Exonic Variants Research Articles

A likely pathogenic homozygous frameshift variant in BLOC1S6 associated with a rare form of congenital Hermansky-Pudlak syndrome 9

Hermansky-Pudlak syndrome (HPS) is a collection of autosomal recessive multisystemic disorders with at least 11 different types, categorized on the basis of involved genes. The disease is mostly characterized by tyrosinase-positive oculocutaneous albinism (OCA), platelet storage deficiency, absence of platelet dense bodies, and immune deficiency. Here we described a 2-month-old female infant with generalized hypotonia, recurrent infections, bilateral optic atrophy, nystagmus, cerebral atrophy, and elevated liver enzymes. Unlike her parents, she had chestnut colored hair, fair skin, and brown eyes. Parents had a consanguineous marriage, and their first child had died with similar symptoms at the age of 5 months. Whole exome sequencing (WES) was performed on DNA extracted from the patient's peripheral blood. Following the bioinformatics analysis, a likely pathogenic novel variant in the fifth exon of the BLOC1S6 gene (NM_001311255: c.506dupT: p. L169Ffs*33) was introduced by the Sadra Medical Genetic Laboratory. This variant was confirmed in the patient and segregated in both parents by Sanger sequencing.This report presented the first congenital case of HPS-9 worldwide that might have led to early neonatal death. Our current patient shows new considerable features related to the BLOC1S6 gene variant and HPS-9, which is a valuable source for future research, prediction, clinical management, genetic counseling, and prenatal diagnosing.

Patient-derived induced pluripotent stem cells to study non-canonical splicing variants associated with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy and a leading cause of sudden death. Genetic testing and familial cascade screening play a pivotal role in the clinical management of HCM patients. However, conventional genetic tests primarily focus on the detection of exonic and canonical splice site variation. Oversighting intronic non-canonical splicing variants potentially contributes to a proportion of HCM patients remaining genetically undiagnosed. Here, using a non-integrative reprogramming strategy, we generated induced pluripotent stem cell (iPSC) lines from four individuals carrying one of two variants within intronic regions of MYBPC3: c.1224-52G > A and c.1898-23A > G. Upon differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), mis-spliced mRNAs were identified in cells harbouring these variants. Both abnormal mRNAs contained a premature termination codon (PTC), fitting the criteria for activation of nonsense mediated decay (NMD). However, the c.1898-23A > G transcripts escaped this mRNA quality control mechanism, while the c.1224-52G > A transcripts were degraded. The newly generated iPSC lines represent valuable tools for studying the functional consequences of intronic variation and for translational research aimed at reversing splicing abnormalities to prevent disease progression.

Novel Biallelic Synonymous Exonic Variant in VPS13A Affecting mRNA Splicing

Novel Biallelic Synonymous Exonic Variant in <i>VPS13A</i> Affecting mRNA Splicing

Beyond HRD status: Unraveling Genetic Variants Impacting PARP Inhibitor Sensitivity in Advanced Ovarian Cancer.

The management of advanced ovarian cancer (AOC) has undergone significant advancements with the emergence of molecular diagnostics, particularly in predicting responses to poly(ADP-ribose) polymerase inhibitors (PARPi) based on homologous recombination deficiency (HRD) status. However, understanding sensitivity and resistance beyond HRD status remains elusive. This study aims to explore molecular factors that may elucidate why HRD status does not consistently predict PARPi sensitivity. Therefore, we conducted a post hoc translational analysis of formalin-fixed paraffin-embedded tumor samples from the ENGOT-ov24/NSGO-AVANOVA part 1 and 2 trial (AVANOVA1&2; NCT02354131), focusing on alterations pertaining radiological response and progression-free survival (PFS). DNA sequencing was performed using the TruSight Oncology 500 HT gene panel, with variants classified according to recent guidelines. HRD status had been assessed by Myriad MyChoice® CDx. We identified, among 92 patients in the AVANOVA1&2 trial, 151 pathogenic or likely pathogenic variants across 81 samples. PARPi sensitizing variants were found in two out of ten HRDneg samples from patients with clinical benefit (PFS ≥ 12 months), while three out of ten HRDpos samples from patients having no benefit (PFS ≤ 6 months) harbored variants associated with PARPi resistance. Additionally, analysis of BRCA1 variants revealed that truncating variants in exon 11 correlated with clinical benefit when niraparib was combined with bevacizumab. Conclusively, our findings highlight the complexity of PARPi response in AOC and underscore the importance of exploring somatic variants beyond HRD status. Further investigation into exon 11 variants of BRCA1 and the potential of combination treatment is warranted.

