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BACKGROUND: Pancreatic neuroendocrine tumors (pNETs) are genomically diverse tumors. The management of newly diagnosed well-differentiated pNETs is limited by a lack of sensitivity of existing biomarkers for prognostication. Our goal was to investigate the potential utility of genetic markers as a predictor of progression-free survival (PFS) and recurrence-free survival (RFS). METHODS: Whole-exome sequencing of resected well-differentiated, low and intermediate-grade (G1 and G2) pNETs and normal adjacent tissue from patients who underwent resection from 2005 to 2015 was performed. Genetic alterations were classified using pan-genomic and oncogenic pathway classifications. Additional samples with genetic and clinicopathologic data available were obtained from the publicly available International Cancer Genome Consortium (ICGC) database and included in the analysis. The prognostic relevance of these genomic signatures on PFS and RFS was analyzed. RESULTS: Thirty-one patients who underwent resection for pNET were identified. Genomic analysis of mutational, copy number, cytogenetic, and complex phenomena revealed similar patterns to prior studies of pNETs with relatively few somatic gene mutations but numerous instances of copy number changes. Analysis of genomic and clinicopathologic outcomes using the combined data from our study as well as the ICGC pNET cohort (n = 124 patients) revealed that the recurrent pattern of whole chromosome loss (RPCL) and metastatic disease were independently associated with disease progression. When evaluating patients with local disease at the time of resection, RPCL and alterations in the TGFβ oncogenic pathway were independently associated with the risk of recurrence. CONCLUSIONS: Well-differentiated pNETs are genomically diverse tumors. Pathway signatures may be prognostic for predicting disease progression and recurrence.

AbstractBackgroundMaterial and methodsResultsConclusionGenes associated with global developmental delay (GDD) and intellectual disability (ID) are increasingly being identified through next-generation sequencing (NGS) technologies. This study aimed to identify novel mutations in GDD/ID phenotypes through whole-exome sequencing (WES) and additional in silico analyses.WES was performed on 27 subjects, among whom 18 were screened for potential novel mutations. In silico analyses included protein-protein interactions (PPIs), gene-miRNA interactions (GMIs), and enrichment analyses. The identified novel variants were further modelled using I-Tasser-MTD and SWISS-MODEL, with structural superimposition performed.Novel mutations were detected in 18 patients, with 10 variants reported for the first time. Among these, three were classified as pathogenic (DNMT1:c.856dup, KCNQ2:c.1635_1636insT, and TMEM94:c.2598_2599insC), and six were likely pathogenic. DNMT1 and MRE11 were highlighted as key players in PPIs and GMIs. GMIs analysis emphasised the roles of hsa-miR-30a-5p and hsa-miR-185-5p. The top-scoring pathways included the neuronal system (R-HSA-112316, p = 7.73E-04) and negative regulation of the smooth muscle cell apoptotic process (p = 3.37E-06). Homology modelling and superimposition revealed a significant functional loss in the mutated DNMT1 enzyme structure.This study identified 10 novel pathogenic/likely pathogenic variants associated with GDD/ID, supported by clinical findings and in silico analyses focused on DNMT1 mutations. [ABSTRACT FROM AUTHOR]

Background: Arthrogryposis multiplex congenita (AMC) is a congenital disorder characterized by multiple joint involvement, primarily affecting limb mobility and leading to various tissue contractures. Variations in the RIPK4 gene may impact connective tissues, thereby resulting in a spectrum of malformations. This study aimed to identify the genetic etiologies of AMC patients and provide genetic testing information for further diagnosis and treatment of AMC. Methods: We recruited a Chinese female patient with hand-related AMC and her family members. Whole-exome sequencing (WES) was employed to determine the genetic etiologies of the patient's disease. The pathogenic mechanisms of the identified variations were analyzed using protein tolerance profiling and modeling. Results: We identified two novel RIPK4 variants (c.1354G > A, p.E452K; c.1558A > T, p.T520S). Pathogenicity studies indicated that the c.1354G > A, p.E452K variant changed the charge from negative to positive and altered the chemical properties from acidic to alkaline, potentially significantly affecting protein function. Conclusions: We reported the discovery of two novel RIPK4 variants (c.1354G > A, p.E452K; c.1558A > T, p.T520S) in a Chinese AMC female patient's family. Our study enhances the genetic repository for AMC and highlights the pathogenicity of RIPK4 variants, underscoring the significance of comprehensive management for genetic-related diseases, particularly the critical roles of prenatal diagnosis and genetic counseling. Trial registration: The research protocol received approval from the Ethics Review Committee of Xiangya Hospital of Central South University in China (approval number: 202103427), registered in March 2021, with all participants providing duly signed informed consent forms. [ABSTRACT FROM AUTHOR]

Introduction: Usually, patients with sY84 or sY86 deficiency present with azoospermia, but recent studies have shown that some males with partial AZFa deletions, including sY84 or sY86, exhibit normal fertility. Here, we reported a rare case of AZF deletion in a family, where both father and son exhibited a deletion at the sY86 site in the AZFa region and a partial deletion in the AZFc region. Methods and Results: Detection was performed using classical multiplex polymerase chain reaction and the "Male AZF Full-region Detection" Panel, revealing specific deletions in AZFa: Yq11.21 (14,607,372–14,637,973), 30.6 kb; AZFc: Yq11.223–11.23 (25,848,831–27,120,665), 1.3 M for the father; and Yq11.223–11.23 (25,505,378–27,120,665), 1.6 M for the son. Notably, although the son's sperm motility parameters showed no significant abnormalities, there was a history of failed pregnancies for twice, with sperm exhibiting a high rate of head defect. Discussion: Given the complexities of the reproductive phenotype following AZF region deletions, additional extended genetic testing is necessary when partial deletions in the AZF region are detected, thus providing more accurate predictions of the spermatogenesis in patient. This study provides valuable insights and guidance for clinical decision-making and the implementation of assisted reproductive technologies in such cases. [ABSTRACT FROM AUTHOR]

