Sequence Variants Research Articles

Breast cancer screening in BRCA1/2 pathogenic sequence variant carriers during pregnancy and lactation

ObjectivesWomen harboring germline BRCA1/BRCA2 pathogenic sequence variants (PSVs) are at an increased risk for breast cancer. There are no established guidelines for screening during pregnancy and lactation in BRCA carriers. The aim of this study was to evaluate the utility of whole-breast ultrasound (US) screening in pregnant and lactating BRCA PSV carriers. MethodsData were retrospectively collected from medical records of BRCA PSV carriers between 2014 and 2020, with follow-up until 2021. Associations between imaging intervals, number of examinations performed and pregnancy-associated breast cancers (PABCs) were examined. PABCs and cancers diagnosed at follow-up were evaluated and characteristics were compared between the two groups. ResultsOverall 212 BRCA PSV carriers were included. Mean age was 33.6 years (SD 3.93, range 25–43 years). During 274 screening periods at pregnancy and lactation, eight (2.9 %) PABCs were diagnosed. An additional eight cancers were diagnosed at follow-up. Three out of eight (37.5 %) PABCs were diagnosed by US, whereas clinical breast examination (n = 3), mammography (n = 1) and MRI (n = 1) accounted for the other PACB diagnoses. One PABC was missed by US. The interval from negative imaging to cancer diagnosis was significantly shorter for PABCs compared with cancers diagnosed at follow-up (3.96 ± 2.14 vs. 11.2 ± 4.46 months, P = 0.002). ConclusionIn conclusion, pregnant BRCA PSV carriers should not delay screening despite challenges like altered breast tissue and hesitancy towards mammography. If no alternatives exist, whole-breast ultrasound can be used. For lactating and postpartum women, a regular screening routine alternating between mammography and MRI is recommended.

Swarm and UNOISE outperform DADA2 and Deblur for denoising high-diversity marine seafloor samples.

The performance of sequence variant resolution analytic tools for metabarcoding has not yet been adequately benchmarked for high-diversity environmental samples. We therefore evaluated the sequence variant tools DADA2, Deblur, Swarm, and UNOISE, using high-diversity seafloor samples, resulting in comparisons of 1800 sequence variant tables. The evaluation was based on 30 sediment grab samples, for which 3 replica samples were collected. Each replica sample was extracted using 5 common DNA extraction kits, resulting in 450 DNA extracts which were 16S rRNA gene sequenced (V3-V4), using Illumina. Assessments included variation across replica samples, extraction kits, and denoising methods, in addition to applying prior knowledge about alpha diversity correlations toward the cosmopolitan marine archaeon Nitrosopumilus with high diversity and the sulfide oxidizing Sulfurovum with low diversity. DADA2 displayed the highest variance between replicates (Manhattan distance 1.14), while Swarm showed the lowest variance (Manhattan distance 0.93). For the analysis based on prior biological knowledge, UNOISE displayed the highest alpha diversity (Simpson's D) correlation toward Nitrosopumilus (Spearman rho = 0.85), while DADA2 showed the lowest (Spearman rho = 0.10). Deblur completely eliminated Nitrosopumilus from the dataset. For Sulfurovum, on the other hand, all the methods showed comparable results. In conclusion, our evaluations show that Swarm and UNOISE performed better than DADA2 and Deblur for high-diversity seafloor samples.

Unraveling noncoding DNA variants and epimutations: a paradigm shift in hereditary cancer research.

Exhaustive efforts have been dedicated to uncovering genomic aberrations linked to cancer susceptibility. Noncoding sequence variants and epigenetic alterations significantly influence gene regulation and could contribute to cancer development. However, exploring noncoding regions in hereditary cancer susceptibility demands cutting-edge methodologies for functionally characterizing genomic discoveries. Additionally, comprehending the impact on cancer development of variants in noncoding DNA and the epigenome necessitates integrating diverse data through bioinformatic analyses. As novel technologies and analytical methods continue to advance, this realm of research is rapidly gaining traction. Within this mini-review, we delve into future research domains concerning aberrations in noncoding DNA regions, such as pseudoexons, promoter variants and cis-epimutations.

