Next-generation Sequencing Research Articles

Evaluation of Risk Factors and a Gene Panel as a Tool for Unexplained Infertility Diagnosis by Next-Generation Sequencing

Background and Objective: Unexplained infertility is a major challenge in reproductive medicine and requires advanced diagnostic approaches to identify the underlying factors accurately. This study aims to evaluate the utility of risk factor analysis and a gene panel in diagnosing unexplained infertility using the next-generation sequencing (NGS) technology. Our study aimed to characterize and identify risk and genetic factors associated with unexplained infertility. Materials and methods: A cohort of patients with unexplained infertility was comprehensively screened for risk factors and genetic variations using a targeted gene panel (10 couples with unexplained infertility (UI) and 36 fertile couples). 108 articles were selected (58 on female infertility and 50 on male infertility) presenting genes that may be associated with unexplained infertility. A gene panel for unexplained infertility was compiled based on the literature data. A customized virtual panel was created from the exome sequencing data. Results: In the female group, controls had a higher mean age, while in the male patients, both groups were similar in terms of age. Both gender groups had comparable BMI values. No significant associations (p > 0.05) between risk factors and unexplained infertility were found when evaluating anthropometric parameters and other sociodemographic characteristics. In two male patients (20%), a molecular defect was detected in NGS variants classified aspossible benign and probably benign In particular, missense variants were identified in the UGT2B7 and CATSPER2 genes, A molecular defect classified as probably damaging was found in five female patients (50%). In particular, missense variants were identified in the CAPN10, MLH3, HABP2, IRS1, GDF9, and SLC19A1 genes. Conclusions: The study emphasizes that unexplained infertility is often related to mechanisms beyond causative mutations and highlights the need for integrative genomic research involving broader gene panels and multi-faceted approaches, including transcriptomics and epigenetics, to uncover latent genetic predispositions.

A ribodepletion and tagging protocol to multiplex samples for RNA-seq based virus detection: application to the cassava virome

Abstract Background Cassava (Manihot esculenta, Crantz), is a staple food and the main source of calories for many populations in Africa, but the plant is beset by several damaging viruses. So far, eight families of virus infecting cassava have been identified; the Geminiviridae (ssDNA viruses responsible for cassava mosaic disease, CMD) and Potyviridae (ssRNA + viruses responsible for cassava brown streak disease, CBSD) families being the most damaging to cassava in Africa. In several cassava-growing regions, the co-existence of species and strains from these two families results in a complex epidemiological situation making it difficult to correctly identify the viruses in circulation and delaying the implementation of disease management schemes. Nevertheless, the development of next generation sequencing (NGS) methods has revolutionized plant virus detection and identification. One NGS method that has been successfully used in virus detection and identification is ribodepleted RNA sequencing. Unfortunately, the relatively high cost makes it difficult to upscale this method to large epidemiological surveys and limits its adoption as a diagnostic tool. Results Here, we develop a high-throughput sequencing protocol, named Ribo-M-Seq, that combines plant rRNA ribodepletion, cDNA synthesis, tagging with a 96 multiplexing scheme and Illumina sequencing. We evaluated the protocol on a series of cassava samples with a known assemblage of viruses. After confirming that the protocol was suitable for ribodepletion, we demonstrated it was possible to detect RNA and DNA viruses via identification of near full-size genomes. Additional phylogenetic analyses confirmed the presence of begomoviruses and ipomoviruses responsible for CMD and CBSD, respectively. We also detected a recently described ampelovirus (Manihot esculenta-associated virus) that was not detected in previous analyses. Conclusions The use of the Ribo-M-Seq protocol will pave the way for large-scale sample analyses of collections with potentially complex viromes, such as those collected in the West African cassava integrated pest management program.

