High-coverage whole-genome sequencing of the expanded 1000 Genomes Project cohort including 602 trios.

The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

The genetic landscape of age-related hearing loss.

Age-related hearing loss (ARHL) is a prevalent concern in the elderly population. Recent genome-wide and phenome-wide association studies (GWASs and PheWASs) have delved into the identification of causative variants and the understanding of pleiotropy, highlighting the polygenic intricacies of this complex condition. While recent large-scale GWASs have pinpointed significant SNPs and risk variants associated with ARHL, the detailed mechanisms, encompassing both genetic and epigenetic modifications, remain to be fully elucidated. This review presents the latest advances in association studies, integrating findings from both human studies and model organisms. By juxtaposing historical perspectives with contemporary genomics, we aim to catalyze innovative research and foster the development of novel therapeutic strategies for ARHL.

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline ATM sequence variants.

The ClinGen Hereditary Breast, Ovarian, and Pancreatic Cancer (HBOP) Variant Curation Expert Panel (VCEP) is composed of internationally recognized experts in clinical genetics, molecular biology, and variant interpretation. This VCEP made specifications for the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines for the ataxia telangiectasia mutated (ATM) gene according to the ClinGen protocol. These gene-specific rules for ATM were modified from the ACMG/AMP guidelines and were tested against 33 ATM variants of various types and classifications in a pilot curation phase. The pilot revealed a majority agreement between the HBOP VCEP classifications and the ClinVar-deposited classifications. Six pilot variants had conflicting interpretations in ClinVar, and re-evaluation with the VCEP's ATM-specific rules resulted in four that were classified as benign, one as likely pathogenic, and one as a variant of uncertain significance (VUS) by the VCEP, improving the certainty of interpretations in the public domain. Overall, 28 of the 33 pilot variants were not VUS, leading to an 85% classification rate. The ClinGen-approved, modified rules demonstrated value for improved interpretation of variants in ATM.

Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Enhancing clinical genomic accuracy with panelGC: a novel metric and tool for quantifying and monitoring GC biases in hybridization capture panel sequencing.

Accurate assessment of fragment abundance within a genome is crucial in clinical genomics applications such as the analysis of copy number variation (CNV). However, this task is often hindered by biased coverage in regions with varying guanine-cytosine (GC) content. These biases are particularly exacerbated in hybridization capture sequencing due to GC effects on probe hybridization and polymerase chain reaction (PCR) amplification efficiency. Such GC content-associated variations can exert a negative impact on the fidelity of CNV calling within hybridization capture panels. In this report, we present panelGC, a novel metric, to quantify and monitor GC biases in hybridization capture sequencing data. We establish the efficacy of panelGC, demonstrating its proficiency in identifying and flagging potential procedural anomalies, even in situations where instrument and experimental monitoring data may not be readily accessible. Validation using real-world datasets demonstrates that panelGC enhances the quality control and reliability of hybridization capture panel sequencing.

scPanel: a tool for automatic identification of sparse gene panels for generalizable patient classification using scRNA-seq datasets.

Single-cell RNA sequencing (scRNA-seq) technologies can generate transcriptomic profiles at a single-cell resolution in large patient cohorts, facilitating discovery of gene and cellular biomarkers for disease. Yet, when the number of biomarker genes is large, the translation to clinical applications is challenging due to prohibitive sequencing costs. Here, we introduce scPanel, a computational framework designed to bridge the gap between biomarker discovery and clinical application by identifying a sparse gene panel for patient classification from the cell population(s) most responsive to perturbations (e.g. diseases/drugs). scPanel incorporates a data-driven way to automatically determine a minimal number of informative biomarker genes. Patient-level classification is achieved by aggregating the prediction probabilities of cells associated with a patient using the area under the curve score. Application of scPanel to scleroderma, colorectal cancer, and COVID-19 datasets resulted in high patient classification accuracy using only a small number of genes (<20), automatically selected from the entire transcriptome. In the COVID-19 case study, we demonstrated cross-dataset generalizability in predicting disease state in an external patient cohort. scPanel outperforms other state-of-the-art gene selection methods for patient classification and can be used to identify parsimonious sets of reliable biomarker candidates for clinical translation.