Mutational Landscape of KIT Proto-Oncogene Coding Sequence in 62 Canine Cutaneous and Subcutaneous Mast Cell Tumors

Canine mast cell tumors (MCTs) are common skin neoplasms with varying biological behaviors. The KIT proto-oncogene plays a key role in the development of these tumors, and internal tandem duplications on exon 11 are usually associated with more aggressive behavior, increased local recurrence, and decreased survival time. However, apart from exons 8–11 and 17, there is limited understanding of the overall KIT mutational landscape in canine MCTs. This work aims to analyze the entire KIT coding sequence (21 exons) in a cohort of 62 MCTs, which included 38 cutaneous and 24 subcutaneous tumors, and potentially identify new variants. In addition to confirming previously reported activating KIT mutations in exons 8, 9, and 11, we identified new variants in exons 2, 3, 5, 16, and the 3′ untranslated region (UTR). Notably, these last variants include an amino acid change (Asp/His) in exon 16. Additionally, we confirmed a differential prevalence of KIT variants in cutaneous and subcutaneous MCTs. These findings enhance our understanding of the KIT proto-oncogene coding sequence and provide valuable information for future confirmatory studies.

Report of a novel missense TDP1 variant in a Pakistani family affected with an extremely rare disorder congenital spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1)

BackgroundSpinocerebellar ataxia with axonal neuropathy type 1 (OMIM: 607250) is an extremely rare autosomal recessive disorder caused by a mutation in the tyrosyl-DNA phosphodiesterase 1 (TDP1) gene. Only a single missense variant (p.His493Arg) in this gene has been reported. This variant was found in three Arab families with a possible common founder effect.Methods and ResultsWe report a female patient born to a consanguineous Pakistani family segregating autosomal recessive spinocerebellar ataxia with axonal neuropathy type 1. The patient presents additional clinical features distinct from previously reported Arab families including congenital onset of the disease. We performed whole exome sequencing with the patient’s DNA and identified a novel missense variant (NC_000014.9:g.89991982C > T; p.His478Tyr) in exon 13 of the TDP1 gene. Sanger sequencing was performed to verify the autosomal recessive segregation of the p.His478Tyr variant in the family.ConclusionThe current study expands both the clinical and mutation spectrum of the TDP1 associated spinocerebellar ataxia with axonal neuropathy type 1 and increases the body of evidence that supports the pathogenic role of TDP1 in cerebellar ataxias with peripheral neuropathy.

Recurrent BMP4 variants in exon 4 cause non-HFE-associated hemochromatosis via the BMP/SMAD signaling pathway

BackgroundHereditary hemochromatosis (HH) is an iron overload disorder and can be caused by variants in non-HFE genes in Chinese patients. However, there is still a considerable proportion of patients suffering from unexplained iron overload. In our previous study, we had identified the p.R269Q variant in exon 4 of the Bone morphogenetic protein 4 (BMP4) gene in Chinese patients with unexplained primary iron overload by Whole Exome sequencing, and then the BMP4 p.H251Y variant was identified by Sanger sequencing in a Chinese patient with secondary iron overload. Our study aimed to explore the pathogenicity and underlying mechanism of BMP4 p.H251Y and BMP4 p.R269Q variants in patients with iron overload.MethodsSanger sequencing was conducted to identify the novel variants in the BMP4 gene of patients with unexplained iron overload. MRI and liver biopsy were used to display iron overload in the liver of the patient harboring the BMP4 p.H251Y variant. The BMP4 and hepcidin levels in BMP4 knockdown and BMP4 variant cells were examined by enzyme-linked immunosorbent assay. The effects of BMP4 p.H251Y and BMP4 p.R269Q variants on the hepcidin-regulation pathway were studied.ResultsOne of 54 HH patients (1.85%) harbored the BMP4 p.R269Q variant. One of 148 patients (0.68%) with secondary hemochromatosis harbored the BMP4 p.H251Y variant, and these two variants were not found in 100 Chinese general population. For the patient harboring the BMP4 p.H251Y variant, abdominal MRI and Perl's staining of liver tissue displayed iron overload in the liver. Cells transfected with the BMP4 p.H251Y and p.R269Q variants showed down-regulation of hepcidin level and BMP/SMAD pathway compared with cells transfected with the wild-type BMP4 vector.ConclusionThe BMP4 p.H251Y and p.R269Q variants can downregulate hepcidin levels by inhibiting the BMP/SMAD axis, suggesting they may play pathogenic roles in iron overload.