A Robust and Comprehensive Study of the Molecular and Genetic Basis of Neurodevelopmental Delay in a Sample of 3244 Patients, Evaluated by Exome Analysis in a Latin Population

Background and Objectives: Neurodevelopmental disorders (NDDs), including developmental delay (DD), autism spectrum disorder (ASD), intellectual disability (ID), attention-deficit/hyperactivity disorder (ADHD), and specific learning disorders, affect 15% of children and adolescents worldwide. Advances in next-generation sequencing, particularly whole exome sequencing (WES), have improved the understanding of NDD genetics. Methodology: This study analyzed 3244 patients undergoing WES (single, duo, trio analyses), with 1028 meeting inclusion criteria (67% male; aged 0–50 years). Results: Pathogenic (P) or likely pathogenic (LP) variants were identified in 190 patients, achieving a diagnostic yield of 13.4% (singleton), 14% (duo), and 21.2% (trio). A total of 207 P/LP variants were identified in NDD-associated genes: 38% were missense (48 de novo), 29% frameshift (26 de novo), 21% nonsense (14 de novo), 11% splicing site (14 de novo), and 1% inframe (1 de novo). De novo variants accounted for 49.8% of cases, with 86 novels de novo variants and 27 novel non de novo variants unreported in databases like ClinVar or scientific literature. Conclusions: This is the largest study on WES in Colombian children with NDDs and one of the largest in Latino populations. It highlights WES as a cost-effective first-tier diagnostic tool in low-income settings, reducing diagnostic timelines and improving clinical care. These findings underscore the feasibility of implementing WES in underserved populations and contribute significantly to understanding NDD genetics, identifying novel variants with potential for further research and clinical applications.

Daratumumab plus bortezomib, lenalidomide and dexamethasone for transplant-ineligible or transplant-deferred newly diagnosed multiple myeloma: the randomized phase 3 CEPHEUS study

Abstract Frontline daratumumab-based triplet and quadruplet standard-of-care regimens have demonstrated improved survival outcomes in newly diagnosed multiple myeloma (NDMM). For patients with transplant-ineligible NDMM, triplet therapy with either daratumumab plus lenalidomide and dexamethasone (D-Rd) or bortezomib, lenalidomide and dexamethasone (VRd) is the current standard of care. This phase 3 trial evaluated subcutaneous daratumumab plus VRd (D-VRd) in patients with transplant-ineligible NDMM or for whom transplant was not planned as the initial therapy (transplant deferred). Some 395 patients with transplant-ineligible or transplant-deferred NDMM were randomly assigned to eight cycles of D-VRd or VRd followed by D-Rd or Rd until progression. The primary endpoint was overall minimal residual disease (MRD)-negativity rate at 10− 5 by next-generation sequencing. Major secondary endpoints included complete response (CR) or better (≥CR) rate, progression-free survival and sustained MRD-negativity rate at 10− 5. At a median follow-up of 58.7 months, the MRD-negativity rate was 60.9% with D-VRd versus 39.4% with VRd (odds ratio, 2.37; 95% confidence interval (CI), 1.58–3.55; P < 0.0001). Rates of ≥CR (81.2% versus 61.6%; P < 0.0001) and sustained MRD negativity (≥12 months; 48.7% versus 26.3%; P < 0.0001) were significantly higher with D-VRd versus VRd. Risk of progression or death was 43% lower for D-VRd versus VRd (hazard ratio, 0.57; 95% CI, 0.41–0.79; P = 0.0005). Adverse events were consistent with the known safety profiles for daratumumab and VRd. Combining daratumumab with VRd produced deeper and more durable MRD responses versus VRd alone. The present study supports D-VRd quadruplet therapy as a new standard of care for transplant-ineligible or transplant-deferred NDMM. ClinicalTrials.gov registration: NCT03652064.

Clonal dynamics of chronic myelomonocytic leukemia progression: paired-sample comparison.

