Functional evaluation allows ACMG/AMP-based re-classification of CNGA3 variants associated with achromatopsia.

PURPOSE: CNGA3 encoding the main subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is one of the major disease-associated genes for achromatopsia. Most CNGA3 variants are missense variants with the majority being functionally uncharacterized and therefore hampering genetic diagnosis. In light of potential gene therapy, objective variant pathogenicity assessment is essential. METHODS: We established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant re-classification. RESULTS: We provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology-based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%. CONCLUSION: Our aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.

Genome-based reclassification of Bacillus acidicola, Bacillus pervagus and the genera Heyndrickxia, Margalitia and Weizmannia.

In the present study, the taxonomic positions of Bacillus acidicola, Bacillus pervagus and members of the genera Heyndrickxia, Margalitia and Weizmannia were evaluated. The 16S rRNA gene sequence similarity between Bacillus acidicola DSM 14745T, Bacillus pervagus DSM 23947T and members of the genera Heyndrickxia and Margalitia were above the cut-off level (>95 %) for genus delineation. Amino acid identity (AAI) values and the results of phylogenomic analysis suggested that B. acidicola and the members of the genera Heyndrickxia, Margalitia and Weizmannia belong to the same genus. Furthermore, the AAI and phylogenomic results also differentiate B. pervagus from B. acidicola and the members of the genera Heyndrickxia, Margalitia and Weizmannia. Based on the results, we propose to transfer Bacillus acidicola, Margalitia and Weizmannia to the genus Heyndrickxia. We also propose the reclassification of B. pervagus into a new genus Oikeobacillus gen. nov., with the type species Oikeobacillus pervagus comb. nov.

Transfer of Bacillus tepidiphilus Narsing Rao et al. 2021 to the genus Peribacillus as Peribacillus tepidiphilus comb. nov.

In the present study, the taxonomic position of Bacillus tepidiphilus was re-evaluated. Bacillus tepidiphilus (B. tepidiphilus) SYSU G01002T showed the highest 16S rRNA gene sequence similarity with the type strain Peribacillus alkalitolerans (P. alkalitolerans) (97.7%). In the phylogenetic (based on 16S rRNA sequence) and phylogenomic (based on 71 bacterial single-copy genes) trees, B. tepidiphilus SYSU G01002T clade with the members of the genus Peribacillus. The amino acid identity (AAI) value of B. tepidiphilus SYSU G01002T was highest with P. alkalitolerans KCTC 33631T (73.6%). The AAI value between B. tepidiphilus SYSU G01002T and P. alkalitolerans KCTC 33631T was above the cutoff level for genus delineation. The average nucleotide identity (ANI) between B. tepidiphilus and P. alkalitolerans KCTC 33631T was 74.1%, which was below the ANI value (95-96%) for species delineation. Based on the phylogenetic, phylogenomic, AAI, and ANI analysis, Bacillus tepidiphilus is proposed to transfer to the genus Peribacillus as Peribacillus tepidiphilus comb. nov. The type strain is SYSU G01002T (= KCTC 43131T = CGMCC 1.17491T).

Scytalidium candidum 3C is a new name for the Geotrichum candidum Link 3C strain.

In the 1970s, the strain Geotrichum candidum Link 3C was isolated from rotting rope and since then has been extensively studied as a source of cellulose and xylan-degrading enzymes. The original identification of the strain was based only on morphological characters of the fungal mycelium in culture. Recent comparison of the internal transcribed spacer (ITS) fragments derived from the draft genome published in 2015 did not show its similarity to G. candidum species. Given the value of the strain 3C in lignocellulosic biomass degradation, we performed morphological and molecular studies to find the appropriate taxonomic placement for this fungal strain within the Ascomycota phylum. ITS, 18S rDNA, 28S rDNA sequences, and RPB2 encoding genes were used to construct phylogenetic trees with Maximum likelihood and Bayesian inference methods. Based on sequence comparison and multiple gene sequencing, we conclude that the fungal strain designated as Geotrichum candidum Link 3C should be placed into the genus Scytalidium (Pezizomycotina, Leotiomycetes) and is redescribed herein as Scytalidium candidum 3C comb. nov.