A fundamental event in the formation of heart valves involves the transformation of endocardial cells within the outflow tract (OFT) and atrioventricular canal (AVC) cushions through a process known as endothelial-to-mesenchymal transition (EndMT). Aberrant EndMT is a primary cause of congenital valvular malformations. Manic Fringe (MFNG) has been previously associated with cardiovascular development, although its role in heart valve development remains underexplored. In this study, we seek to enhance our understanding of MFNG's involvement in valve formation and its association with EndMT. Staining results of histological section revealed the expression of MFNG in the AVC and OFT from embryonic day 9.5 to 10.5 (E9.5–E10.5), when EndMT takes place. Cellular data demonstrated that MFNG exerts a positive regulatory influence on the EndMT process, promoting endothelial cell (EC) migration by enhancing the activity of the Notch signalling pathway. MFNG knockdown mediated by antisense morpholino oligonucleotides (MO) injection caused abnormal development of the heart and valves in zebrafish. Furthermore, through whole-exome sequencing (WES), we identified a heterozygous MFNG mutation in patients diagnosed with tetralogy of Fallot-pulmonary valve stenosis (TOF-PS). Cellular and molecular assays confirmed that this deleterious mutation reduced MFNG expression and hindered the EndMT process. In summary, our study verifies that MFNG plays a role in promoting EndMT mediated by the Notch signalling pathway during the heart and valve development. The MFNG deleterious variant induces MFNG loss of function, potentially elucidating the underlying molecular mechanisms of MFNG's involvement in the pathogenesis of congenital heart valve defects. These observations contribute to our current genetic understanding of congenital heart valve disease and may provide a potential target for prenatal diagnosis and treatment. Key messages: Our examination revealed, for the first time, that MFNG exhibited high expression levels during EndMT of heart valve development in mice. Our findings provide compelling evidence that MFNG plays a role in promoting EndMT mediated by the Notch signalling pathway. Our results identified, for the first time, a deleterious MFNG p. T77M variant that inhibited the EndMT process by downregulating the activity of the Notch signalling pathway, thereby preventing the normal valve formation. MFNG may serve as an early diagnostic marker and an effective therapeutic target for the clinical treatment of congenital heart valve defects. [ABSTRACT FROM AUTHOR]

Sudden unexplained death (SUD) can affect apparently healthy adolescents and young adults with no prior clinical symptoms and no clear diagnostic findings at autopsy. Although primary cardiac arrhythmias have been shown to be the direct cause of death in the majority of SUD cases, the genetic predisposition contributing to SUD remains incompletely understood. Currently, molecular autopsy is considered to be an effective diagnostic tool in the multidisciplinary management of SUD, but the analysis focuses mainly on the coding region and the significance of many identified variants remains unclear. Recent studies have demonstrated the strong association between human disease and genetic variants in untranslated regions (UTRs), highlighting the potential role of UTR variants in the genetic predisposition to SUD. In this study, we searched for UTR variants with likely functional effects in the exome data of 45 SUD cases. Among 244 genes associated with cardiac diseases, three candidate variants with high confidence of pathogenicity were identified in the UTRs of SCO2, CALM2 and TBX3 based on a rigorous filtering strategy. A functional assay further validated the effect of these candidate variants on gene transcriptional activity. In addition, the constraint metrics, intolerance indexes, and dosage sensitivity scores of genes affected by the candidate variants were considered when estimating the consequence of aberrant gene expression. In conclusion, our study presents a practical strategy for UTR variant prioritization and functional annotation, which could improve the interpretation of molecular autopsy findings in SUD cohorts. [ABSTRACT FROM AUTHOR]

Background: Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive disorder presenting with cerebellar ataxia, sensory-motor axonal neuropathy, oculomotor apraxia, cerebellar atrophy and high alpha-fetoprotein (AFP) serum level. AOA2 is due to coding mutations of the SETX gene, mapped to chromosome 9q34. Seldom noncoding mutations affecting RNA processing have been reported too. To date psychiatric symptoms have never been reported in AOA2. Case presentation: A 19 years-old man came to our attention for progressive gait ataxia debuted five years earlier. His past medical history was unremarkable, while his parents were consanguineous. On neurological examination, he had bilateral horizontal gaze-evoked nystagmus with hypometric saccades and saccadic horizontal smooth pursuit, appendicular ataxia, limbs and trunk myoclonic involuntary movements with hands' dystonic postures and dance of the tendons. Psychological evaluation described intrusive and obsessive thoughts experienced by the patient, then diagnosed as obsessive-compulsive disorder. Blood tests detected an elevated AFP level. Brain MRI showed cerebellar atrophy, while electroneuromyography revealed an axonal sensory-motor polyneuropathy. In the suspicion of a pathology belonging to the autosomal recessive cerebellar ataxias (ARCA) spectrum disorder, a direct search of point mutations by whole-exome sequencing was performed revealing a novel biallelic variant in SETX gene (c.6208+2dupT), which was classified as likely pathogenic. Conclusion: The present case expands the genotypic and phenotypic spectrum of AOA2, reporting a novel likely pathogenic SETX mutation (c.6208+2dupT) and highlighting an early psychiatric involvement in AOA2, suggesting the need for psychiatric assessment in these neurologic patients. [ABSTRACT FROM AUTHOR]

We studied a family with three male individuals across two generations affected by common variable immune deficiency (CVID). We identified a novel missense heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives. Functional studies of the mutant allele in an overexpression system and of the patients’ cells confirmed the deleteriousness of the NFKB2 variant and genotype, respectively, on the activation of the non-canonical NF-κB signaling pathway. Impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity. The three patients’ plasma contained autoantibodies that neutralized IFN-α2 and/or IFN-ω, accounting for the severe or recurrent viral diseases of the patients, including influenza pneumonia in one sibling, and severe COVID-19 and recurrent herpes labialis in another. Our results confirm that NFKB2 alleles that are IκBδ GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases. [ABSTRACT FROM AUTHOR]