Bacterial community of ticks (Acari: Ixodidae) and mammals from Arauca, Colombian Orinoquia

Ticks are obligate hematophagous ectoparasites of vertebrates and are relevant worldwide due to the number of bacterial and other pathogens they can transmit. To date, the knowledge about the microorganisms that ticks harbor and transmit to their hosts is incipient. In this study, 24 samples of mammals belonging to four taxonomic orders and ticks of the genera Amblyomma and Rhipicephalus from the Orinoco region of Colombia were analyzed to described and compare the bacterial microbiome. Genetic extraction was performed, and the V3–V4 region of the 16S rRNA gene was amplified by PCR. Libraries were created, and those samples with adequate quality indices were sequenced using Illumina MiSeq technology. Bacterial taxonomic assignment analyses were conducted through Amplicon Sequence Variants (ASVs) and Operational Taxonomic Units (OTUs). The results correspond to 16 samples that passed the quality filters, with 3218 OTUs (415 families). Although a considerable number of unknown bacteria was found, Enterobacteriaceae, Beijerinckiaceae, Moraxellaceae, and Burkholderiaceae are the most prevalent families, and the presence of the genera Coxiella, Escherichia-Shigella, Enterobacter, which can harbor pathogenic species was confirmed. In individuals of Amblyomma mixtum found actively feeding on Hydrochoerus hydrochaeris, bacteria of the genera Escherichia-Shigella and Enterobacter were documented. Similarly, Rhipicephalus microplus found actively feeding on Odocoileus virginianus cariacou shared Escherichia-Shigella. Ralstonia was shared among the blood samples of H. hydrochaeris, while Anaplasma and Eubacterium were shared in blood and liver samples of O. v. cariacou. Shared bacteria between A. mixtum and R. microplus included Bacillus, Coxiella, and Escherichia-Shigella. The results highlight the need of additional studies in other natural regions of Colombia and other American countries where tick-borne diseases have been detected. Likewise, the recorded data are the first at the level of bacterial communities in ticks of the family Ixodidae and provide valuable knowledge for the understanding host-tick and pathogen interactions.

Temporal and spatial diversity and abundance of cryptophytes in San Diego coastal waters.

Cryptophytes (class Cryptophyceae) are bi-flagellated eukaryotic protists with mixed nutritional modes and cosmopolitan distribution in aquatic environments. Despite their ubiquitous presence, their molecular diversity is understudied in coastal waters. Weekly 18S rRNA gene amplicon sequencing at the Scripps Institution of Oceanography pier (La Jolla, California) in 2016 revealed 16 unique cryptophyte amplicon sequence variants (ASVs), with two dominant "clade 4" ASVs. The diversity of cryptophytes was lower than what is often seen in other phytoplankton taxa. One ASV represented a known Synechococcus grazer, while the other one appeared not to have cultured representatives and an unknown potential for mixotrophy. These two dominant ASVs were negatively correlated, suggesting possible niche differentiation. The cryptophyte population in nearby San Diego Bay was surveyed in 2019 and showed the increasing dominance of a different clade 4 ASV toward the back of the bay where conditions are warmer, saltier, and shallower relative to other areas in the bay. An ASV representing a potentially chromatically acclimating cryptophyte species also suggested that San Diego Bay exerts differing ecological selection pressures than nearby coastal waters. Cryptophyte and Synechococcus cell abundance at the SIO Pier from 2011 to 2017 showed that cryptophytes were consistently present and had a significant correlation with Synechococcus abundance, but no detectable seasonality. The demonstrated mixotrophy of some cryptophytes suggests that grazing on these and perhaps other bacteria is important for their ecological success. Using several assumptions, we calculated that cryptophytes could consume up to 44% (average 6%) of the Synechococcus population per day. This implies that cryptophytes could significantly influence Synechococcus abundance.

Metagenomic evaluation of peanut rhizosphere microbiome from the farms of Saurashtra regions of Gujarat, India

The narrow zone of soil around the plant roots with maximum microbial activity termed as rhizosphere. Rhizospheric bacteria promote the plant growth directly or indirectly by providing the nutrients and producing antimicrobial compounds. In this study, the rhizospheric microbiota of peanut plants was characterized from different farms using an Illumina-based partial 16S rRNA gene sequencing to evaluate microbial diversity and identify the core microbiome through culture-independent (CI) approach. Further, all rhizospheric bacteria that could grow on various nutrient media were identified, and the diversity of those microbes through culture-dependent method (CD) was then directly compared with their CI counterparts. The microbial population profiles showed a significant correlation with organic carbon and concentration of phosphate, manganese, and potassium in the rhizospheric soil. Genera like Sphingomicrobium, Actinoplanes, Aureimonas _A, Chryseobacterium, members from Sphingomonadaceae, Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae family, and Bacilli class were found in the core microbiome of peanut plants. As expected, the current study demonstrated more bacterial diversity in the CI method. However, a higher number of sequence variants were exclusively present in the CD approach compared to the number of sequence variants shared between both approaches. These CD-exclusive variants belonged to organisms that are more typically found in soil. Overall, this study portrayed the changes in the rhizospheric microbiota of peanuts in different rhizospheric soil and environmental conditions and gave an idea about core microbiome of peanut plant and comparative bacterial diversity identified through both approaches.