This study investigated the clonal evolution of chronic myelomonocytic leukemia (CMML) progression to secondary acute myeloid leukemia (sAML) by next-generation sequencing and pyrosequencing for variant allele frequency (VAF) of gene mutations and SNP microarray for copy neutral loss of heterozygosity (CN-LOH) in 38 paired samples from CMML/sAML patients of Taiwanese origin. The median interval between CMML and sAML samples collection was 14.9 months (1.0-89.6). RUNX1 (57%), TET2 (46%), SRSF2 (37%), and ASXL1 (28%) mutations were frequent at CMML diagnosis. Baseline VAF in epigenetic regulator genes was high (>35%) in 83% of mutational events at the CMML phase, remained stable in 78% (VAF changes <10%), and increased in 20% (increased VAF > 10%) during progression to sAML. Transcription factor genes showed high VAF (>35%) in 51% at the CMML phase, and stable VAF in 60% during progression. VAF of spliceosome genes was high (>35%) in 70% at CMML phase, and stable in 61% during progression. Activated signaling genes exhibited acquisition or loss during progression. TET2 mutations were often founding clones, and SRSF2, ASXL1, DNMT3A, EZH2, or spliceosome genes also acted as ancestral mutations. RUNX1 mutations were typically later events and occasionally ancestral hits or germline mutations. Acquisition of cytogenetic changes, signaling pathways genes (PTPN11, FLT3, NRAS, CBL), or AML-defined genes (NPM1, CEBPA, CBFB::MYH11) by linear or branching evolution occurred during sAML progression. CN-LOH was noted in EZH2, CBL, TET2, and DNMT3A genes. CEBPA mutation and concurrent biallelic TET2 with NRAS mutations at CMML diagnosis were risk factors for time to AML progression and overall survival. A characteristic ASXL1MT/RUNX1MT/SpliceosomeMT/signalingWT genetic profile was associated with monocyte counts of 0.5-1.0 × 109/l. This study highlights the complexity and heterogeneity of dynamic changes in clonal architecture during CMML progression, emphasizing its importance in pathogenesis, phenotype, risk stratification, and therapeutic strategy. © 2025 The Pathological Society of Great Britain and Ireland.

Lactiplantibacillus plantarum, the Integral Member of Vegetable Fermentations

Lactiplantibacillus plantarum is omnipresent in vegetable fermentations. Its large metabolic capacity and its ability to adapt to the fermenting microenvironment enable this species, in many cases, to dominate the microecosystem and drive the fermentation. In addition, its metabolic capacity enables it to produce bioactive compounds of great interest for human health. These attributes have directed research for many decades. The widespread application of next-generation sequencing approaches has enabled the genotypic verification of the phenotypically assessed attributes and supplemented them with novel insights, justifying the characterization of a multifunctional tool that has been awarded to this species. However, there are still issues that need to be properly addressed in order to improve our understanding of the microecosystem functionality and to enhance our knowledge regarding the capacities of this species. The aim of the present article is to collect and critically discuss the available information on Lp. plantarum subsistence in vegetable fermentations.

A mutation in DNA polymerase γ harbors a shortened lifespan and high sensitivity to mutagens in the filamentous fungus Neurospora crassa.

Hyphal elongation is the vegetative growth of filamentous fungi, and many species continuously elongate their hyphal tips over long periods. The details of the mechanisms for maintaining continuous growth are not yet clear. A novel short lifespan mutant of N. crassa that ceases hyphal elongation early was screened and analyzed to better understand the mechanisms for maintaining hyphal elongation in filamentous fungi. The mutant strain also exhibited high sensitivity to mutagens such as hydroxyurea and ultraviolet radiation. Based on these observations, we named the novel mutant "mutagen sensitive and short lifespan 1 (ms1)." The mutation responsible for the short lifespan and mutagen sensitivity in the ms1 strain was identified in DNA polymerase γ (mip-1:NCU00276). This mutation changed the amino acid at position 814 in the polymerase domain from leucine to arginine (MIP-1 L814R). A dosage analysis by next-generation sequencing reads suggested that mitochondrial DNA (mtDNA) sequences are decreased nonuniformly throughout the genome of the ms1 strain. This observation was confirmed by quantitative PCR for 3 representative loci and restriction fragment length polymorphisms in purified mtDNA. Direct repeat-mediated deletions, which had been reported previously, were not detected in the mitochondrial genome by our whole-genome sequencing analysis. These results imply the presence of novel mechanisms to induce the nonuniform decrease in the mitochondrial genome by DNA polymerase γ mutation. Some potential reasons for the nonuniform distribution of the mitochondrial genome are discussed in relation to the molecular functions of DNA polymerase γ.