A rare type of autosomal recessive skeletal disorder, known as microcephalic osteodysplastic primordial dwarfism (MOPD) type II, causes a wide range of clinical abnormalities, including skeletal dysplasia, microcephaly, abnormal skin pigmentation, insulin resistance, typical facial features, and severe tooth deformities. Given the diverse manifestations of MOPD disorders and the overlapping clinical characteristics among primordial dwarfism (PD) subtypes, mutation analysis is crucial for accurate diagnosis and confirmation of MOPD II. In this study, whole-exome sequencing (WES) and GAP-PCR were employed to identify relevant genetic variants in three patients suspected of having MOPD. The clinical characteristics of three Iranian patients exhibiting hallmark features of MOPD were assessed. All patients were the offspring of consanguineous marriages and were referred from various provinces of Iran. WES was performed, and the identified variants were prioritized according to the standard filtration criteria. In the next step, Sanger sequencing was conducted to validate the candidate variants identified through WES in patients and their parents. Finally, GAP-PCR was implemented to resolve conflicting results between WES and Sanger sequencing for one of the patients. Analysis of three distinct cases revealed a novel homozygous copy number variation (CNV) in Case 3, consisting of a 490 bp deletion harboring exon 19 in the PCNT gene. Additionally, a nonsense homozygous variant in the PCNT gene (c.2812 C > T, p.Gln 938*) was found in Cases 1 and 2. This pathogenic variant has been previously documented in the literature. Reporting a novel deletion in the PCNT gene improves genetic testing services, including PND and pre-implantation genetic diagnosis (PGD) for MOPD II. [ABSTRACT FROM AUTHOR]

Background: Darier's disease (DD) is a skin disorder caused by mutations in the ATP2A2 gene. Researchers have been investigating the correlation between genotype and phenotype in DD. Understanding the genotype–phenotype relationship in DD can enhance our comprehension of the genetic background and phenotypic characteristics of the condition, as well as the relationship between its systemic and localized manifestations. Objectives: This study aimed to investigate the molecular pathogenesis of DD in a Chinese family, and to elucidate the genotype–phenotype correlation in DD by summarizing relevant literature. Methods: Gene mutations associated with DD were screened by whole-exome sequencing and verified using Sanger sequencing. Genetic analysis assessed the potential impact of these mutations. Genotype–phenotype correlation was obtained by chi-square analysis using literature search test. Results: (1) A novel ATP2A2 Missense mutation, c.2560T>C (p.W854R), was identified and confirmed by Sanger sequencing. Annotation analysis with the ANNOVAR tool indicated that this mutation disrupts normal protein function and is linked to DD clinical manifestations. (2) Genotype–phenotype analysis showed a significant correlation between the prevalence of DD-related mental disorders and geographic regions (P = 0.00), but no association between mutation type and mental disorder prevalence (P = 0.324). The age of onset varied between sporadic and familial cases (P = 0.032), averaging 33 years in sporadic cases and 16 years in familial cases. Conclusion: By analyzing the genotype–phenotype correlation, we aim to enhance our understanding of the genetic basis of DD. This research could improve early diagnosis, intervention, and the development of personalized long-term health management plans. [ABSTRACT FROM AUTHOR]

Context X-linked hypophosphatemia (XLH) is the most common form of inherited hypophosphatemic rickets (HR), caused by pathogenic variants in the PHEX gene. Genetic diagnosis of XLH facilitates early treatment optimization, especially for patients suitable for burosumab, a recombinant anti-fibroblast growth factor-23 monoclonal antibody. Objective This study aimed to use whole-exome sequencing (WES) and pedigree analysis to identify patients with XLH. Methods Medical records at a single center in Saudi Arabia were screened between 2014 and 2024 to identify patients with suggested HR. Of the 800 patients identified, 27 had had suspected XLH. The genetic study comprised 100 patients drawn from these 27 families. Results Clinical manifestations were widespread and variable within families. Severe disease was reported in 55% of children and 25% of adults. At presentation, all children were receiving either conventional therapy (60%) or burosumab (40%); however, 53% of adults were not treated. WES provided a genetic diagnosis in 23 families: alterations in the PHEX gene (20 families), with homozygous ENPP1 and DMP1 variants detected in 2 and 1 families, respectively. Pathogenic/likely pathogenic variants were detected in 23 families (diagnostic yield 85%). Ten novel likely pathogenic variants were detected. Pedigree analysis provided information to support disease-specific patient management. Conclusion WES detected a diagnostic molecular abnormality in 85% of families with HR phenotypes; PHEX variants were the most common. Combined use of WES and pedigree analysis highlighted the underdiagnosis of adult XLH in this population, with most family members being diagnosed after the pedigree analysis. [ABSTRACT FROM AUTHOR]

Background: CRMP1 is a key protein involved in brain development. Methods: We performed genetic testing through whole-exome sequencing (WES) in an individual with a neurodevelopmental disorder. Results: We identified a de novo heterozygous CRMP1 NM_001014809.3:c.1755del (p.Lys586fs) variant in the affected individual. This mutation was submitted to ClinVar (SCV005196589). Conclusion: Currently, the CRMP1 gene has no clear disease phenotype association in the Online Mendelian Inheritance in Man (OMIM) database. Our report may provide evidence for an association between the CRMP1 gene and neurodevelopmental disorders (NDDs). [ABSTRACT FROM AUTHOR]

Primary ciliary dyskinesia (PCD) is a clinically rare, genetically and phenotypically heterogeneous condition characterized by chronic respiratory tract infections, male infertility, tympanitis, and laterality abnormalities. PCD is typically resulted from variants in genes encoding assembly or structural proteins that are indispensable for the movement of motile cilia. Here, we identified a novel nonsense mutation, c.466G>T, in cilia- and flagella-associated protein 300 (CFAP300) resulting in a stop codon (p.Glu156*) through whole-exome sequencing (WES). The proband had a PCD phenotype with laterality defects and immotile sperm flagella displaying a combined loss of the inner dynein arm (IDA) and outer dynein arm (ODA). Bioinformatic programs predicted that the mutation is deleterious. Successful pregnancy was achieved through intracytoplasmic sperm injection (ICSI). Our results expand the spectrum of CFAP300 variants in PCD and provide reproductive guidance for infertile couples suffering from PCD caused by them. [ABSTRACT FROM AUTHOR]