Effect of prebiotic fiber on physical function and gut microbiota in adults, mostly women, with knee osteoarthritis and obesity: a randomized controlled trial.

Obesity is a primary risk factor for knee osteoarthritis (OA). Prebiotics enhance beneficial gut microbes and can reduce body fat and inflammation. Our objective was to examine if a 6-month prebiotic intervention improved physical function in adults with knee osteoarthritis and obesity. We also measured knee pain, body composition, quality of life, gut microbiota, inflammatory markers, and serum metabolomics. Adults (n = 54, mostly women) with co-morbid obesity (BMI > 30kg/m2) and unilateral/bilateral knee OA were randomly assigned to prebiotic (oligofructose-enriched inulin; 16g/day; n = 31) or isocaloric placebo (maltodextrin; n = 21) for 6months. Performance based-tests, knee pain, quality of life, serum metabolomics and inflammatory markers, and fecal microbiota and short-chain fatty acids were assessed. Significant between group differences were detected for the change in timed-up-and-go test, 40m fast paced walk test, and hand grip strength test from baseline that favored prebiotic over placebo. Prebiotic also reduced trunk fat mass (kg) at 6months and trunk fat (%) at 3months compared to placebo. There was a trend (p = 0.059) for reduced knee pain at 6months with prebiotic versus placebo. In gut microbiota analysis, a total of 37 amplicon sequence variants differed between groups. Bifidobacterium abundance was positively correlated with distance walked in the 6-min walk test and hand grip strength. At 6months, there was a significant separation of serum metabolites between groups with upregulation of phenylalanine and tyrosine metabolism with prebiotic. Prebiotics may hold promise for conservative management of knee osteoarthritis in adults with obesity and larger trials are warranted. Clinicaltrials.gov/study/NCT04172688.

Microbial community and extracellular polymeric substance dynamics in arid–zone temporary pan ecosystems

Microbial extracellular polymeric substances (EPS) are an important component in sediment ecology. However, most research is highly skewed towards the northern hemisphere and in more permanent systems. This paper investigates EPS (i.e., carbohydrates and proteins) dynamics in arid Austral zone temporary pans sediments. Colorimetric methods and sequence–based metagenomics techniques were employed in a series of small temporary pan ecosystems characterised by alternating wet and dry hydroperiods. Microbial community patterns of distribution were evaluated between seasons (hot–wet and cool–dry) and across depths (and inferred inundation period) based on estimated elevation. Carbohydrates generally occurred in relatively higher proportions than proteins; the carbohydrate:protein ratio was 2.8:1 and 1.6:1 for the dry and wet season respectively, suggesting that EPS found in these systems was largely diatom produced. The wet– hydroperiods (Carbohydrate mean 102 μg g−1; Protein mean 65 μg g−1) supported more EPS production as compared to the dry– hydroperiods (Carbohydrate mean 73 μg g−1; Protein mean 26 μg g−1). A total of 15,042 Unique Amplicon Sequence Variants (ASVs) were allocated to 51 bacterial phyla and 1127 genera. The most abundant genera had commonality in high temperature tolerance, with Firmicutes, Actinobacteria and Proteobacteria in high abundances. Microbial communities were more distinct between seasons compared to within seasons which further suggested that the observed metagenome functions could be seasonally driven. This study's findings implied that there were high levels of denitrification by mostly nitric oxide reductase and nitrite reductase enzymes. EPS production was high in the hot–wet season as compared to relatively lower rates of nitrification in the cool–dry season by ammonia monooxygenases. Both EPS quantities and metagenome functions were highly associated with availability of water, with high rates being mainly associated with wet– hydroperiods compared to dry– hydroperiods. These data suggest that extended dry periods threaten microbially mediated processes in temporary wetlands, with implications to loss of biodiversity by desiccation.

Genetic Testing Resources and Practice Patterns Among Pediatric Cardiomyopathy Programs.