Risk prediction tool for grade re-classification in men with favourable-risk prostate cancer on active surveillance.

OBJECTIVE: To create a nomogram for men on active surveillance (AS) for prediction of grade re-classification (GR) above Gleason score 6 (Grade group >2) at surveillance biopsy. PATIENTS AND METHODS: From a cohort of men enrolled in an AS programme, a multivariable model was used to identify clinical and pathological parameters predictive of GR. Nomogram performance was assessed using receiver operating characteristic curves, calibration, and decision curve analysis. RESULTS: Of 1 374 men, 254 (18.50%) were re-classified to Gleason ≥7 on surveillance prostate biopsy. Variables predictive of GR were earlier year of diagnosis [≤2004 vs ≥2005; odds ratio (OR) 2.16, P < 0.001], older age (OR 1.05, P < 0.001), higher prostate-specific antigen density [OR 1.19 (per 0.1 unit increase), P = 0.04], bilateral disease (OR 2.86, P < 0.001), risk strata (low-risk vs very-low-risk, OR 1.79, P < 0.001), and total number of biopsies without GR (OR 0.68, P < 0.001). On internal validation, a nomogram created using the multivariable model had an area under the curve of 0.757 (95% confidence interval 0.730-0.797) for predicting GR at the time of next surveillance biopsy. CONCLUSION: The nomogram described is currently being used at each return visit to assess the need for a surveillance biopsy, and could increase retention in AS.

Accuracy of death certificates of cardiovascular disease in a community intervention in Sweden.

AIM: The aim was to investigate the possibility to evaluate the mortality pattern in a community intervention programme against cardiovascular disease by official death certificates. METHODS: For all deceased in the intervention area (Norsjö), the accuracy of the official death certificates were compared with matched controls in the rest of Västerbotten. The official causes of death were compared with new certificates, based on the last clinical record, issued by three of the authors, and coded by one of the authors, all four accordingly blinded. RESULTS: The degree of agreement between the official underlying causes of death in "cardiovascular disease" (CVD) and the re-evaluated certificates was not found to differ between Norsjö and the rest of Västerbotten. The agreement was 87% and 88% at chapter level, respectively, but only 55% and 55% at 4-digit level, respectively. The reclassification resulted in a 1% decrease of "cardiovascular deaths" in both Norsjö and the rest of Västerbotten. CONCLUSIONS: The disagreements in the reclassification of cause of death were equal but large in both directions. The official death certificates should be used with caution to evaluate CVD in small community intervention programmes, and restricted to the chapter level and total populations.

Relevance of next generation sequencing (NGS) data re-analysis in the diagnosis of monogenic diseases leading to organ failure.

BACKGROUND: In 2018, our center started a program to offer genetic diagnosis to patients with kidney and liver monogenic rare conditions, potentially eligible for organ transplantation. We exploited a clinical exome sequencing approach, followed by analyses of in silico gene panels tailored to clinical suspicions, obtaining detection rates in line with what reported in literature. However, a percentage of patients remains without a definitive genetic diagnosis. This work aims to evaluate the utility of NGS data re-analysis for those patients with an inconclusive or negative genetic test at the time of first analysis considering that (i) the advance of alignment and variant calling processes progressively improve the detection rate, limiting false positives and false negatives; (ii) gene panels are periodically updated and (iii) variant annotation may change over time. METHODS: 114 patients, recruited between 2018 and 2020, with an inconclusive or negative NGS report at the time of first analysis, were included in the study. Re-alignment and variant calling of previously generated sequencing raw data were performed using the GenomSys Variant Analyzer software. RESULTS: 21 previously not reported potentially causative variants were identified in 20 patients. In most cases (n = 19), causal variants were retrieved out of the re-classification from likely benign to variants of unknown significance (VUS). In one case, the variant was included because of inclusion in the analysis of a newly disease-associated gene, not present in the original gene panel, and in another one due to the improved data alignment process. Whenever possible, variants were validated with Sanger sequencing and family segregation studies. As of now, 16 out of 20 patients have been analyzed and variants confirmed in 8 patients. Specifically, in two pediatric patients, causative variants were de novo mutations while in the others, the variant was present also in other affected relatives. In the remaining patients, variants were present also in non-affected parents, raising questions on their re-classification. CONCLUSIONS: Overall, these data indicate that periodic and systematic re-analysis of negative or inconclusive NGS data reports can lead to new variant identification or reclassification in a small but significant proportion of cases, with benefits for patients' management.