Introduction: The sodium/iodide symporter (NIS) mediates active iodide accumulation in the thyroid follicular cell. Biallelic loss-of-function variants in the NIS-coding SLC5A5 gene cause congenital dyshormonogenic hypothyroidism due to a defect in the accumulation of iodide, which is required for thyroid hormonogenesis. Objective: We aimed to identify, and if so to functionally characterize, novel pathogenic SLC5A5 gene variants in a patient diagnosed with severe congenital dyshormonogenic hypothyroidism characterized by undetectable radioiodide accumulation in a eutopic thyroid gland, as well as in the salivary glands. Methods: The coding region of the SLC5A5 gene was sequenced using whole-exome sequencing. In silico analysis and in vitro functional characterization of missense SLC5A5 gene variants were performed. Results: Proposita's whole-exome sequencing revealed a novel pair of compound heterozygous missense variants in the SLC5A5 gene, c.1,627G>A (p.G543R) and c.1,684T>A (p.L562M). The parents were heterozygous carriers of the variants as determined by Sanger sequencing of the SLC5A5 gene. The p.G543R variant in the homozygous state has previously been associated with congenital hypothyroidism. The novel p.L562M variant was not reported in the Genome Aggregation Consortium dataset. In silico analysis of the pathogenic impact of the p.L562M variant yielded inconclusive results. Functional in vitro studies showed that the p.L562M variant reduces iodide accumulation due to defective expression of the mutant NIS protein at the plasma membrane. Notably, the aliphatic residue Leu at position 562 in the carboxy terminus of the protein, which is highly conserved in NIS orthologues, is required for NIS plasma membrane expression. Conclusions: We report novel compound heterozygous missense SLC5A5 gene variants causing defective iodide accumulation, thus leading to congenital dyshormonogenic hypothyroidism. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint inhibitors (ICIs) are effective in a subset of patients with metastatic solid tumors. However, the patients who would benefit most from ICIs in biliary tract cancer (BTC) are still controversial. Materials and methods: We molecularly characterized tissues and blood from 32 patients with metastatic BTC treated with the ICI pembrolizumab as second-line therapy. Results: All patients had microsatellite stable (MSS) type tumors. Three of the 32 patients achieved partial response (PR), with an objective response rate (ORR) of 9.4% (95% confidence interval [CI], 2.0–25.2) and nine showed stable disease (SD), exhibiting a disease control rate (DCR) of 37.5% (95% CI, 21.1–56.3). For the 31 patients who had access to PD-1 ligand 1 (PD-L1) combined positive score (CPS) testing (cut-off value ≥1%), the ORR was not different between those who had PD-L1-positive (PD-L1+; 1/11, 9.1%) and PDL1-(2/20, 10.0%) tumors (p = 1.000). The tumor mutational burden (TMB) of PD-L1+ BTC was comparable to that of PD-L1-BTC (p = 0.630). TMB and any exonic somatic mutations were also not predictive of pembrolizumab response. Molecular analysis of blood and tumor samples demonstrated a relatively high natural killer (NK) cell proportion in the peripheral blood before pembrolizumab treatment in patients who achieved tumor response. Moreover, the tumors of these patients presented high enrichment scores for NK cells, antitumor cytokines, and Th1 signatures, and a low enrichment score for cancer-associated fibroblasts. Conclusions: This study shows the molecular characteristics associated with the efficacy of pembrolizumab in BTC of the MSS type. [ABSTRACT FROM AUTHOR]

Short stature, joint hyperextension, ocular hypotension, Rieger abnormalities, and delayed tooth eruption (SHORT) syndrom is a rare primary autosomal dominant genetic disorder mainly caused by pathogenic loss-of-function variants in the phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) gene. We report the case of a Chinese adult female patient with SHORT syndrome, carrying a PIK3R1 gene variant (c.1945C > T), who developed abnormal glucose metabolism and severe postprandial insulin resistance over 9 years. Although there are currently no established treatment guidelines for insulin resistance in patients with SHORT syndrome, we implemented a comprehensive treatment plan, including lifestyle interventions, metformin, and voglibose for glucose control. After 6 months of continuous observation, the patient's blood glucose levels and insulin resistance improved significantly. This case study provides useful insights for future treatment strategies. [ABSTRACT FROM AUTHOR]

The complex and heterogeneous genomic landscape of multiple myeloma (MM) and many of its clinical and prognostic implications remains to be understood. In other cancers, such as breast cancer, using whole-exome sequencing (WES) and molecular signatures in clinical practice has revolutionized classification, prognostic prediction, and patient management. However, such integration is still in its early stages in MM. In this study, we analyzed WES data from 35 MM patients to identify potential mutational signatures and driver mutations correlated with clinical and cytogenetic characteristics. Our findings confirm the complex mutational spectrum and its impact on previously described ontogenetic and epigenetic pathways. They show TYW1 as a possible new potential driver gene and find no significant associations of mutational signatures with clinical findings. Further studies are needed to strengthen the role of mutational signatures in the clinical context of patients with MM to improve patient management. [ABSTRACT FROM AUTHOR]

Summary: The role of imatinib in PDGFRA/B‐negative hypereosinophilic syndromes (HES) is controversial because of the heterogeneity of HES and the scarcity of prospective studies. We conducted a phase II clinical trial to evaluate the efficacy of imatinib in PDGFRA/B‐negative HES. Thirty‐two patients were treated with imatinib (100–400 mg daily), and the molecular basis of their response was identified using whole‐exome sequencing (WES) and whole‐transcriptome sequencing (WTS). The haematological response rate was 46.9%, with a complete haematological response (CHR) rate of 18.8%. The median time to response was 1.5 months. Among the six patients who achieved CHR, five maintained it until the 24th cycle of imatinib and one lost response after 20 months. The median progression‐free survival was 4.3 months. WES and WTS were conducted for 11 patients. The number of non‐silent mutations did not differ between responders and non‐responders. Nine differentially expressed genes, including SNORD15A, were downregulated in responders. STAT5B::RARA, PAK2::PIGX, and FIP1L1::CHIC2 fusions were identified in patients with sustained responses, and RNF130::BRAF and WNK1::KDM5A fusions were identified in non‐responders. Imatinib, along with an appropriate biomarker, could be a promising option for PDGFRA/B‐negative HES. [ABSTRACT FROM AUTHOR]