A patient with P369S/R408Q variants in the MEFV gene presented with clinical features of Kikuchi disease and Mollaret meningitis, successfully treated with colchicine

BackgroundThis case report presents the case of a patient with P369S and R408Q variants in the MEFV gene who exhibited clinical features of Kikuchi disease and Mollaret meningitis. Furthermore, it discusses colchicine as a new potential treatment option for Kikuchi disease-associated meningitis.Case presentationA 41-year-old Japanese woman presented with fever and headache. She had nuchal rigidity and bilateral cervical lymphadenopathies. Her past medical history included multiple episodes of aseptic meningitis and cervical lymphadenopathy for more than twenty years. Lumbar puncture showed increased lymphocytes and IL-6 level and pathognomonic Mollaret cells. Excisional lymph node biopsy revealed histiocytic necrotizing lymphadenitis, confirming the diagnosis of Kikuchi disease. Subsequently, her recurrent Kikuchi disease and meningitis were successfully treated with colchicine. Furthermore, genetic analysis of the MEFV gene revealed heterozygous P369S/R408Q variants in exon 3.ConclusionMollaret meningitis can be associated with Kikuchi disease, and recurrence of both conditions may be suppressed by colchicine when these two coexist.

Bidirectional Risk Modulator and Modifier Variant of Dilated and Hypertrophic Cardiomyopathy in BAG3

The genetic factors that modulate the reduced penetrance and variable expressivity of heritable dilated cardiomyopathy (DCM) are largely unknown. BAG3 genetic variants have been implicated in both DCM and hypertrophic cardiomyopathy (HCM), nominating BAG3 as a gene that harbors potential modifier variants in DCM. To interrogate the clinical traits and diseases associated with BAG3 coding variation. This was a cross-sectional study in the Penn Medicine BioBank (PMBB) enrolling patients of the University of Pennsylvania Health System's clinical practice sites from 2014 to 2023. Whole-exome sequencing (WES) was linked to electronic health record (EHR) data to associate BAG3 coding variants with EHR phenotypes. This was a health care population-based study including individuals of European and African genetic ancestry in the PMBB with WES linked to EHR phenotypes, with replication studies in BioVU, UK Biobank, MyCode, and DCM Precision Medicine Study. Carrier status for BAG3 coding variants. Association of BAG3 coding variation with clinical diagnoses, echocardiographic traits, and longitudinal outcomes. In PMBB (n = 43 731; median [IQR] age, 65 [50-76] years; 21 907 female [50.1%]), among 30 324 European and 11 198 African individuals, the common C151R variant was associated with decreased risk for DCM (odds ratio [OR], 0.85; 95% CI, 0.78-0.92) and simultaneous increased risk for HCM (OR, 1.59; 95% CI, 1.25-2.02), which was confirmed in the replication cohorts. C151R carriers exhibited improved longitudinal outcomes compared with noncarriers as assessed by age at death (hazard ratio [HR], 0.85; 95% CI, 0.74-0.96; median [IQR] age, 71.8 [63.1-80.7] in carriers and 70.3 [61.6-79.2] in noncarriers) and heart transplant (HR, 0.81; 95% CI, 0.66-0.99; median [IQR] age, 56.7 [46.1-63.1] in carriers and 55.6 [45.2-62.9] in noncarriers). C151R was associated with reduced risk of DCM (OR, 0.42; 95% CI, 0.24-0.74) and heart failure (OR, 0.27; 95% CI, 0.14-0.50) among individuals harboring truncating TTN variants in exons with high cardiac expression (n = 358). BAG3 C151R was identified as a bidirectional modulator of risk along the DCM-HCM spectrum, as well as an important genetic modifier variant in TTN-mediated DCM. This work expands on the understanding of the etiology and penetrance of DCM, suggesting that BAG3 C151R is an important genetic modifier variant contributing to the variable expressivity of DCM, warranting further exploration of its mechanisms and of genetic modifiers in DCM more broadly.

