden.

Introduction. There has been a recent trend toward a gradual increase in the incidence of rectal cancer and a decrease in the average age of patients. These changes interpret the need to personalize treatment in each case.Objective – to evaluate the association of polymorphic variants of some genes with the results of neoadjuvant chemoradiotherapy of rectal cancer.Materials and methods. We analyzed polymorphic variants of MTHFR, XPD, XRCC1, XRCC1, P53, VEGF, EGFR, TNF, CHEK2 and MMP1 genes in 76 patients with rectal cancer who underwent preoperative chemoradiation therapy followed by surgical treatment. Genotyping was performed by DNA isolation from venous blood leukocytes of the subjects followed by polymerase chain reaction with electrophoretic detection of the result.Results. Statistical analysis of the association of polymorphic variants of the studied genes with the treatment pathomorphosis revealed significance in relation to the MMP1-1607 gene (1G >2G) (p = 0.033). There was also an association of co-carrying polymorphic variants of TNF (G / A) + MMP1 (2G / 2G) genes with grade III–IV therapeutic pathomorphosis (p = 0.007).Conclusion. Carriage of recessive allele of MMP1 gene can be a predictor of favorable prognosis of preoperative chemoradiotherapy in patients with rectal cancer.

Background Malaria patients can have two or more haplotypes in their blood sample making it challenging to identify which haplotypes they carry. In addition, there are challenges in measuring the type and frequency of resistant haplotypes in populations. This study presents a novel statistical method Gibbs sampler algorithm to investigate this issue. Results The performance of the algorithm is evaluated on simulated datasets consisting of patient blood samples characterized by their multiplicity of infection (MOI) and malaria genotype. The simulation used different resistance allele frequencies (RAF) at each Single Nucleotide Polymorphisms (SNPs) and different limit of detection (LoD) of the SNPs and the MOI. The Gibbs sampler algorithm presents higher accuracy among high LoD of the SNPs or the MOI, validated, and deals with missing MOI compared to previous related statistical approaches. Conclusions The Gibbs sampler algorithm provided robust results when faced with genotyping errors caused by LoDs and functioned well even in the absence of MOI data on individual patients.

Objective Behcet's disease is a complex systemic inflammatory vasculitis of incompletely understood etiology. This study was undertaken to investigate genetic associations with Behcet's disease in a diverse multiethnic population. Methods A total of 9,444 patients and controls from 7 different populations were included in this study. Genotyping was performed using an Infinium ImmunoArray-24 v.1.0 or v.2.0 BeadChip. Analysis of expression data from stimulated monocytes, and epigenetic and chromatin interaction analyses were performed. Results We identified 2 novel genetic susceptibility loci for Behcet's disease, including a risk locus in IFNGR1 (rs4896243) (odds ratio [OR] 1.25; P = 2.42 x 10(-9)) and within the intergenic region LNCAROD/DKK1 (rs1660760) (OR 0.78; P = 2.75 x 10(-8)). The risk variants in IFNGR1 significantly increased IFNGR1 messenger RNA expression in lipopolysaccharide-stimulated monocytes. In addition, our results replicated the association (P < 5 x 10(-8)) of 6 previously identified susceptibility loci in Behcet's disease: IL10, IL23R, IL12A-AS1, CCR3, ADO, and LACC1, reinforcing the notion that these loci are strong genetic factors in Behcet's disease shared across ancestries. We also identified >30 genetic susceptibility loci with a suggestive level of association (P < 5 x 10(-5)), which will require replication. Finally, functional annotation of genetic susceptibility loci in Behcet's disease revealed their possible regulatory roles and suggested potential causal genes and molecular mechanisms that could be further investigated. Conclusion We performed the largest genetic association study in Behcet's disease to date. Our findings reveal novel putative functional variants associated with the disease and replicate and extend the genetic associations in other loci across multiple ancestries., National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) [R01AR070148]; NIH [U54GM104938, U19AI082714, UM1AI144292, P30AR053483, P30AR073750], Supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) grant number R01AR070148 to Dr. Sawalha. Recruitment and genotyping of the EuropeanAmerican controls was supported by NIH grants number U54GM104938, U19AI082714, UM1AI144292, P30AR053483, and P30AR073750 to Drs. Guthridge and James. This work was supported by the use of study data downloaded from the dbGaP web site, under dbGaP accession phs000272. v1.p1.