Comparative genome analysis, predicted lifestyle and antimicrobial strategies of Lactococcus carnosus and Lactococcus paracarnosus isolated from meat.

Strains of the psychrotrophic bacterium Lactococcus piscium have gained increasing attention as potentially bioprotective cultures due to their assertiveness against fish and meat spoilage bacteria as well as pathogenic bacteria. Recently, we have described two novel species within the genus Lactococcus (Lc.) namely Lc. carnosus (TMW 2.1612T) and Lc. paracarnosus (TMW 2.1615T) isolated from modified atmosphere packaged meat. Within this study, we compared the genomes of two Lc. carnosus strains, two Lc. paracarnosus strains and 16 Lc. piscium strains from our laboratory and five publicly available genomes previously affiliated to the species Lc. piscium. Our phylogenetic analysis supports reclassification of 20 of the strains to either Lc. carnosus or Lc. paracarnosus, so far limiting the Lc. piscium type strain (DSM 6634T) as sole representative of this species. Comparative genomics approach was conducted to predict underlying mechanisms involved in interspecies competition strategies of Lc. carnosus and Lc. paracarnosus against meat spoilers and predict their lifestyle in meat environments. In general, strains of the three species were highly similar regarding metabolic pathways for most of the relevant meat-derived substrates. In silico analyses enabled prediction of homolactic hexose fermentation by Lc. carnosus, Lc. paracarnosus and Lc. piscium. Further, genes required for the heterofermentative metabolism of hexoses and pentoses were only found in the Lc. pisicum type strain (DSM 6634T). We predict a low spoilage potential for Lc. carnosus and Lc. paracarnosus strains. No genes for decarboxylation of amino acids yielding biogenic amines were found in the genomes. Regarding their antimicrobial mechanisms against spoilers, we found a strain-specific putative polymorphic toxin system predictively delivered by the type VIIb secretion system, enabling cell-to-cell contact-dependent growth inhibition. Furthermore, we found additional genes predictively involved to the suppression of spoilers within the food microbiome (prophages, lytic domains, bacteriocins, metabolites).

T Cells With Activated STAT4 Drive the High-Risk Rejection State to Renal Allograft Failure After Kidney Transplantation.

In kidney transplantation, deteriorated progression of rejection is considered to be a leading course of postoperative mortality. However, the conventional histologic diagnosis is limited in reading the rejection status at the molecular level, thereby triggering mismatched pathogenesis with clinical phenotypes. Here, by applying uniform manifold approximation and projection and Leiden algorithms to 2,611 publicly available microarray datasets of renal transplantation, we uncovered six rejection states with corresponding signature genes and revealed a high-risk (HR) state that was essential in promoting allograft loss. By identifying cell populations from single-cell RNA sequencing data that were associated with the six rejection states, we identified a T-cell population to be the pathogenesis-triggering cells associated with the HR rejection state. Additionally, by constructing gene regulatory networks, we identified that activated STAT4, as a core transcription factor that was regulated by PTPN6 in T cells, was closely linked to poor allograft function and prognosis. Taken together, our study provides a novel strategy to help with the precise diagnosis of kidney allograft rejection progression, which is powerful in investigating the underlying molecular pathogenesis, and therefore, for further clinical intervention.

Treatment target re-classification of subjects comparing estimation of low-density lipoprotein cholesterol by the Friedewald equation and direct measurement of LDL-cholesterol.