A NOVEL NOTCH3 VARIANT LEADING TO LATERAL MENINGOCELE SYNDROME: PRENATAL DIAGNOSIS AND POSSIBLE EXPANSION OF THE PHENOTYPE

Introduction: NOTCH3, one of the four mammalian Notch receptors, acts as a transcriptional activator in a variety of tissues. Variants in NOTCH3 lead to distinct phenotypes, depending on variant type and location. Truncating variants in the last exon generate a protein lacking the PEST domain, responsible for degradation, leading to a gain-of-function effect and causing Lateral Meningoceles syndrome (LMS), characterized by dysmorphisms and variable cardiac, skeletal, and connective tissue abnormalities; motor delay may occur, but the cognitive function is usually normal. Case presentation: We report the first case of prenatal molecular diagnosis of LMS, which was made using prenatal exome sequencing after an ultrasound with findings of fetal cystic hygroma, mild bilateral ventriculomegaly, and facial dysmorphisms. After birth, magnetic resonance imaging (MRI) confirmed the presence of lateral meningoceles. A complete clinical evaluation was performed and unexpected biliary anomalies were found. Conclusion: The occurrence of biliary anomalies has not been previously reported in LMS, but may have biological plausibility. Expression of NOTCH3 has been demonstrated in biliary development and is thought to play a role in the differentiation of hepatoblasts into biliary epithelial cells, and also in liver regeneration and repair. We hypothesize that the findings reported here might expand the phenotype of LMS.

Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation.

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN(acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE.In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygousframeshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Updating probability of pathogenicity for RYR1 and CACNA1S exon variants in individuals without malignant hyperthermia after exposure to triggering anesthetics

Objectives We aimed to classify genetic variants in RYR1 and CACNA1S associated with malignant hyperthermia using biobank genotyping data in patients exposed to triggering anesthetics without malignant hyperthermia phenotype. Methods We identified individuals who underwent surgery and were exposed to triggering anesthetics without malignant hyperthermia phenotype and who had RYR1 or CACNA1S genotyping data available in our biobank. We classified all variants in the cohort using a Bayesian framework of the American College of Medical Genetics and Genomics and the Association of Molecular Pathologists guidelines for variant classification and updated the posterior probabilities from this model with the new information from our biobank cohort. Results We identified 253 patients with 95 RYR1 variants and 12 CACNA1S variants. After applying a Bayesian framework, we classified 17 variants as benign (B), 31 as likely benign (LB), 57 as uncertain (VUS), and 2 as likely pathogenic (LP). When we incorporated evidence about unique exposures to malignant hyperthermia triggering anesthetic agents, 48 of 107 (45%) variants were downgraded (9 to B, 37 to LB, and 2 to VUS). Notably, 41 (72%) of 57 VUSs were downgraded to B or LB. When repeat anesthetics in the same individual were counted as one exposure, 42 of 107 (39%) of variants were downgraded (5 to B, 35 to LB, and 2 to VUS). Specifically, 37 (65%) of 57 VUSs were downgraded to LB. Conclusion Deidentified biorepositories linked with anesthetic data offer a new method of integrating clinical evidence into the assessment of variant probability of pathogenicity.

Exon nomenclature and classification of transcripts database (ENACTdb): a resource for analyzing alternative splicing mediated proteome diversity.