Background: Infiltrative follicular variant of papillary thyroid carcinoma (IFVPTC) exhibits nuclear characteristics typical of papillary thyroid carcinoma (PTC) but demonstrates a follicular growth pattern. The diagnosis of IFVPTC presenting with atypical nuclear features of PTC poses challenges for both preoperative cytopathology and postoperative histopathology. In such cases, molecular markers are needed to serve as diagnostic aids. Given the limited knowledge of IFVPTC's genomic features, this study aimed to characterize its genetic alterations and identify clinically relevant molecular markers. Methods: Whole‐exome sequencing of 50 IFVPTC tumor–normal pairs identified single‐nucleotide variants, somatic copy number alterations (sCNAs), and subclonal architecture. Key mutations were verified via polymerase chain reaction and Sanger sequencing, whereas valuable biomarkers were validated via immunohistochemistry (IHC). Results: This study found that endogenous processes rather than exogenous mutagens dominated the shaping of the genome of IFVPTC during tumorigenesis. BRAF V600E was the only common trunk mutation and significantly mutated gene in IFVPTC. Subcloning analysis found that most IFVPTC samples harbored two or more coexisting clones. sCNA analysis revealed that human leukocyte antigen C (HLA‐C) and HLA‐A were significantly amplified. Subsequent IHC investigations indicated that HLA‐C shows promise in averting the misclassification of challenging‐to‐interpret IFVPTC and invasive encapsulated follicular variant of PTC (I‐EFVPTC) as noninvasive follicular thyroid neoplasm with papillary‐like nuclear features (NIFTP). Although there were several similarities between classic PTC and IFVPTC, they differed significantly in their sCNA patterns. Conclusions: This study provides valuable insights into IFVPTC's genetic alterations and highlights the potential of HLA‐C IHC to distinguish challenging‐to‐interpret IFVPTC and I‐EFVPTC from NIFTP, which will enhance the understanding of its molecular features for improved diagnosis and management. This study characterizes the genetic alterations of infiltrative follicular variant of papillary thyroid carcinoma (IFVPTC) via whole‐exome sequencing, which identified BRAF V600E as the only common trunk mutation and emphasized the significance of human leukocyte antigen C immunohistochemistry staining in distinguishing challenging‐to‐interpret IFVPTC and invasive encapsulated follicular variant of papillary thyroid carcinoma from noninvasive follicular thyroid neoplasm with papillary‐like nuclear features. These findings enhance the understanding of IFVPTC's molecular characteristics and contribute to advancements in its diagnosis and management. [ABSTRACT FROM AUTHOR]

Background: Ovarian failure (OF) is a multifactorial, complex disease presented by up to 1% of women under 40 years of age. Despite 90% of patients being diagnosed with idiopathic OF, the underlying molecular mechanisms remain unknown, making it difficult to personalize treatments for these patients in the clinical setting. Studying the presence and/or accumulation of SNVs at the gene/pathway levels will help describe novel genes and characterize disrupted biological pathways linked with ovarian failure. Methods: Ad-hoc case-control SNV screening conducted from 2020 to 2023 of 150 VCF files WES data included Spanish IVF patients with (n = 118) and without (n = 32) OF (< 40 years of age; mean BMI 22.78) along with GnomAD (n = 38,947) and IGSR (n = 1,271; 258 European female VCF) data for pseudo-control female populations. SNVs were prioritized according to their predicted deleteriousness, frequency in genomic databases, and proportional differences across populations. A burden test was performed to reveal genes with a higher presence of SNVs in the OF cohort in comparison to control and pseudo-control groups. Systematic in-silico analyses were performed to assess the potential disruptions caused by the mutated genes in relevant biological pathways. Finally, genes with orthologues in Drosophila melanogaster were considered to experimentally validate the potential impediments to ovarian function and reproductive potential. Results: Eighteen genes had a higher presence of SNVs in the OF population (FDR < 0.05). AK2, CDC27, CFTR, CTBP2, KMT2C, and MTCH2 were associated with OF for the first time and their silenced/knockout forms reduced fertility in Drosophila. We also predicted the disruption of 29 sub-pathways across four signalling pathways (FDR < 0.05). These sub-pathways included the metaphase to anaphase transition during oocyte meiosis, inflammatory processes related to necroptosis, DNA repair mismatch systems and the MAPK signalling cascade. Conclusions: This study sheds light on the underlying molecular mechanisms of OF, providing novel associations for six genes and OF-related infertility, setting a foundation for further biomarker development, and improving precision medicine in infertility. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial complex V deficiency refers to a shortage (deficiency) of a protein complex called complex V or a loss of its function. The TMEM70 gene provides the blueprint for mitochondrial ATP synthase, a protein essential for cellular energy production through oxidative phosphorylation. The diagnostic method based on whole-exome sequencing (WES) provides more appropriate genetic counseling by saving time and money. Methods: This study investigated three patients presenting with similar clinical symptoms using WES. After DNA extraction, WES was performed, and a novel mutation (c.311T > G: p.V104G) in the TMEM70 gene was identified. Sanger sequencing was used to confirm the variant in the parents. Results: The novel TMEM70 mutation (c.311T > G: p.V104G) was identified as a potential cause of the disease in these patients. Conclusions: This study highlights the utility of WES in the rapid and cost-effective identification of pathogenic variants associated with mitochondrial disorders. The identification of a novel TMEM70 mutation underscores the importance of this gene in mitochondrial function and further emphasizes the need for comprehensive genetic screening in individuals presenting with clinical symptoms consistent with mitochondrial ATP synthase deficiency. Further research is needed to elucidate the specific functional consequences of this mutation and to investigate the prevalence of TMEM70 mutations in various populations. [ABSTRACT FROM AUTHOR]