Genotype imputation accuracy and the quality metrics of the minor ancestry in multi-ancestry reference panels.

Large-scale imputation reference panels are currently available and have contributed to efficient genome-wide association studies through genotype imputation. However, whether large-size multi-ancestry or small-size population-specific reference panels are the optimal choices for under-represented populations continues to be debated. We imputed genotypes of East Asian (180k Japanese) subjects using the Trans-Omics for Precision Medicine reference panel and found that the standard imputation quality metric (Rsq) overestimated dosage r2 (squared correlation between imputed dosage and true genotype) particularly in marginal-quality bins. Variance component analysis of Rsq revealed that the increased imputed-genotype certainty (dosages closer to 0, 1 or 2) caused upward bias, indicating some systemic bias in the imputation. Through systematic simulations using different template switching rates (θ value) in the hidden Markov model, we revealed that the lower θ value increased the imputed-genotype certainty and Rsq; however, dosage r2 was insensitive to the θ value, thereby causing a deviation. In simulated reference panels with different sizes and ancestral diversities, the θ value estimates from Minimac decreased with the size of a single ancestry and increased with the ancestral diversity. Thus, Rsq could be deviated from dosage r2 for a subpopulation in the multi-ancestry panel, and the deviation represents different imputed-dosage distributions. Finally, despite the impact of the θ value, distant ancestries in the reference panel contributed only a few additional variants passing a predefined Rsq threshold. We conclude that the θ value substantially impacts the imputed dosage and the imputation quality metric value.

Serum Proteomics Identifies Biomarkers Associated With the Pathogenesis of Idiopathic Pulmonary Fibrosis.

The heterogeneity of idiopathic pulmonary fibrosis (IPF) limits its diagnosis and treatment. The association between the pathophysiological features and the serum protein signatures of IPF currently remains unclear. The present study analyzed the specific proteins and patterns associated with the clinical parameters of IPF based on a serum proteomic dataset by data-independent acquisition using MS. Differentiated proteins in sera distinguished patients with IPF into three subgroups in signal pathways and overall survival. Aging-associated signatures by weighted gene correlation network analysis coincidently provided clear and direct evidence that aging is a critical risk factor for IPF rather than a single biomarker. Expression of LDHA and CCT6A, which was associated with glucose metabolic reprogramming, was correlated with high serum lactic acid content in patients with IPF. Cross-model analysis and machine learning showed that a combinatorial biomarker accurately distinguished patients with IPF from healthy individuals with an area under the curve of 0.848 (95% CI = 0.684-0.941) and validated from another cohort and ELISA assay. This serum proteomic profile provides rigorous evidence that enables an understanding of the heterogeneity of IPF and protein alterations that could help in its diagnosis and treatment decisions.

Clinical and molecular correlates of the Index of Severity for Eosinophilic Esophagitis.

BACKGROUND: The Index of Severity for Eosinophilic Esophagitis (I-SEE) is a new expert-defined clinical tool that classifies disease severity of eosinophilic esophagitis (EoE). OBJECTIVE: We aimed to determine whether I-SEE is associated with patient characteristics, molecular features of EoE, or both. METHODS: We analyzed a prospective cohort of patients with EoE from the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR). Associations between I-SEE and clinical and molecular features (assessed by an EoE diagnostic panel [EDP]) were assessed. RESULTS: In 318 patients with chronic EoE (209 adults, 109 children), median total I-SEE score was 7.0, with a higher symptoms and complications score in children than adults (4.0 vs 1.0; P < .001) and higher inflammatory and fibrostenotic features scores in adults than children (3.0 vs 1.0 and 3.0 vs 0, respectively; both P < .001). Total I-SEE score had a bimodal distribution with the inactive to moderate categories and severe category. EDP score correlated with total I-SEE score (r = -0.352, P < .001) and both inflammatory and fibrostenotic features scores (r = -0.665, P < .001; r = -0.446, P < .001, respectively), but not with symptoms and complications scores (r = 0.047, P = .408). Molecular severity increased from inactive to mild and moderate, but not severe, categories. Longitudinal changes of modified I-SEE scores and inflammatory and fibrostenotic features scores reflected histologic and molecular activity. CONCLUSIONS: I-SEE score is associated with select clinical features across severity categories and with EoE molecular features for nonsevere categories, warranting further validation.