AIMS: To compare low-density lipoprotein cholesterol (LDL-C) values calculated by the Friedewald equation with direct LDL-C in patient samples and assess the possible impact on re-classification of LDL-C target values for primary prevention or high cardiovascular disease (CVD) risk (<2.5 mmol/L) and secondary prevention or very high CVD risk (<1.8 mmol/L). LDL-C is an important CVD risk factor. Over the last decade, there has been a change in laboratory methodology from indirectly calculated LDL-C with the Friedewald equation to direct LDL-C measurements (dLDL-C). METHODS: Reported results for plasma triglycerides, total cholesterol, high-density lipoprotein-cholesterol, and dLDL-C from 34,981 samples analyzed in year 2014 were extracted from the laboratory information system, Uppsala University Hospital, Uppsala, Sweden. RESULTS: dLDL-C was approximately 10% lower than the corresponding LDL-C results calculated by the Friedewald equation in both men and women. In subjects with triglyceride concentrations above 4 mmol/L (n = 1250) the same discordant pattern was seen as for the entire study population. Altogether 5469 out of 18,051 men (30.3%) and 4604 out of 16,928 women (27.2%) were down-classified at least one CVD risk category. A very small number of subject was up-classified, in total 37 out of 18,051 men (0.2%) and 28 out of 16,928 women (0.2%). CONCLUSIONS: The two LDL-C methods had a high concordance, but the direct LDL-C measurement consistently gave approx. 10% lower values, and this caused one-third of subjects to be re-classified as having a lower cardiovascular disease risk in relation to recommended LDL-C target values and decision limits.

Influence of sex on risk stratification with stress myocardial perfusion Rb-82 positron emission tomography: Results from the PET (Positron Emission Tomography) Prognosis Multicenter Registry.

OBJECTIVES: The aim of the current analysis was to compare sex differences in the prognostic accuracy of stress myocardial perfusion rubidum-82 (Rb-82) positron emission tomography (PET). BACKGROUND: The diagnostic evaluation of women presenting with suspected cardiac symptoms is challenging with reported reduced accuracy, attenuation artifact, and more recent concerns regarding radiation safety. Stress myocardial perfusion Rb-82 PET is a diagnostic alternative with improved image quality and radiation dosimetry. Currently, the prognostic accuracy of stress Rb-82 PET in women has not been established. METHODS: A total of 6,037 women and men were enrolled in the PET Prognosis Multicenter Registry. Patients were followed for the occurrence of coronary artery disease (CAD) mortality, with a median follow-up of 2.2 years. Cox proportional hazards modeling was used to estimate CAD mortality. The net re-classification improvement index (NRI) was calculated. RESULTS: The 5-year CAD mortality was 3.7% for women and 6.0% for men (p < 0.0001). Unadjusted CAD mortality ranged from 0.9% to 12.9% for women (p < 0.0001) and from 1.5% to 17.4% for men (p < 0.0001) for 0% to ≥15% abnormal myocardium at stress. In multivariable models, the percentage of abnormal stress myocardium was independently predictive of CAD mortality in women and men. An interaction term of sex by the percentage of abnormal stress myocardium was nonsignificant (p = 0.39). The categorical NRI when Rb-82 PET data was added to a clinical risk model was 0.12 for women and 0.17 for men. Only 2 cardiac deaths were reported in women <55 years of age; accordingly the percentage of abnormal myocardium at stress was of borderline significance (p = 0.063), but it was highly significant for women ≥55 years of age (p < 0.0001), with an increased NRI of 0.21 (95% confidence interval: 0.09 to 0.34), including 17% of CAD deaths and 3.9% of CAD survivors that were correctly re-classified in this older female subset. CONCLUSIONS: Stress Rb-82 PET provides significant and clinically meaningful effective risk stratification of women and men, supporting this modality as an alternative to comparative imaging modalities. Rb-82 PET findings were particularly helpful at identifying high-risk, older women.