The swift updates of public databases and advancements in next-generation sequencing (NGS) technologies have enhanced the genetic identification capacities of epilepsy clinics. This study aimed to evaluate the diagnostic efficacy of NGS in pediatric epilepsy patients as a whole and to present the data obtained in the whole exome sequence analysis. We enrolled 40 children with suspected childhood epilepsy in this study. All patients underwent evaluation by a clinical geneticist or pediatric neurologist and the molecular genetic analysis of those children was performed by whole-exome sequencing (WES). Out of the 40 patients, 12 (30%) received a genetic diagnosis, involving 14 mutations across 13 genes. The cumulative positive diagnostic yield was 30%. Twelve of these patients were identified to have 5 variants previously documented as pathogenic, 9 variants classified as likely pathogenic, and 5 novel variants that have not been reported before. The outcomes indicate that whole-exome sequencing offers great benefits in clinical patient diagnosis, particularly in terms of detecting diagnostic variants. This study underscored the significance of whole exome sequencing (WES) studies, where only a broad gene set is examined in epilepsy patients. This approach has the potential to establish gene-specific phenotypic profiles, particularly by uncovering novel candidate genes in epilepsy patients with well-defined phenotypes. Additionally, conducting validation studies on variants of uncertain clinical significance could enhance the outcome yield. [ABSTRACT FROM AUTHOR]

Background: Runs of homozygosity (ROHs), continuous homozygous regions across the genome, are often linked to consanguinity, with their size and frequency reflecting shared parental ancestry. Homozygosity mapping (HM) leverages ROHs to identify genes associated with autosomal recessive diseases. Whole-exome sequencing (WES) improves HM by detecting ROHs and disease-causing variants. Methods: To streamline personalized multigene panel creation, using WES and ROHs, we developed a methodology integrating ROHMMCLI and HomozygosityMapper algorithms, and, optionally, Human Phenotype Ontology (HPO) terms, implemented in a Django Web application. Resorting to a dataset of 12,167 WES, we performed the first ROH profiling of the Portuguese population. Clustering models were applied to predict consanguinity from ROH features. Results: These resources were applied for the genetic characterization of two siblings with epilepsy, myoclonus and dystonia, pinpointing the CSTB gene as disease-causing. Using the 2021 Census population distribution, we created a representative sample (3941 WES) and measured genome-wide autozygosity (FROH). Portalegre, Viseu, Bragança, Madeira, and Vila Real districts presented the highest FROH scores. Multidimensional scaling showed that ROH count and sum were key predictors of consanguinity, achieving a test F1-score of 0.96 with additional features. Conclusions: This study contributes with new bioinformatics tools for ROH analysis in a clinical setting, providing unprecedented population-level ROH data for Portugal. [ABSTRACT FROM AUTHOR]

Key Clinical Message: This study reports a Chinese male patient with a novel MeCP2 p.Lys254*variant. Upon birth, the patient presented with typical symptoms, such as abnormal electroencephalogram, immature sleep rhythm, hypotonia, feeding difficulties, pulmonary fluid accumulation, horizontal fissures in the lungs, hypoventilation, and heart defects. MeCP2 is a gene located on the X chromosome and the main pathogenic gene responsible for Rett syndrome, which mainly occurs in females. Herein, we identified a male patient with a novel MeCP2 p.Lys254* variant through whole‐exome sequencing, although both parents are wild type. Upon birth, the patient presented with typical symptoms, such as abnormal electroencephalogram, immature sleep rhythm, hypotonia, feeding difficulties, pulmonary fluid accumulation, horizontal fissures in the lungs, hypoventilation, and other symptoms. Period of breathing support, but also found that the boy had a heart defect and horizontal fissure in the lungs. Our discovery of a new spontaneous MeCP2 nonsense mutation enriches the understanding of Rett syndrome and provides a reference for its early diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Auditory neuropathy spectrum disorder (ANSD) is often missed by standard hearing tests, accounting for up to 10% of hearing impairments (HI) and commonly linked to variants in 23 genes. We assessed 122 children with HI, including 102 with sensorineural hearing loss (SNHL) and 20 with ANSD. SNHL patients were genotyped for common GJB2 variants using qPCR, while ANSD patients underwent whole exome sequencing, with variants analyzed across 249 genes. Homozygous GJB2 variants were found in 54.9% of SNHL patients. In 60% of ANSD patients, variants were detected in OTOF (25%), CDH23, TMC1, COL11A1, PRPS1, TWNK, and HOMER2 genes, including eight novel variants. Transient evoked otoacoustic emissions testing revealed differences at 4000 Hz (p = 0.0084) between the ANSD and SNHL groups. The auditory steady-state response (ASSR) test showed significant differences at 500 Hz (p = 2.69 × 10−4) and 1000 Hz (p = 0.0255) compared to pure-tone audiometry (PTA) in ANSD patients. Our questionnaire shows that the parents of children with SNHL often report an improved quality of life with hearing aids or cochlear implants, while parents of children with ANSD frequently experience uncertainty about outcomes (p = 0.0026), leading to lower satisfaction. [ABSTRACT FROM AUTHOR]

Objective Several monogenic autoinflammatory disorders and primary immunodeficiencies can present early in life with features that may be mistaken for Behçet's disease (BD). We aimed to develop a genetic analysis workflow to identify rare monogenic BD-like diseases and establish the contribution of HLA haplotype in a cohort of patients from the UK. Methods Patients with clinically suspected BD were recruited from four BD specialist care centres in the UK. All participants underwent whole-exome sequencing (WES), and genetic analysis thereafter by (i) examining genes known to cause monogenic immunodeficiency, autoinflammation or vasculitis by virtual panel application; (ii) scrutiny of variants prioritized by Exomiser using Human Phenotype Ontology (HPO); (iii) identification of copy number variants using ExomeDepth; and (iv) HLA-typing using OptiType. Results Thirty-one patients were recruited: median age 15 (4–52), and median disease onset age 5 (0–20). Nine/31 (29%) patients had monogenic disease mimicking BD: five cases of Haploinsufficiency of A20 with novel TNFAIP3 variants (p.T76I, p. M112Tfs*8, p. S548Dfs*128, p. C657Vfs*14, p. E661Nfs*36); one case of ISG15 deficiency with a novel nonsense variant (ISG15 : p.Q16X) and 1p36.33 microdeletion; one case of common variable immune deficiency (TNFRSF13B : p.A181E); and two cases of TNF receptor-associated periodic syndrome (TNFRSF1A : p.R92Q). Of the remaining 22 patients, eight (36%) were HLA-B*51 positive. Conclusion We describe a novel genetic workflow for BD, which can efficiently detect known and potentially novel monogenic forms of BD, whilst additionally providing HLA-typing. Our results highlight the importance of genetic testing before BD diagnosis, as this has an impact on choice of therapy, prognosis and genetic counselling. [ABSTRACT FROM AUTHOR]