The use of genetic testing has enhanced the diagnostic accuracy of heritable genetic cardiomyopathies. However, it remains unclear how genetic information is interpreted and incorporated into clinical practice for children with cardiomyopathy. The primary aim of this study was to understand how clinical practice differs regarding sequence variant classifications amongst pediatric cardiologists who treat children with cardiomyopathy. A secondary aim was to understand the availability of genetic testing and counseling resources across participating pediatric cardiomyopathy programs. An electronic survey was distributed to pediatric heart failure, cardiomyopathy, or heart transplantation physicians between August and September 2022. A total of 106 individual providers from 68 unique centers responded to the survey. Resources for genetic testing and genetic counseling vary among large pediatric cardiomyopathy programs. A minority of centers reported having a geneticist (N = 16, 23.5%) or a genetic counselor (N = 21, 31%) on faculty within the division of pediatric cardiology. A total of 9 centers reported having both (13%). Few centers (N = 13, 19%) have a formal process in place to re-engage patients who were previously discharged from cardiology follow-up if variant reclassification would alter clinical management. Clinical practice patterns were uniform in response to pathogenic or likely pathogenic variants but were more variable for variants of uncertain significance. Efforts to better incorporate genetic expertise and resources into the clinical practice of pediatric cardiomyopathy may help to standardize the interpretation of genetic information and better inform clinical decision-making surrounding heritable cardiomyopathies.

Sequence variants of unknown significance in small fiber neuropathy : Characterization of a heterogeneous patient population

In almost half of patients suffering from small fiber neuropathies (SFN), the etiology remains elusive. For these patients with "idiopathic SFN", symptomatic analgesic therapy is the only option. Reports on apotential genetic background of neuropathic pain syndromes are increasing and particularly in SFN patients, several genetic variants were found mainly located in genes encoding voltage-gated sodium channels. Although up to 30% of SFN patients show genetic alterations, most of these remain of "unknown pathogenic significance" and little is known about "genetic SFN". The study aimed to determine clinical characteristics of SFN patients carrying arare genetic variant of unknown significance in pain-associated genes. From 2015 to 2020, 66patients with primarily idiopathic SFN were examined and rare gene variants of unknown significance detected in 13/66 (20%) of these. Adetailed medical history with focus on pain was recorded and patients filled in standardized questionnaires to assess physical and emotional burden due to pain. The authors found 13/66 (20%) patients with rare variants of unknown significance located in pain-associated genes who reported pain refractory to analgesic treatment, ahigher number of external factors influencing clinical symptoms, and ahigher level of physical impairment and emotional stress due to pain compared with patients without such genetic variants. Early genetic assessment is recommended to optimize the management of patients with potentially hereditary SFN. Early access to rehabilitation and mental support as well as aconsequent elimination of external triggering factors should be granted.

Orodental malformations associated with human MSX1 sequence variants

BackgroundMSX1 sequence variants have been known to cause human tooth agenesis (TA) with or without orofacial clefts. However, their roles during the whole processes of tooth development are not fully understood. This study aimed to characterize a 4-membered family with TA carrying a novel MSX1 pathogenic variant and investigate the disease mechanism. MethodsThe authors conducted whole exome analysis to define the disease-causing sequence variant. They performed microcomputed tomography, morphometric analyses, transcriptome profiling, and molecular characterization to study the affected teeth and the gene variant. ResultsThe authors identified an MSX1 pathogenic variant, p.Glu232∗, in affected family members with TA and concomitant orodental anomalies, namely, prominent maxillary labial frenum, central incisor diastema, median maxillary anterior alveolar cleft, tooth fusion, mandibular molar dysmorphology, thin dentin layer, and slender dental roots. MSX1-defective teeth were not apparently microdontic but had thin dentin layers. The mandibular molars showed a homeotic transformation to maxillary counterparts. Genes involved in extracellular matrix organization and dentinogenesis, such as DMP1 and MMP20, were downregulated in dental pulp tissues of MSX1-defective teeth. The p.Glu232∗-truncated MSX1 properly localized to the nucleus but partially lost its transactivation ability. Analyzing reported cases indicated that truncation sequence variants within the homeobox domain of MSX1 caused a more severe TA phenotype than those outside of the homeobox domain, probably due to dominant negativity compared with haploinsufficiency. ConclusionsThis study provides in vivo evidence that MSX1 contributes to developmental processes of various orodental tissues in humans. Practical ImplicationsClinically, hypertrophic labial frenum, incisor diastema, and median maxillary anterior alveolar cleft might be considered diagnostic for MSX1-associated TA.