Background/Aims: 17β-hydroxysteroid dehydrogenase 13 (HSD17B13) variants were recently reported to have significantly lower odds of non-alcoholic fatty liver disease (NAFLD). This is a two-part study that aimed to evaluate the association of HSD17B13 variants with NAFLD and its histological severity, and to identify the association of the variants with clinical outcomes in a cohort of biopsy-proven NAFLD patients.Methods: Consecutive biopsy-proven NAFLD patients and controls without fatty liver were recruited for this study between 2009 and 2014. Genotyping for HSD17B13 variants was performed using rhAmp assays. A total of 165 patients with NAFLD were monitored up until August 2019. Clinical outcomes were recorded.Results: HSD17B13 rs72613567 TA allele and rs6834314 G allele were associated with lower odds of non-alcoholic steatohepatitis (NASH) in the overall cohort and among ethnic Chinese, but not among ethnic Malays or Indians (P

Purpose We previously developed Haploseek, a method for comprehensive preimplantation genetic testing (PGT). However, some key features were missing, and the method has not yet been systematically validated. Methods We extended Haploseek to incorporate DNA from embryo grandparents and to allow testing of variants on chromosome X or in regions where parents share common haplotypes. We then validated Haploseek on 151 embryo biopsies from 27 clinical PGT cases. We sequenced all biopsies to low coverage (0.2×), and performed single-nucleotide polymorphism (SNP) microarray genotyping on the embryos' parents and siblings/grandparents. We used the extended Haploseek to predict chromosome copy-number variants (CNVs) and relevant variant-flanking haplotypes in each embryo. We validated haplotype predictions for each clinical sample against polymerase chain reaction (PCR)-based PGT case results, and CNV predictions against established commercial kits. Results For each of the 151 embryo biopsies, all Haploseek-derived haplotypes and CNVs were concordant with clinical PGT results. The cases included 17 autosomal dominant, 5 autosomal recessive, and 3 X-linked monogenic disorders. In addition, we evaluated 1 Robertsonian and 2 reciprocal translocations, and 17 cases of chromosome copy-number counting were performed. Conclusion Our results demonstrate that Haploseek is clinically accurate and fit for all standard clinical PGT applications.

An outbreak of blackleg and soft rot of potato, caused primarily by the bacterial pathogen Dickeya dianthicola, has resulted in significant economic losses in the northeastern United States since 2015. The spread of this seedborne disease is highly associated with seed distribution; therefore, the pathogen likely spread with seed tubers. To describe the blackleg epidemic and track inoculum origins, a total of 1,183 potato samples were collected from 11 states associated with blackleg outbreak from 2015 to 2019. Of these samples, 39.8% tested positive for D. dianthicola. Seventeen isolates of D. dianthicola were recovered from these samples and the genetic diversity of these isolates was examined. Fingerprinting with BOX-A1R-based repetitive extragenic palindromic PCR and phylogenetic analysis based on sequences of the 16S rRNA and gapA genes indicated that D. dianthicola isolates were divided into three genotypes, denoted types I, II, and III. Ninety-five percent of samples from Maine were type I. Type II was found in Maine only in 2015 and 2018. Type II was present throughout the 5 years in some states at a lower percentage than type I. Type III was found in Pennsylvania, New Jersey, and Massachusetts, but not in Maine. Therefore, type I appears to be associated with Maine, but type II appeared to be distributed throughout the northeastern United States. The type II and rarer type III strains were closer to the D. dianthicola type strain isolated from the United Kingdom. This work provides evidence that the outbreak of blackleg of potato in the northeastern United States was caused by multiple strains of D. dianthicola. The geographic origins of these strains remain unknown.

Aim: Methadone exhibits significant variability in clinical response. This study explores the genetic influence of variable methadone pharmacokinetics. Methods: This is a prospective study of methadone in children undergoing major surgery. CYP2B6 genotyping, plasma methadone and metabolite levels were obtained. Clinical outcomes include pain scores and postoperative nausea and vomiting (PONV). Results: CYP2B6 poor metabolizers (*6/*6) had >twofold lower methadone metabolism compared with normal/rapid metabolizers. The incidence of PONV was 4.7× greater with CYP2B6 rs1038376 variant. AG/GG variants of rs2279343 SNP had 2.86-fold higher incidence of PONV compared with the wild variant ( AA). Nominal associations between rs10500282, rs11882424, rs4803419 and pain scores were observed. Conclusion: We have described novel associations between CYP2B6 genetic variants and perioperative methadone metabolism, and associations with pain scores and PONV.

The Human Leukocyte Antigen (HLA) system has a critical role in immunorecognition, transplantation, and disease association. Early typing techniques provided the foundation for genotyping methods that revealed HLA as one of the most complex, polymorphic regions of the human genome. Next Generation Sequencing (NGS), the latest molecular technology introduced in clinical tissue typing laboratories, has demonstrated advantages over other established methods. NGS offers high-resolution sequencing of entire genes in time frames and price points considered unthinkable just a few years ago, contributing a wealth of data informing histocompatibility assessment and standards of clinical care. Although the NGS platforms share a high-throughput massively parallel processing model, differing chemistries provide specific strengths and weaknesses. Research-oriented Third Generation Sequencing and related advances in bioengineering continue to broaden the future of NGS in clinical settings. These diverse applications have demanded equally innovative strategies for data management and computational bioinformatics to support and analyze the unprecedented volume and complexity of data generated by NGS. We discuss some of the challenges and opportunities associated with NGS technologies, providing a comprehensive picture of the historical developments that paved the way for the NGS revolution, its current state and future possibilities for HLA typing.