Persistent esophageal changes after histologic remission in eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by persistent or relapsing allergic inflammation, and both clinical and histologic features of esophageal inflammation persist over time in most individuals. Mechanisms contributing to EoE relapse are not understood, and chronic EoE-directed therapy is therefore required to prevent long-term sequelae. OBJECTIVE: We investigated whether EoE patients in histologic remission have persistent dysregulation of esophageal gene expression. METHODS: Esophageal biopsy samples from 51 pediatric and 52 adult subjects with EoE in histopathologic remission (<15 eosinophils per high-power field [eos/hpf]) and control (48 pediatric and 167 adult) subjects from multiple institutions were subjected to molecular profiling by the EoE diagnostic panel, which comprises a set of 94 esophageal transcripts differentially expressed in active EoE. RESULTS: Defining remission as <15 eos/hpf, we identified 51 and 32 differentially expressed genes in pediatric and adult EoE patients compared to control individuals, respectively (false discovery rate < 0.05). Using the stringent definition of remission (0 eos/hpf), the adult and pediatric cohorts continued to have 18 and 25 differentially expressed genes (false discovery rate < 0.05). Among 6 shared genes between adults and children, CDH26 was upregulated in both children and adults; immunohistochemistry demonstrated increased cadherin 26 staining in the epithelium of EoE patients in remission compared to non-EoE controls. In the adult cohort, POSTN expression correlated with the endoscopic reference system score (Spearman r = 0.35, P = .011), specifically correlating with the rings' endoscopic reference system subscore (r = 0.53, P = .004). CONCLUSION: We have identified persistent EoE-associated esophageal gene expression in patients with disease in deep remission. These data suggest potential inflammation-induced epigenetic mechanisms may influence gene expression during remission in EoE and provide insight into possible mechanisms that underlie relapse in EoE.

Precision Diagnostics in Myeloid Malignancies: Development and Validation of a National Capture-Based Gene Panel.

Gene panel sequencing has become a common diagnostic tool for detecting somatically acquired mutations in myeloid neoplasms. However, many panels have restricted content, provide insufficient sensitivity levels, or lack clinically validated workflows. We here describe the development and validation of the Genomic Medicine Sweden myeloid gene panel (GMS-MGP), a capture-based 191 gene panel including mandatory genes in contemporary guidelines as well as emerging candidates. The GMS-MGP displayed uniform coverage across all targets, including recognized difficult GC-rich areas. The validation of 117 previously described somatic variants showed a 100% concordance with a limit-of-detection of a 0.5% variant allele frequency (VAF), achieved by utilizing error correction and filtering against a panel-of-normals. A national interlaboratory comparison investigating 56 somatic variants demonstrated highly concordant results in both detection rate and reported VAFs. In addition, prospective analysis of 323 patients analyzed with the GMS-MGP as part of standard-of-care identified clinically significant genes as well as recurrent mutations in less well-studied genes. In conclusion, the GMS-MGP workflow supports sensitive detection of all clinically relevant genes, facilitates novel findings, and is, based on the capture-based design, easy to update once new guidelines become available. The GMS-MGP provides an important step toward nationally harmonized precision diagnostics of myeloid malignancies.

Prevalence and Reclassification of Genetic Variants in South African Populations with Breast Cancer.