Background and purpose: The penetrance of the RNF213 p.R4810K, a founder mutation of moyamoya disease (MMD), is estimated to be only 1/150–1/300. However, the factors affecting its penetrance remain unclear. Therefore, our study aims to identify modifier genes associated with the incomplete penetrance of this founder mutation. Methods: Whole‐exome sequencing (WES) was performed on 36 participants, including 22 MMD patients and 14 non‐MMD controls with RNF213 p.R4810K mutation. Fisher's exact test was used to assess the presence of genetic variants that differed significantly between MMD patients and non‐MMD controls. In order to exclude false‐positive results, another 55 carriers were included to perform Fisher's exact test for the selected sites in the WES discovery stage. Subsequently, human brain microvascular endothelial cells were transfected with wild‐type and mutant HAPLN3 for tube formation assays and western blotting to explore the impact of candidate genes on angiogenesis. Results: Analysis of variants from WES data revealed a total of 12 non‐synonymous variants. Through bioinformatics analysis, the focus was on the HAPLN3 p.T34A variant with a significant p value of 0.00731 in Fisher's exact test. Validation through Sanger sequencing confirmed the presence of this variant in the WES data. In vitro experiments revealed that silencing HAPLN3 and transfecting HAPLN3 p.T34A significantly enhanced tube formation and increased the relative protein expression of vascular endothelial growth factor in endothelial cells. Conclusions: These results suggest that HAPLN3 may function as a modifier gene of RNF213 p.R4810K, promoting the development of MMD and contributing to the incomplete penetrance of MMD founder mutations. [ABSTRACT FROM AUTHOR]

Inborn errors of immunity (IEI) are defined as genetic disorders affecting the immune system and resulting in diverse clinical signs and symptoms. Despite the lack of diagnosis and unavailability of IEI estimation in the Pakistani population, consanguinity is exacerbating its prevalence. The current study focuses on severe combined immunodeficiency (SCID) and leukocyte adhesion deficiency type 1 (LAD1). SCID is associated with the life-threatening symptoms developing at post-birth. LAD1 is clinically characterized by recurrent bacterial infections related to the skin, mouth, and respiratory tract owing to impaired leukocytes. Herein, in six consanguineous families, flow cytometry was used to evaluate the patient's immune status. Whole-exome sequencing (WES) was then conducted to search for the causative variations in immunodeficiency genes. Sanger sequencing was used to assess the segregation of the variants with the disorder within the families. Sequence analysis revealed five homozygous variants in four different causative genes. This included four novel nonsense variants in CD70 p.(Thr126Profs*33), CD3e p.(Trp151*), IL7R p.(Val138Ilefs*10), and ITGB2 p.(Ser627Valfs*61), and one previously reported in ITGB2 p.(Cys62*). In one of the families, two variants in two different genes, including DNAH6 p.(Tyr2653His) and NIPAL4 p.(Gly121Ser), were detected in an unclassified patient. All the identified variants were found in a homozygous state in the patient but in a heterozygous state in the available parents. The study will facilitate the diagnosis and management of IEI patients. [ABSTRACT FROM AUTHOR]

We report the case of a Spanish pediatric patient with developmental delay, hypotonia, feeding difficulties, visual problems, and hyperkinetic movements. Whole-exome sequencing uncovered a new heterozygous de novo Synaptotagmin 1 (SYT1) missense variant, NM_005639.3:c.930T>A (p.Asp310Glu), in a female proband. This gene encodes the synaptotagmin-1 (SYT1) protein, which is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. Pathogenic SYT1 variants have been associated with Baker–Gordon syndrome (BAGOS), an autosomal dominant neurodevelopmental disorder. Although up to 30 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with mitochondrial respiratory chain deficiencies and rod–cone dysfunction. In conclusion, our data expand both the genetic and phenotypic spectrum associated with SYT1 variants. [ABSTRACT FROM AUTHOR]

Amyloidosis are a group of diseases in which soluble proteins aggregate and deposit in fibrillar conformation extracellularly in tissues. The effectiveness of therapeutic strategies depends on the specific protein involved, being crucial to accurately determine its nature. Moreover, following the diagnosis, the search for the mutation within relatives allows the clinical advice. Here we report the precise diagnosis and explored the possible reasons of the structural pathogenicity for a renal amyloidosis related to a fibrinogen Aα‐chain variant. Whole‐exome sequencing and GATK calling pipeline were leveraged to characterize the protein variant present in a patient with kidney failure. Bioinformatics strategies were applied to suggest potential explanations of the variants aggregation. Our pipeline allowed the identification of a single‐point variant of fibrinogen Aα‐chain, which opened the possibility of curative transplantation. In silico structural analysis suggested that the pathogenicity of the variant may be attributed to a heightened susceptibility to yield a peptide prone to deposit as an oligomer with a β‐sheet structure. Exploiting the comprehensive coverage of whole‐genome sequencing, we managed to fill a vacant stage in the diagnosis of hereditary amyloidosis and to stimulate the advancement in biomedicine. [ABSTRACT FROM AUTHOR]

Background: Rare movement disorders often have a genetic etiology. New technological advances have increased the odds of achieving genetic diagnoses: next-generation sequencing (NGS) (whole-exome sequencing—WES; whole-genome sequencing—WGS) and long-read sequencing (LRS). In 2017, we launched a WES program for patients with rare movement disorders of suspected genetic etiology. We aim to describe the accumulated experience of a modern movement disorder genetic clinic, highlighting how different available genetic tests might be prioritized according to the clinical phenotype and pattern of inheritance. Methods: Participants were studied through WES analysis. Descriptive statistics, including the mean, standard deviation, counts, and percentages, were used to summarize demographic and clinical characteristics in all subjects and with each type of result [pathogenic or likely pathogenic, variants of uncertain significance (VUS), negative]. Results: We studied 88 patients (93.2% Caucasian, 5.72% African American, and 1.08% Hispanic or Latino). After excluding six family members from four index participants, the diagnostic yield of WES reached 27% (22/82 probands). The age at onset was significantly lower in patients with pathogenic/likely pathogenic variants. The most common clinical phenotypes were ataxia and parkinsonism. Dystonia, ataxia, leukoencephalopathy, and parkinsonism were associated with most genetic diagnoses. Conclusions: We propose a comprehensive protocol with decision tree testing for WGS and LRS, a return of results, and a re-analysis of inconclusive genetic data to increase the diagnostic yield of patients with rare neurogenetic disorders. [ABSTRACT FROM AUTHOR]