226 Oral and rumen microbiome characterization to predict host phenotypes

Abstract Studies have utilized rumen microbiome composition and function to predict animal phenotypes. Yet, sample collection for rumen microbial community analysis can be difficult and invasive and hinders the possibility of sampling large animal populations. In this study, we investigated the development of the bovine rumen microbiome over time, to identify the potential of the oral microbiome as a proxy for the rumen microbiome, and to test the applicability of using the oral microbiome for predicting phenotypes in beef cattle. We employed 16S rRNA amplicon sequencing to characterize the rumen and oral bacterial community structure over time and collected both birth and weaning weights in a population of 220 animals. Our findings show that both the oral and rumen microbiomes are established early, and a significant portion of the taxa persists even after weaning. We identified a group of core successional species shared between the oral and rumen bacterial communities, which are established early and persist over time. The oral bacterial community was dominated by phyla Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria, while the rumen bacterial community was dominated by phyla Bacteroidota, Firmicutes, Verrucomicrobia, Fibrobacteria, and Spirochaetota. Furthermore, we observed that 38% of Amplicon Sequence Variants (ASVs) were shared between the oral and rumen microbiomes at 50 to 120 d of age. At 170 to 240 d of age, 37% of ASVs were shared, and at 295 to 365 d, 33% of ASVs were shared. Finally, at 400 to 468 d, 21% of ASVs were shared. Based on weighted Unifrac analysis, notable differences and distinct clustering was observed between oral and rumen microbiome communities at different sampling ages (P = 0.001; R2 = 0.50969, 50 to 120; P = 0.001; R2 = 0.66247, 170 to 240; P = 0.001; R2 = 0.62036, 295 to 365; and P = 0.001, R2 = 0.66559, 400 to 468). Microbiability estimates varied across different stages and microbial environments. For the oral bacterial community at 50 to 120, the estimates were 0.029%, 0.032%, 0.029%, and 0.028% for birth weight, weaning weight, intramuscular fat (IMF), and ribeye area (REA), respectively. In contrast, the rumen microbiome exhibited microbiability values of 0.034%, 0.058, 0.059, and 0.069% for birth weight, weaning weight, IMF, and REA respectively during the same period. As the animal aged the microbiability increased. At 170 to 240, oral microbiomes showed microbiability estimates of 0.051%, 0.060%, 0.069%, and 0.21% for birth weight, weaning weight, IMF, and REA respectively. In comparison, the rumen microbiome displayed greater microbiability estimates of 0.12%, 0.124%, 0.20%, and 0.26% for birth weight, weaning weight, IMF, and REA respectively. In conclusion, the oral microbiome is not a good proxy for the rumen microbiome composition; however, there is potential to use the oral microbiome for phenotype prediction.

3 The impact of early-life cecal microbiota transplantation on injurious behaviors in egg-laying chickens

Abstract Injurious behaviors in laying hens are a critical issue facing the poultry egg industry. Recent studies have shown that the gut microbiota influences host brain function and behavioral characteristics in humans and various animals. In laying hens, it has been reported that injurious behaviors (such as aggression, feather pecking and cannibalism) are associated with dysregulation of the microbiota-gut-brain axis. To further interrogate the role of the microbiota-gut-brain axis on bird behavior, we used two inbred layer chicken lines, 63 and 72 (line 72 displays more aggressive behavior), and transferred pooled cecal contents from either line to day of hatch chicks. Cecal microbiota transplantation (CMT) from each line was conducted once daily, by gavage, from d 1 to 10, then boosted once weekly from wk 3 to 5. We hypothesized that the cecal microbiota composition and animal behavior in recipient birds would be similar to that of their donor animals because of the microbiota-gut-brain axis. Microbial communities were compared using 16S rRNA gene amplicon sequencing, behavior was recorded in home cages as well as in paired aggression tests, and physiological measurements were taken from brain tissue and blood to measure levels of neurotransmitters and immune function. When examining the differences in the donor birds, hypothalamic serotonin and tryptophan concentrations were greater for line 63 compared with line 72 birds (P < 0.05). Plasma corticosterone, heterophil/lymphocyte ratios, and central norepinephrine concentrations were less for line 63 birds (P < 0.05). Cecal microbial diversity was not statistically different between the lines, but many bacterial taxa were differentially abundant between the two lines. After cecal transplantation, 63-CMT (recipient) birds displayed less aggressive behavior with a greater hypothalamic serotonergic activity at wk 5, similar to donor birds. Correspondingly, two amplicon sequence variants (ASVs) belonging to Lachnospiraceae and one Ruminococcaceae UCG-005 ASV were positively correlated with the concentrations of brain tryptophan and serotonin, respectively. Along with greater aggression, 72-CMT birds seemed to have greater physical growth traits (increased body weight, ileal villus/crypt; P ≤ 0.05), but decreased concentrations of brain norepinephrine and dopamine, and the greatest ileal serotonin turnover in the ileum at wk 5 (P < 0.05). ASVs belonging to Mollicutes RF39 and GCA-900066225 in 72-CMT birds were negatively correlated with the brain 5-hydroxyindoleacetic acid (5-HIAA) at wk 5, and one Bacteroides ASV was negatively correlated with plasma serotonin at wk 16. Meanwhile, 63-CMT birds potentially had better gut health and immune function (greater concentrations of ileal mucosal secretory IgA, plasma IL-10; P < 0.05). These results suggest linkages among serotonergic activity, stress response, innate immunity, and gut microbiota populations and that CMT could be a novel strategy for reducing aggressive behavior through regulating signaling along the microbiota-gut-brain axis.