Concurrent testing of numerous genes for hereditary breast cancer (BC) is available but can result in management difficulties. We evaluated use of an expanded BC gene panel in women of diverse South African ancestries and assessed use of African genomic data to reclassify variants of uncertain significance (VUS). A total of 331 women of White, Black African, or Mixed Ancestry with BC had a 9-gene panel test, with an additional 75 genes tested in those without a pathogenic/likely pathogenic (P/LP) variant. The proportion of VUS reclassified using ClinGen gene-specific allele frequency (AF) thresholds or an AF > 0.001 in nonguidelines genes in African genomic data was determined. The 9-gene panel identified 58 P/LP variants, but only two of the P/LP variants detected using the 75-gene panel were in confirmed BC genes, resulting in a total of 60 (18.1%) in all participants. P/LP variant prevalence was similar across ancestry groups, but VUS prevalence was higher in Black African and Mixed Ancestry than in White participants. In total, 611 VUS were detected, representing 324 distinct variants. 10.8% (9/83) of VUS met ClinGen AF thresholds in genomic data while 10.8% (26/240) in nonguideline genes had an AF > 0.001. Overall, 27.0% of VUS occurrences could potentially be reclassified using African genomic data. Thus, expanding the gene panel yielded few clinically actionable variants but many VUS, particularly in participants of Black African and Mixed Ancestry. However, use of African genomic data has the potential to reclassify a significant proportion of VUS.

Potential causal and temporal relationship between plasma triglyceride levels and circulating leukocyte.

BACKGROUND: Circulating triglyceride (TG) and leukocytes, the main components of the vascular system, may impact each other and co-fuel atherosclerosis. While the causal relationship between plasma TG levels and leukocyte counts remains unclear. METHODS: Bidirectional mendelian randomisation (MR) analysis was conducted to investigate the potential causal relationship between TG levels and the counts of leukocytes and their subtypes. A cross-lagged panel model (CLPM) using a longitudinal healthy screening data (13,389 adults with an follow-up of 4 years) was fitted to examine the temporal relationship between them. RESULTS: Genetically predicted plasma TG levels were positively associated with total leukocyte counts (TLC) [ß(se)=0.195(0.01)], lymphocyte counts (LC) [ß(se)=0.196(0.019)], and neutrophill counts (NC) [ß(se)=0.086(0.01)], which remained significant after adjusting for several confounders. Inversely, the genetically predicted TLC [ß(se)=0.033(0.008)], LC [ß(se)=0.053(0.008)], and NC [ß(se)=0.034(0.008)] were positively associated with plasma TG levels. However, when all three of them were put into the MR model adjusted for each other, only LC was significantly associated with TG levels. There was no association between genetically predicted TG levels and monocyte counts (MC), basophil counts, and eosinophill counts. The results of CLPM showed that the temporal effect of elevated TLC, MC, LC, and NC on plasma TG levels were stronger than the inverse effect. CONCLUSION: Our findings suggesting a causal associations of plasma TG levels with TLC, LC, and NC. In turn, LC was positively associated with plasma TG levels. Additionally, elevated circulating LC may precede high plasma TG levels.

A common resequencing-based genetic marker data set for global maize diversity.

Maize (Zea mays ssp. mays) populations exhibit vast ranges of genetic and phenotypic diversity. As sequencing costs have declined, an increasing number of projects have sought to measure genetic differences between and within maize populations using whole-genome resequencing strategies, identifying millions of segregating single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). Unlike older genotyping strategies like microarrays and genotyping by sequencing, resequencing should, in principle, frequently identify and score common genetic variants. However, in practice, different projects frequently employ different analytical pipelines, often employ different reference genome assemblies and consistently filter for minor allele frequency within the study population. This constrains the potential to reuse and remix data on genetic diversity generated from different projects to address new biological questions in new ways. Here, we employ resequencing data from 1276 previously published maize samples and 239 newly resequenced maize samples to generate a single unified marker set of approximately 366 million segregating variants and approximately 46 million high-confidence variants scored across crop wild relatives, landraces as well as tropical and temperate lines from different breeding eras. We demonstrate that the new variant set provides increased power to identify known causal flowering-time genes using previously published trait data sets, as well as the potential to track changes in the frequency of functionally distinct alleles across the global distribution of modern maize.