Pharmaco-virological Outcomes and Genotypic Resistance Profiles Among Children and Adolescents Receiving a Dolutegravir-Based Regimen in Togo.

Few data are available on the real-world efficacy of receiving tenofovir-lamivudine-dolutegravir (-DTG) as human immunodeficiencyvirus (HIV) treatment, particularly among young people in West Africa. Here, we evaluated pharmaco-virological outcomes and resistance profiles among Togolese children and adolescents. A cross-sectional study was conducted in Lomé, Togo, enrolling antiretroviral-treated people with HIV aged from 18 months to 24 years. Plasma HIV-1 viral load and antiretroviral concentrations were measured. Next-generation sequencing of protease, reverse transcriptase (RT), and integrase was performed on all samples with viral loads >200 copies/mL. Drug resistance mutations (DRMs) were identified and interpreted using the ANRS-MIE algorithm. 264 participants were enrolled (median age, 17 years); 226 received a DTG-based regimen for a median of 20.5 months. Among them, there was virological suppression at the 200-copies/mL threshold in 80.0% of the participants. Plasma DTG concentrations were adequate (ie, >640 ng/mL), suboptimal, and below the limit of quantification in 74.1%, 6.7%, and 19.2% of participants receiving DTG, respectively. Overall, viruses resistant to any of nucleoside RT inhibitors, non-NRTIs, and protease inhibitors were found in 52%, 66%, and 1.6% of participants, respectively. A major integrase inhibitor DRM was observed in 9.4% (n = 3/32; R263K, E138A-G140A-Q148R, and N155H) of participants with a viral load >200 copies/mL. These first findings in a large series of adolescents in a low-income country showed a good virological response of 80% and the presence of an integrase DRM in 9.4% of virological failures, supporting the need to monitor DTG drug resistance to reduce the risk of resistance acquisition.

Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.

Unveiling the intriguing relationship: oncogenic KRAS, morphological shifts, and mutational complexity in pancreatic mucinous cystic neoplasms.

Pancreatic ductal adenocarcinoma (PDAC) often arises from preexisting cystic lesions such as intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN). This study investigated the molecular heterogeneity and mutational landscape of MCN in relation to PDAC, highlighting the significance of KRAS mutations in tumor progression. Utilizing targeted next-generation sequencing on low-grade MCN and invasive PDAC samples, we identified a substantial overlap in mutational profiles, particularly mutations in KRAS, TP53, and FBXW7. Specifically, 69.2% of MCN exhibited somatic mutations, with KRAS mutations being a predominant oncogenic driver. The characterization of mutant versus wildtype KRAS variant allele frequencies (VAF) indicated higher mutation levels in PDAC compared to MCN, suggesting an evolutionary trajectory toward malignancy. Further histological analysis of 12 additional MCN cases revealed significant intratumor heterogeneity, with variant KRAS mutation distributions correlating with distinct cellular morphologies and dysplastic features. Additionally, we explored the potential of liquid biopsies, demonstrating a concordance rate of 71.4% for KRAS mutation detection in circulating tumor DNA (ctDNA) relative to tissue biopsies across cohorts. Our findings underscore the relevance of evaluating KRAS mutations-herein referred to as VAF per microdissected region-as they relate to histopathological markers of dysplasia, contributing to improved stratification of pancreatic lesions and facilitating personalized treatment strategies. In conclusion, this comprehensive analysis of MCN highlights the importance of KRAS as a crucial biomarker for both malignant progression and therapeutic decision-making in pancreatic pathology. Ultimately, our study suggests that characterizing the mutational landscape and histological features of MCN can enhance early detection and intervention strategies for at-risk patients. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Deterministic genetic barcoding for multiplexed behavioral and single-cell transcriptomic studies.