Aims: Recent‐onset dilated cardiomyopathy (RODCM) is characterized by heterogeneous aetiology and diverse clinical outcomes, with scarce data on genotype–phenotype correlates. Our aim was to correlate individual RODCM genotypes with left ventricular reverse remodelling (LVRR) and clinical outcomes. Methods and results: In this prospective study, a total of 386 Czech RODCM patients with symptom duration ≤6 months underwent genetic counselling and whole‐exome sequencing (WES). The presence of pathogenic (class 5) or likely pathogenic (class 4) variants in a set of 72 cardiomyopathy‐related genes was correlated with the occurrence of all‐cause death, heart transplantation, or implantation of a ventricular assist device (primary outcome) and/or ventricular arrhythmia event (secondary outcome). LVRR was defined as an improvement of left ventricular ejection fraction to >50% or ≥10% absolute increase, with a left ventricular end‐diastolic diameter ≤33 mm/m2 or ≥10% relative decrease. Median follow‐up was 41 months. RODCM was familial in 98 (25%) individuals. Class 4–5 variants of interest (VOIs) were identified in 125 (32%) cases, with 69 (18%) having a single titin‐truncating variant (TTNtv) and 56 (14%) having non‐titin (non‐TTN) VOIs. The presence of class 4–5 non‐TTN VOIs, but not of TTNtv, heralded a lower probability of 12‐month LVRR and proved to be an independent baseline predictor both of the primary and the secondary outcome. The negative result of genetic testing was a strong protective baseline variable against occurrence of life‐threatening ventricular arrhythmias. Detection of class 4–5 VOIs in genes coding nuclear envelope proteins was another independent predictor of both study outcomes at baseline and also of life‐threatening ventricular arrhythmias after 12 months. Class 4–5 VOIs of genes coding cytoskeleton were associated with an increased risk of life‐threatening ventricular arrhythmias after baseline assessment. A positive family history of dilated cardiomyopathy alone only related to a lower probability of LVRR at 12 months and at the final follow‐up. Conclusions: RODCM patients harbouring class 4–5 non‐TTN VOIs are at higher risk of progressive heart failure and life‐threatening ventricular arrhythmias. Genotyping may improve their early risk stratification at baseline assessment. [ABSTRACT FROM AUTHOR]

Background: Retinitis pigmentosa (RP) is a genetically heterogeneous disease. RP 79 has been associated with heterozygous variants of hexokinase 1 (HK1). Only two missense HK1 variants have been reported in 11 families. Objective: To discover the molecular pathogenic mechanism of RP and validate the biological harm of HK1 through in vitro experiments. Methods: We conducted a genetic analysis of a 3-year-old female patient with RP and her family. We also evaluated the ocular phenotypes caused by HK1 (the identified variant). Peripheral blood samples were collected from the patient, her parents, and her brother, and trio whole-exome sequencing was performed. A protein structure analysis was performed to assess the functional impact of the variant, and a mutant plasmid was constructed for the quantitative polymerase chain reaction (qPCR) and western blot (WB) analysis of the effects of the variant on transcription and protein translation. Results: The patient harbored the NM_000188.3: c.613del (p.Ala205Leufs*3) variant, which is a heterozygous variant of HK1. Sanger sequencing confirmed the presence of this variant in the patient; however, the patient's parents and brother had the wild-type variant. The protein structure analysis indicated that the variant resulted in a truncated protein caused by premature termination of amino acid coding. The qPCR results indicated that the variant may not have affected the transcription process. However, the WB analysis demonstrated that the mutant HK-1 protein was not expressed and that the wild-type group exhibited normal expression. Conclusions: Our patient had a loss-of-function (LoF) variant of HK1, which may be the genetic cause of typical features of RP that are observed at an early age. These findings expand the spectrum of HK1 variants and phenotypes and suggest that LoF variants of HK1 may represent a specific pathogenic mechanism of RP. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB) is currently the standard of care for metastatic renal cell carcinoma (RCC), but treatment responses remain unpredictable. Aristolochic acid (AA), a prevalent supplement additive in Taiwan, has been associated with RCC and induces signature mutations, although its effect on the tumor‐immune microenvironment (TIME) is unclear. We aimed to investigate the immune profile of AA‐positive RCCs and explore its potential role as a susceptible candidate for ICB. Tissue samples from 22 patients with clear cell RCC (ccRCC) were collected for whole‐exome sequencing to determine the genetic features and AA mutational signature (the discovery cohort). The corresponding RNA was sent for NanoString PanCancer IO 360 gene expression analysis to explore the immunological features. The formalin‐fixed, parafilm‐embedded slides of ccRCCs were sent for multiplex immunohistochemistry/immunofluorescence stain using Vectra system to evaluate the TIME. Tissues from two patients with metastatic RCC demonstrating complete response to ICB were sent for studies to validate the findings (the index patients). The results showed that AA mutational signatures with high tumor mutational burden (TMB) were present in 31.81% of the tumors in the discovery cohort. Three distinct clusters were observed through NanoString analysis. Clusters 1 and 3 were composed mainly of AA‐positive RCCs. Cluster 3 RCCs exhibited higher tumor inflammation signature scores and higher immune cell type scores. Vectra analysis revealed a higher percentage of CD15+ and BATF3+ cells in cluster 1, whereas the percentage of CD8+ cells was potentially higher in cluster 3. Strong AA mutational signatures were found in the tumors of two index patients, and both were grouped to cluster 3. In conclusion, AA may induce higher TMB and alter the immune microenvironment in RCCs, which makes the tumors more susceptible to ICB. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]