Identification of TP53 germline variants in pediatric patients undergoing tumor testing: strategy and prevalence.

TP53 alterations are common in certain pediatric cancers, making identification of putative germline variants through tumor genomic profiling crucial for disease management. We analyzed TP53 alterations in 3123 tumors from 2788 pediatric patients sequenced using tumor-only or tumor-normal paired panels. Germline confirmatory testing was performed when indicated. Somatic and germline variants were classified based on published guidelines. In 248 tumors from 222 patients, 284 tier 1/2 TP53 sequence and small copy number variants were detected. Following germline classification, 86.6% of 142 unique variants were pathogenic or likely pathogenic. Confirmatory testing on 118 patients revealed germline TP53 variants in 28 of them (23 pathogenic or likely pathogenic and 5 of uncertain significance), suggesting a minimum Li-Fraumeni syndrome incidence of 0.8% (23/2788) in this cohort, 10.4% (23/222) in patients with TP53 variant-carrying tumors, and 19.5% (23/118) with available normal samples. About 25% (7/28) of patients with germline TP53 variants did not meet Li-Fraumeni syndrome diagnostic or testing criteria, while 20.9% (28/134) with confirmed or inferred somatic origins did. TP53 biallelic inactivation occurred in 75% of germline carrier tumors and was also prevalent in other groups, causing an elevated tumor-observed variant allelic fraction. Somatic evidence, however, including low variant allele fraction correctly identified only 27.8% (25/90) of patients with confirmed somatic TP53 variants. The high incidence and variable phenotype of Li-Fraumeni syndrome in this cohort highlights the importance of assessing germline status of TP53 variants identified in all pediatric tumors. Without clear somatic evidence, distinguishing somatic from germline origins is challenging. Classifying germline and somatic variants should follow appropriate guidelines.

Impact of root canal disinfection on the bacteriome present in primary endodontic infection: A next generation sequencing study.

To investigate the bacteriome present in teeth with primary endodontic infection (PEI) and apical periodontitis (AP) and to determine quantitatively and qualitatively the impact of chemomechanical preparation (CMP) using 2.5% sodium hypochlorite NAOCl on the bacteriome found in PEI with AP using the Illumina MiSeq platform. Thirty-six paired samples from 18 patients were successfully sequenced and analysed. Samples were collected at two sampling times: before (s1) and after (s2) CMP using 2.5% NaOCl. The DNA was extracted from s1 and s2 samples and quantified using quantitative PCR (qPCR). All 36 samples were sequenced using the Illumina MiSeq platform. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants (ASVs). Alpha diversity metrics representing abundance (Chao1) and diversity and evenness (Shannon, Simpson) were computed. The paired-sample Wilcoxon's test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The PERMANOVA method (with 999 permutations) was applied to compare community composition between sample types (s1 versus s2) and between patient IDs. ALDEx2 (ANOVA-like differential expression tool for high-throughput sequencing data) to investigate differentially abundant taxa between s1 and s2. A paired-sample Wilcoxon's test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The qPCR counts were significantly higher in s1 compared to s2 (p = .0007). The Chao1 index indicated no difference in alpha diversity (p < .7019); whereas Shannon (p = .0056) and Simpson (p = .02685) indexes showed higher values in s2. The PERMANOVA test using Adonis2 showed a significant effect of sample time on community composition (R2 = .0630, p = .012). Patient ID also showed a significant effect on community composition (R2 = .6961, p = .001). At the genus level, Dialister, Mogibacterium, Prevotella, and Olsenella were differentially enriched at s1, while Actinomyces, Stenotrophomonas_unclassified, Enterococcus_unclassified, and Actinomyces_unclassified were differentially enriched in s2. The bacteriome present in teeth with PEI with AP is complex and diverse. CMP using 2.5% NaOCl showed a high quantitatively and qualitatively disinfectant impact on the bacteriome present in PEI with AP.