Advances in single-cell sequencing technologies have provided novel insights into the dynamics of gene expression and cellular heterogeneity within tissues and have enabled the construction of transcriptomic cell atlases. However, linking anatomical information to transcriptomic data and positively identifying the cell types that correspond to gene expression clusters in single-cell sequencing data sets remains a challenge. We describe a straightforward genetic barcoding approach that takes advantage of the powerful genetic tools in Drosophila to allow in vivo tagging of defined cell populations. This method, called Targeted Genetically-Encoded Multiplexing (TaG-EM), involves inserting a DNA barcode just upstream of the polyadenylation site in a Gal4-inducible UAS-GFP construct so that the barcode sequence can be read out during single-cell sequencing, labeling a cell population of interest. By creating many such independently barcoded fly strains, TaG-EM enables positive identification of cell types in cell atlas projects, identification of multiplet droplets, and barcoding of experimental timepoints, conditions, and replicates. Furthermore, we demonstrate that TaG-EM barcodes can be read out using next-generation sequencing to facilitate population-scale behavioral measurements. Thus, TaG-EM has the potential to enable large-scale behavioral screens in addition to improving the ability to multiplex and reliably annotate single-cell transcriptomic experiments.

Microbiota of Punctuated Snake Eel Ophichthus remiger (Valenciennes, 1842) Reared in Recirculation System Is Dominated by Latilactobacillus

Research on microbiota has underscored the crucial influence of microbial communities on numerous biological functions that yield positive outcomes for the host, such as digestion, nutrient metabolism, resistance against pathogen invasion, and growth performance. Concurrently, numerous variables, including the host’s diet, genetics, and physiological condition and environmental factors, influence the gut microbiota. Our study aims to characterize the bacterial community composition of the common snake eel (Ophichthus remiger), captured wild and then reared under controlled conditions. We employed a 16S rRNA gene-based approach facilitated by next-generation sequencing to conduct this analysis. The gut microbiota of the snake eel was highly dominated by bacteria from the phylum Firmicutes, comprising over 80% of the relative abundance, with Lactilactobacillus being the most important genus. The results suggest that feed-associated bacteria may influence the composition of the microbiota, contributing the most relevant bacteria within the intestinal content. This study provides the first comprehensive analysis of the gut microbiota in Ophichthus remiger, offering novel insights into the potential roles of Firmicutes and Lactilactobacillus in marine eels.

Treatment and reasons for choosing treatment in breast cancer patients who underwent next-generation sequencing test.

Next-generation sequencing (NGS) is commonly used in clinical practice to decide treatment based on genomic information. This study was performed to optimize the proportion of actionable gene profiling and treatment based on genetic alterations in breast cancer at one of cancer centers in Japan. Patients with breast cancer who reported NGS results at one of cancer centers in Japan from August 2019 to December 2023 were retrospectively investigated by reviewing their electronic medical records. Patients were examined using the OncoGuideTM NCC Oncopanel System, FoundationOne® CDx, or FoundationOne® Liquid CDx. The evidence levels for drug recommendation were added for each gene alteration according to the guidelines from three Japanese oncology-related societies. ''Actionable alterations'' were those at evidence levels A-D, including high microsatellite instability and high tumor mutation burden status. "Patients with recommended drug" (approved, investigational, and off-label drugs) were defined as those who were selected by the Molecular Tumor Board. Of the 106 patients, 54 were tested using the NCC Oncopanel System and 50 using FoundationOne CDx. The most frequent alterations were TP53 mutations (52.8%) and PIK3CA mutations (31.1%). Of the 56 patients (52.8%) with recommended drugs, 11 (10.4%) received genome-matched therapy and only three (2.8%) participated in clinical trials. The most common reason for not receiving genome-matched therapy was patient refusal for personal reasons, although clinical trials were available (18 patients). The top reasons for patients not receiving the recommended genome-matched therapy were factors related to the patient, including a number of prior treatments higher than what was allowed by the eligibility criteria of the clinical trials, and poor physical condition. Most patients received four or more regimens of cytotoxic chemotherapy before NGS. NGS is only available at the late phase of treatment in Japan, which would constitute a problem for the treatment of breast cancer.