Gene transcripts are distinguished by the composition of their exons, and this different exon composition may contribute to advancing proteome complexity. Despite the availability of alternative splicing information documented in various databases, a ready association of exonic variations to the protein sequence remains a mammoth task. To associate exonic variation(s) with the protein systematically, we designed the Exon Nomenclature and Classification of Transcripts (ENACT) framework for uniquely annotating exons that tracks their loci in gene architecture context with encapsulating variations in splice site(s) and amino acid coding status. After ENACT annotation, predicted protein features (secondary structure/disorder/Pfam domains) are mapped to exon attributes. Thus, ENACTdb provides trackable exonic variation(s) association to isoform(s) and protein features, enabling the assessment of functional variation due to changes in exon composition. Such analyses can be readily performed through multiple views supported by the server. The exon-centric visualizations of ENACT annotated isoforms could provide insights on the functional repertoire of genes due to alternative splicing and its related processes and can serve as an important resource for the research community. The database is publicly available at https://www.iscbglab.in/enactdb/. It contains protein-coding genes and isoforms for Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens.

Canine Multiple System Degeneration Associated with Sequence Variants in SERAC1.

Canine multiple system degeneration (CMSD) is an early onset, progressive movement disorder affecting Kerry Blue Terriers and Chinese Crested dogs. The associated pathologic lesions include degeneration of the cerebellum, caudate nucleus, and substantia nigra. CMSD is inherited as an autosomal recessive trait in both dog breeds. Previous linkage mapping localized the CMSD locus to a 15 MB region on canine chromosome 1 (CFA1). Next-generation sequencing was used to generate whole-genome sequences from the DNA of an affected dog from each breed. The resulting sequence reads were aligned to the NCBI canine reference genome (build 3.1). Among the homozygous sequence variants within the CFA1 target region, a nonsense variant in exon 15 of SERAC1 was identified in the affected Kerry Blue Terrier, while in the Chinese Crested dog, a 4 bp deletion in the SERAC1 exon 4 acceptor splice site was found. RT-PCR showed that this deletion resulted in exon 4 skipping. Genotyping of large cohorts of Kerry Blue Terriers and Chinese Crested dogs for the respective breed-specific SERAC1 variants showed complete concordance between genotype and disease phenotype. Genotype-phenotype concordance was also observed in offspring generated by cross breeding between SERAC1-heterozygous Kerry Blue Terrier and Chinese Crested dogs, with only the compound heterozygotes exhibiting the disease phenotype, further confirming the recessive inheritance of CMSD. Variants in human SERAC1 are associated with disorders with a range of ages of disease onset and patterns of clinical signs, but that are all characterized by movement abnormalities similar to those of the dogs with CMSD. Canine CMSD could serve as a valuable model to elucidate the mechanisms underlying SERAC1-deficiency disorders and to evaluate potential therapeutic interventions.

Genetic Variability in the CPA1 Gene and Its Impact on Acute Pancreatitis Risk: New Insights from a Large-Scale Study.

Acute pancreatitis (AP) is a common and potentially lethal disease. Over the last 10 years, AP has become one of the most important healthcare problems. On a global scale, the incidence has increased by 63% over the last 20 years. AP is usually caused by gallstones and excessive alcohol consumption and genetic factors play an important role in the development of inflammation. Recent studies involving the CPA1 mutations are ambiguous and dependent on the population studied. In this study, the variability of the CPA1 gene in patients with AP was analyzed. Genetic material was isolated from the blood of 301 patients with AP and 184 healthy individuals. Identification of the variants in exons 5, 6, 8, and 9 with introns was performed using molecular biology methods. Mutations were identified by comparison to the reference sequence (NM_001868.4). Statistical analysis included the identification of mutations correlating with the risk of AP, the etiology of inflammation, and family history. Several novel mutations in the CPA1 gene have been identified, along with a high degree of variability within the coding region of the carboxypeptidase gene. A correlation between mutations CPA1:c.1072 + 84del; c.987 + 57G>A and increased risk of developing AP was found. Two protective mutations, CPA1:c.625A>T, c.1072 + 94del, were identified. The CPA1 gene is characterized by high sequence variability and regions in which mutations lead to an increased risk of developing AP. Single or co-occurring mutations of the CPA1 gene can significantly affect the risk of developing AP.

Slowly progressive autosomal dominant Alport Syndrome due to COL4A3 splicing variant.