Abstract PO1-08-02: Population-based screening of Uruguayan Ashkenazi Jews for recurrent BRCA1 and BRCA2 pathogenic sequence variants

Abstract In Ashkenazi Jews (AJ) three recurring pathogenic sequence variants (PSVs) are detected in ~2.5% of the general population in the BRCA1 (c.68_69del = 185delAG, c.5266dup = 5382insC), and BRCA2 (c.5946del = 6174delT). Population-based screening for these PSVs in AJ women is part of the health basket in Israel. To assess the feasibility and outcome of BRCA genotyping in the Jewish population of Uruguay, AJ in the greater Montevideo area were recruited using ethically approved protocol and without pretest counseling were genotyped for the three predominant AJ PSVs in the BRCA genes. Independently confirmed PSV carriers were counseled, and genetic testing was offered to additional family members. Overall, 327 participants were enrolled: 312 (95%) female, 261 (80%) had all four grandparents AJ, and 14 (4%) women were breast cancer survivors with a mean age ± standard deviation (SD) 50 ± 11.5 years. The BRCA1 c.68_69del PSV was detected in three cancer free participants (0.92%, CI 95% 0.31-2.6), all with a suggestive family history. No carriers of the other two recurrent PSVs were detected. Online oncogenetic counseling was provided for all carriers. In conclusion, the rate of the BRCA1 c.68_69del PSV was similar with the rate in other AJ communities. AJ population BRCA genotyping screens in Uruguay seem feasible and should be promoted. Pedigrees of the carrier cases (a-c) Citation Format: Cecilia Castillo, Natalia Camejo, Nora Artagaveytia, Lucia Brignoni, Yael Laitman, Alfonso Cayota, Gabriel Krygier, Lucia Delgado, Eitan Friedman. Population-based screening of Uruguayan Ashkenazi Jews for recurrent BRCA1 and BRCA2 pathogenic sequence variants [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-08-02.

Salivary microbiome and MRP-8/14 levels in children with gingivitis, healthy children, and their mothers.

Gingivitis is the most common form of periodontal disease among children and adolescents and is associated with disrupted host-microbiome homeostasis. Family is an important factor influencing the prevalence of gingivitis. In the present study, we investigated the salivary microbiome, oral hygiene habits, and the salivary level of myeloid-related protein (MRP)-8/14 in children aged 7-12 years with gingivitis, periodontally healthy children, and their mothers. This study included 24 children with gingivitis (including four sibling pairs) and 22 periodontally healthy children (including two sibling pairs) and their mothers. The whole saliva was collected, DNA was extracted, the variable V3-V4 region of the eubacterial 16S ribosomal RNA gene was amplified, and sample library preparation was performed according to the Illumina protocol. The salivary levels of MRP-8/14 were analyzed by ELISA. Alpha diversity of the salivary microbiome was considerably higher in gingivitis children and mothers of gingivitis children compared to healthy children and their mothers, respectively. Significant differences in beta diversity between healthy and gingivitis children, healthy children and their mothers, and gingivitis children and their mothers were detected. Overall, the number of common core amplicon sequence variants between children and their own mothers was significantly higher than between children and other mothers. The salivary MRP-8/14 levels in children with gingivitis were significantly higher compared to healthy children; a similar tendency was also mentioned for mothers. Our study underlines the importance of family as an essential factor influencing oral health.

Comprehensive profiling of endocrine metabolism identifies a novel signature with robust predictive value in ovarian cancer.