Molecular Review of Suspected Alport Syndrome Patients—A Single-Centre Experience

Background: Alport syndrome (AS) is a clinically and genetically heterogeneous glomerulopathy resulting from pathogenic variants in COL4A3, COL4A4, and COL4A5. Genetic diagnosis is increasingly being conducted using next-generation sequencing (NGS). Methods: Within eight years, we examined a group of 247 Polish individuals and found in total 138 unrelated probands suspected with AS based on clinical course, laboratory findings, and/or family history, as well as the total of 109 family members. We applied a targeted NGS panel to identify the genetic spectrum of AS. Known and novel variants were revealed, and detailed evaluation was performed according to ACMG/AMP guidelines to classify them as pathogenic/likely pathogenic/VUS changes. Identified genotypes were compared with clinical manifestations: hematuria, proteinuria, chronic kidney disease, sensorineural hearing impairment, ocular abnormalities, and hypertension. Results: The molecular background was established in 109/138 probands. Overall, 79 different COL4A3-COL4A5 changes (56 known and 23 novel) were revealed. About 97% were SNVs, and only two COL4A5 CNVs were identified. In total, 11 recurrent COL4A3-COL4A5 variants were observed, including the most frequent COL4A5:p.Gly624Asp, accounting for 31% of X-linked AS. Conclusions: The use of NGS panel has shown considerable promise in the field of AS, increasing diagnostic rate to 79% and reducing time to diagnosis. The phenotype-driven gene panel, specific for genetic diseases in the pediatric population, is an affordable alternative to WGS and WES, offering comparable diagnostic efficacy and supporting its implementation as a first-line genetic test in rare diseases, including AS. Based on the obtained genotype–phenotype correlation, we assessed that NGS allows us to avoid invasive renal biopsy in AS diagnosis. It provides AS confirmation/exclusion, atypical AS identification, symptomatic/asymptomatic monoallelic COL4A3-COL4A5 carrier (especially COL4A5 females) determination, and inheritance pattern establishment. AS diagnosis confirmation enables clinical course prediction and is crucial for the early introduction of renoprotective treatment with renin–angiotensin–aldosterone system blockade, aimed at slowing the disease progression and estimating the risk in family members, which is important for genetic counselling.

Association of pituitary neuroendocrine tumors and neurofibromatosis type 1: assessing causation versus coincidence. Case report

IntroductionPatients with neurofibromatosis type 1 (NF1) are at risk for developing various neoplasms. Since the early twentieth century, multiple cases of pituitary neuroendocrine tumors (PitNETs) occurring in this context have been published. Yet, the role of NF1 (17q11.2) loss-of-function (LOF) variants in pituitary tumorigenesis remains unclear.AimWe report the clinical and molecular characterization of a case of PitNET diagnosed in a patient with NF1. We also review the available data for and against a causal association between NF1 defects and pituitary tumors.MethodsOur patient was recruited via an ongoing prospective study of individuals with neuroendocrine neoplasms. Genetic testing was carried out by means of targeted next generation sequencing (NGS) and Sanger sequencing in blood and tumor DNA, respectively. NF1 expression was analyzed via quantitative polymerase chain reaction (qPCR) in blood and tumor cDNA. Similar cases were searched in the literature.ResultsA 54-year-old-man was incidentally diagnosed with a clinically non-functioning PitNET via brain imaging. He had a personal and family history of NF1 and carried the germline pathogenic variant NF1 (NM_001042492.3): c.147C&amp;gt;A, p.Y49*. Via transsphenoidal surgery, a 16 mm lesion was resected, showing strong granular cytoplasmic immunoreactivity with patchy distribution for NF1 and preserved heterozygosity for the NF1 defect. Additional NGS ruled out germline defects in PitNET-associated genes. By qPCR, NF1 was significantly overexpressed in the tumor when compared with another NF-PitNET, but not when compared with a corticotropinoma. We reviewed twenty-three case reports of PitNETs occurring in patients with either clinical NF1 without genetic study, individuals with NF1 germline variants with or without clinical NF1 or associated with somatic NF1 defects. Predominance of GH-secreting and large PitNETs, with young-onset in around half of the cases, were noticed. Two individuals developed multiple endocrine neoplasia-like phenotypes but tested negative for other relevant genetic defects.ConclusionsAlthough the association of NF1 and PitNETs could be coincidental, the clinical characteristics of the reviewed cases differ from those of typical incidentalomas. NF1 could drive pituitary tumorigenesis via haploinsufficiency, but this hypothesis requires further research. Additional clinical and molecular data from large cohorts of affected individuals should help clarify this question.