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

Genome-wide association study between copy number variation and feeding behavior, feed efficiency, and growth traits in Nellore cattle.

BACKGROUND: Feeding costs represent the largest expenditures in beef production. Therefore, the animal efficiency in converting feed in high-quality protein for human consumption plays a major role in the environmental impact of the beef industry and in the beef producers' profitability. In this context, breeding animals for improved feed efficiency through genomic selection has been considered as a strategic practice in modern breeding programs around the world. Copy number variation (CNV) is a less-studied source of genetic variation that can contribute to phenotypic variability in complex traits. In this context, this study aimed to: (1) identify CNV and CNV regions (CNVRs) in the genome of Nellore cattle (Bos taurus indicus); (2) assess potential associations between the identified CNVR and weaning weight (W210), body weight measured at the time of selection (WSel), average daily gain (ADG), dry matter intake (DMI), residual feed intake (RFI), time spent at the feed bunk (TF), and frequency of visits to the feed bunk (FF); and, (3) perform functional enrichment analyses of the significant CNVR identified for each of the traits evaluated. RESULTS: A total of 3,161 CNVs and 561 CNVRs ranging from 4,973 bp to 3,215,394 bp were identified. The CNVRs covered up to 99,221,894 bp (3.99%) of the Nellore autosomal genome. Seventeen CNVR were significantly associated with dry matter intake and feeding frequency (number of daily visits to the feed bunk). The functional annotation of the associated CNVRs revealed important candidate genes related to metabolism that may be associated with the phenotypic expression of the evaluated traits. Furthermore, Gene Ontology (GO) analyses revealed 19 enrichment processes associated with FF. CONCLUSIONS: A total of 3,161 CNVs and 561 CNVRs were identified and characterized in a Nellore cattle population. Various CNVRs were significantly associated with DMI and FF, indicating that CNVs play an important role in key biological pathways and in the phenotypic expression of feeding behavior and growth traits in Nellore cattle.

Whole exome sequencing in Serbian patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a group of neurodegenerative diseases with a high genetic and clinical heterogeneity. Numerous HSP patients remain genetically undiagnosed despite screening for known genetic causes of HSP. Therefore, identification of novel variants and genes is needed. Our previous study analyzed 74 adult Serbian HSP patients from 65 families using panel of the 13 most common HSP genes in combination with a copy number variation analysis. Conclusive genetic findings were established in 23 patients from 19 families (29%). In the present study, nine patients from nine families previously negative on the HSP gene panel were selected for the whole exome sequencing (WES). Further, 44 newly diagnosed adult HSP patients from 44 families were sent to WES directly, since many studies showed WES may be used as the first step in HSP diagnosis. WES analysis of cohort 1 revealed a likely genetic cause in five (56%) of nine HSP families, including variants in the ETHE1, ZFYVE26, RNF170, CAPN1, and WASHC5 genes. In cohort 2, possible causative variants were found in seven (16%) of 44 patients (later updated to 27% when other diagnosis were excluded), comprising six different genes: SPAST, SPG11, WASCH5, KIF1A, KIF5A, and ABCD1. These results expand the genetic spectrum of HSP patients in Serbia and the region with implications for molecular genetic diagnosis and future causative therapies. Wide HSP panel can be the first step in diagnosis, alongside with the copy number variation (CNV) analysis, while WES should be performed after.

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Respiratory Viruses in Wastewater Compared with Clinical Samples, Leuven, Belgium.

In a 2-year study in Leuven, Belgium, we investigated the use of wastewater sampling to assess community spread of respiratory viruses. Comparison with the number of positive clinical samples demonstrated that wastewater data reflected circulation levels of typical seasonal respiratory viruses, such as influenza, respiratory syncytial virus, and enterovirus D68.