The cell endocrine pathway is a critical physiological process composed of the endoplasmic reticulum, Golgi apparatus and associated vesicles. Loss of enzymes or proteins can cause dysfunction of endoplasmic reticulum and Golgi apparatus and affect secretion pathways leading to a variety of human diseases, including cancer. The single-cell RNA sequencing and single nucleotide variant principal component analysis data of ovarian cancer were retrieved from The Cancer Genome Atlas and Gene Expression Omnibus (GEO) datasets. Eighty-four genes from SECRETORY_PATHWAYs were obtained from the gene set enrichment analysis (GSEA) website. Univariate cox regression analyses and ConsensusClusterPlus were used to identify prognostic genes and molecular subtypes, which were validated using the tumor immune dysfunction and exclusion (i.e. TIDE) analysis and gene mutation analysis. A prognosis model was established by randomForestSRC. Abundant infiltrated immune cells and pathway enrichment analyses were carried out, respectively, through ssGSEA, ESTIMATE, MCP-counter and GSEA. The drug sensitive analysis was performed using pRRophetic package. Immunotherapy datasets and pan-carcinoma analysis were used to examine the performance of prognostic model. Eighteen prognostic genes from SECRETORY_PATHWAYs were found in both TCGA and GEO datasets. Next, two clusters (C1 and C2) were determined, for which C1 with a poor prognosis had higher immune infiltration. Tumor-related pathways, such as PATHWAYS_IN_CANCER and B_CELL_RECEPTOR_SIGNALING_PATHWAY, were enriched in C1. Moreover, C2 was suitable for immunotherapy. A four-gene (DNAJA1, NDRG3, LUZP1 and ZCCHC24) signature was developed and successfully validated. RiskScore of higher levels were significantly associated with worse prognoses. An enhanced immune infiltration, increased pathways score and inappropriate immunotherapy were observed in the high RiskScore group. The high- and low-RiskScore groups had different drug sensitivities. Immunotherapy datasets and pan-carcinoma analysis indicated that the low RiskScore group may benefit from immunotherapy. Based on the perspective of the secretory signaling pathway, a robust prognostic signature with great performances was determined, which may provide clues for clinical precision treatment of ovarian cancer.

Enhancing pathogen detection and characterization of respiratory microbiome signatures of infection through next-generation sequencing

Next-generation sequencing-based (NGS) methods for pathogen detection, which can capture the entire microbial composition in a clinical sample, are used strategically in parallel to culture-based methods. While NGS-based methods cannot confirm organism viability, culture-based methods can but are limited by the number of culture conditions routinely utilized in clinical microbiology laboratories. The relatively wide scope provided by sequencing compensates for the limited scope of culture-based methods but creates obstacles in distinguishing pathogens from an expected body-site-specific microbiome or benign transient colonizers. This study compares NGS-based methods for pathogen detection in respiratory samples to current diagnostics at Texas Children's Hospital in Houston, Texas. Current diagnostics include various culture-based assays, targeted PCR assays, and reference laboratory-generated sequencing. We hypothesize that employing sample metadata (source, type, etc.) and patient data (clinical history, chronic conditions, etc.) during data analysis will help distinguish pathogens from expected flora in respiratory samples. To develop these methods, 27 respiratory samples with suffcient volume for both routine diagnostic testing and this study were collected. Nucleic acid was extracted using the Qiagen DNeasy PowerSoil Pro Kit after collection, and targeted short-read sequencing (16S, 18S/ITS) was performed. These data were then analyzed using a custom bioinformatic pipeline built around Qiime2-2022. In brief, sequences are denoised and quality filtered using DADA2, an algorithm that uses a statistical model for correcting sequencing errors, after which similar amplicon sequence variants (ASVs) are clustered. Next, a feature table of ASVs that are 100% identical is generated, and taxonomy assigned using a naive Bayes taxonomy classifier against the Silva 13.2 database. Finally, unassigned and low abundance ASVs are removed. When species-level identification couldn't be determined through targeted short-read sequencing, untargeted long-read sequencing was used. In one case, we found that a bronchoalveolar lavage sample from the lung of a patient with a history of recurrent pneumonia contained a singular pathogenic bacterium, H. influenzae. This finding agreed with results obtained through reference laboratory-generated sequencing. In another case, we identified &gt;14 unique bacteria at varying concentrations in a wash sample collected from the lung of an asymptomatic patient during a routine bronchoscopy. These findings, however, were determined to be expected respiratory flora. Our NGS-based methods’ enhanced pathogen detection compared to current diagnostics and discriminates between respiratory flora and active infections. Setting thresholds for pathogen detection, evaluating clinical relevance in the context of body site and presentation, and deploying additional molecular techniques when necessary are crucial steps to increase the accuracy of pathogen detection in the clinic. This study was supported by the Texas Children's Hospital Department of Pathology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.