Three Cases of Spinocerebellar Ataxia Type 2 (SCA2) and Pediatric Literature Review: Do Not Forget Trinucleotide Repeat Disorders in Childhood-Onset Progressive Ataxia

Background: Childhood-onset progressive ataxias are rare neurodegenerative disorders characterized by cerebellar signs, sometimes associated with other neurological or extra-neurological features. The autosomal dominant forms, known as spinocerebellar ataxias (SCAs), linked to trinucleotide (i.e., CAG) repeat disorders, are ultra-rare in children. We describe three patients from two unrelated families affected by spinocerebellar ataxia type 2 (SCA2) and present a literature review of pediatric cases. Methods: The patients’ clinical and genetic data were collected retrospectively. Results: The first case was a 9.5-year-old boy, affected by ataxia with oculomotor apraxia and cerebellar atrophy, subcortical myoclonus, and peripheral axonal sensitive polyneuropathy caused by a pathologic expansion in ATXN2, inherited from his asymptomatic father. Two brothers with familial SCA2 presented neurodegeneration leading to early death in one case and progressive ataxia, parkinsonism, and epilepsy with preserved ambulation at age 18 years in the second. To date, 19 pediatric patients affected by SCA2 have been reported, 3 of whom had a phenotype consistent with progressive ataxia with shorter CAG repeats, while 16 had more severe early-onset encephalopathy, with longer alleles. Conclusions: Although they are ultra-rare, trinucleotide repeat disorders must be considered in differential diagnosis of hereditary progressive ataxias in children, especially considering that they require targeted genetic testing and can manifest even before a parental carrier becomes symptomatic. Thus, they must also be taken into account with negative family history and when Next-Generation Sequencing (NGS) results are inconclusive. Notably, the association between cerebellar ataxia and other movement disorders should raise suspicion of SCA2 among differential diagnoses.

Leveraging Artificial Intelligence and Gene Expression Analysis to Identify Some Potential Bovine Coronavirus (BCoV) Receptors and Host Cell Enzymes Potentially Involved in the Viral Replication and Tissue Tropism

Bovine coronavirus (BCoV) exhibits dual tissue tropism, infecting both the respiratory and enteric tracts of cattle. Viral entry into host cells requires a coordinated interaction between viral and host proteins. However, the specific cellular receptors and co-receptors facilitating BCoV entry remain poorly understood. Similarly, the roles of host proteases such as Furin, TMPRSS2, and Cathepsin-L (CTS-L), known to assist in the replication of other coronaviruses, have not been extensively explored for BCoV. This study aims to identify novel BCoV receptors and host proteases that modulate viral replication and tissue tropism. Bovine cell lines were infected with BCoV isolates from enteric and respiratory origins, and the host cell gene expression profiles post-infection were analyzed using next-generation sequencing (NGS). Differentially expressed genes encoding potential receptors and proteases were further assessed using in-silico prediction and molecular docking analysis. These analyses focused on known coronavirus receptors, including ACE2, NRP1, DPP4, APN, AXL, and CEACAM1, to identify their potential roles in BCoV infection. Validation of these findings was performed using the qRT-PCR assays targeting individual genes. We confirmed the gene expression profiles of these receptors and enzymes in some BCoV (+/−) lung tissues. Results revealed high binding affinities of 9-O-acetylated sialic acid and NRP1 to BCoV spike (S) and hemagglutinin-esterase (HE) proteins compared to ACE2, DPP4, and CEACAM1. Additionally, Furin and TMPRSS2 were predicted to interact with the BCoV-S polybasic cleavage site (RRSRR|A), suggesting their roles in S glycoprotein activation. This is the first study to explore the interactions of BCoV with multiple host receptors and proteases. Functional studies are recommended to confirm their roles in BCoV infection and replication.

Constructing a potential HLA haplo-homozygous induced pluripotent stem cell haplobank using data from an umbilical cord blood bank

BackgroundInduced pluripotent stem cells (iPSCs) can differentiate into any type of cell and have potential uses in regenerative medicine for the treatment of many diseases. However, reducing immune rejection is a key problem in the application of iPSCs that can be solved by the development of haplobanks containing specially selected iPSC lines.MethodsTo study the feasibility of constructing an HLA (human leukocyte antigen)-matched induced pluripotent stem cell haplobank in China, 5421 umbilical cord blood samples were randomly collected from the Umbilical Cord Blood Bank of Zhejiang Province, China. The HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci were genotyped using next-generation sequencing. Using HLA genotype data at the high-resolution level, the number of HLA homozygous donors needed to cover a certain percentage of the Chinese population and the feasibility of constructing a high-matching iPSC haplobank were estimated.ResultsThirteen HLA-A, -B, and -DRB1 and 11 HLA-A, -B, -C, -DRB1, and -DQB1 haplotype homozygotes were observed among the stored umbilical CB units which were as HLA zero-mismatched iPSC donors cumulatively matched 37.01% and 32.99% of 5421 potential patients respectively. The analysis showed that 100 distinct HLA-A, -B, and -DRB1 and HLA-A, -B, -C, -DRB1, and -DQB1 homozygous haplotypes would cover 72.74% and 67.87% of Chinese populations, respectively, and 600 HLA-A, -B, -C, -DRB1, and -DQB1 homozygous haplotypes would cover more than 90% of Chinese populations. PCA (principal component analysis) of published HLA data from different populations revealed that the frequency of these haplotypes in Asian populations is different from those in European populations.ConclusionThe results suggested that at least some HLA-homozygous iPSC lines developed from Chinese individuals will not only be useful for covering the Chinese population but will also cover other Asian populations. A high-matching iPSC haplobank generated from umbilical CB units may be an economical and effective option in an allogeneic model of iPSC therapy.

Genome-Wide Association Study Reveals Genetic Architecture of Common Epilepsies.

Epilepsy, affecting approximately 50 million individuals worldwide, exhibits a genetic heritability of 32%. While several genes/loci associated with epilepsy have been identified through candidate and genome-wide association studies (GWAS), exploration of population-specific markers remains underexplored. We conducted the first GWAS in north Indian population (~1500 samples) to identify genetic variants/loci associated with epilepsy risk, validated using targeted next-generation sequencing (NGS). Our GWAS revealed 30 variants across seven loci associated with epilepsy risk, including six novel loci. Subtype analysis based on etiology and seizure types, identified 57 variants across 11 loci, 10 of which are novel. Gene-set analysis unveiled enrichment in genes associated with glutathione synthesis and recycling and regulation of dopaminergic neuron differentiation pivotal in epilepsy pathophysiology. Furthermore, PRS analysis revealed a significant genetic contribution to the epilepsy with an R2 of 0.00573. Additionally, targeted NGS showed ~95% concordance with GSA genotypes. Our study highlights six novel loci rs17031055/4q31.3(DCHS2), rs73182224/3q27.2(DGKG), rs9322462/6q25.2(CNKSR3), rs75328617/8q24.23(RNU1-35P), rs2938010/10q26.13(CTBP2) and rs11652575/17p11.2(SLC5A10) associated with epilepsy risk. These findings offer valuable insights into the genetic landscape of epilepsy in the north Indian population, providing foundation for future exploratory studies.

Diagnostic value of metagenomic next-generation sequencing using bronchoalveolar lavage fluid samples for pathogen detection in children with severe or refractory pneumonia.

This study on 127 patients with severe and refractory pneumonia showed that mNGS was significantly superior to CMTs in terms of bacterial and fungal detection. We also found that multiplex PCR assay was comparable to mNGS for the detection of Mycoplasma pneumoniae and respiratory viruses and may have greater application advantages in combination with CMTs, such as M. pneumoniae IgM. For severe and refractory pneumonia, or when empiric treatment is not effective, collecting BALF for mNGS can help to quickly identify the causative organisms at an early stage. It is also important to choose more appropriate methods or combinations for different pathogens.