Alport syndrome is a rare genetic kidney disease caused by variants in the COL4A3/A4/A5 genes. It's characterised by progressive kidney failure, though therapies targeting Renin-Angiotensin System can delay its progression. Additionally, extrarenal manifestations may sometimes coexist. Recent advances in genetic analysis and the necessity to better clarify genotype-phenotype correlations in affected patients raises the importance of detecting even cryptic splicing variants, lying in both canonical and non-canonical splice sites variants such as last exonic nucleotide variants. These variants, often, do not cause an amino acid change but alter the snRNP proteins binding. We studied a big Italian family with Alport syndrome showing a clear dominant pattern of transmission with younger family members having only haematuria and older individuals presenting with End-Stage Kidney Failure (ESKF). Kidney biopsy showed the typical disease hallmarks. We deeply mined the data for SNV and CNV through exome sequencing on DNA from both peripheral blood samples and patients' podocytes-lineage cells. We identified an already reported synonymous variant, c.765G>A (p.(Thr255Thr)), in the last exonic nucleotide of exon 13 of the COL4A3 gene. Employing the patient's podocytes we demonstrated that this variant results in exon skipping leading to an in-frame deletion of 28 amino acids without leaky effect. According to the pattern of transmission, to the kidney biopsy and to the exome data analysis we provided further evidence that autosomal dominant Alport syndrome is a well-defined clinical entity. We also confirmed the pathogenicity of the synonymous COL4A3 variant for the first time demonstrating its role in a dominant pattern of transmission.

OpenASO: RNA Rescue - designing splice-modulating antisense oligonucleotides through community science.

Splice-modulating antisense oligonucleotides (ASOs) are precision RNA-based drugs that are becoming an established modality to treat human disease. Previously, we reported the discovery of ASOs that target a novel, putative intronic RNA structure to rescue splicing of multiple pathogenic variants of F8 exon 16 that cause hemophilia A. However, the conventional approach to discovering splice-modulating ASOs is both laborious and expensive. Here, we describe an alternative paradigm that integrates data-driven RNA structure prediction and community science to discover splice-modulating ASOs. Using a splicing-deficient pathogenic variant of F8 exon 16 as a model, we show that 25% of the top-scoring molecules designed in the Eterna OpenASO challenge have a statistically significant impact on enhancing exon 16 splicing. Additionally, we show that a distinct combination of ASOs designed by Eterna players can additively enhance the inclusion of the splicing-deficient exon 16 variant. Together, our data suggests that crowdsourcing designs from a community of citizen scientists may accelerate the discovery of splice-modulating ASOs with potential to treat human disease.

Clinical and genetic analysis of a Chinese pedigree affected with Familial focal epilepsy with variable foci due to variant of NPRL3 gene

To explore the clinical manifestations and genetic etiology of a Chinese pedigree affected with Familial focal epilepsy with variable foci (FFEVF). A FFEVF pedigree presented at the Department of Medical Genetics of Linyi Maternal and Child Health Care Hospital on March 14, 2023 was selected as the study subject. The proband was subjected to whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing of the proband and other affected members and bioinformatic analysis. This study was approved by the Linyi Maternal and Child Health Care Hospital (Ethics No. QTL-YXLL-2023048). WES revealed that the proband has harbored a heterozygous c.1642C>T (p.Arg548Cys) missense variant in exon 15 of the NPRL3 gene. Sanger sequencing confirmed that the variant was inherited from the proband's father, and multiple members of the pedigree had also harbored the same variant. Based on guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as variant of unknown significance (PM2_supporting + PP3). The clinical phenotype of FFEVF patients caused by variants of NPRL3 gene is extensive, and the patients may present with neurological abnormality of autism spectrum disorder in addition to seizures.

A Novel Loss of Function Variant in HCN1 Gene Underlies Early Infantile Epileptic Encephalopathy 24 [EIEE24]

Background: Early infantile epileptic encephalopathy (EIEE) is a rare neurological condition characterized by frequent seizures in the early stages of life, resulting in severely impaired cognitive and motor development. Although the specific causes of EIEE remain unknown, one of the primary causes is gene pathogenicity (even in the absence of consanguinity). Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are essential for proper brain function. They are regulated by multiple genes, and mutations in these genes induce channel malfunction, which can result in various severe conditions, including EIEE. Herein, we have reported a patient presenting hallmarks of EIEE. Methods: The patient underwent clinical, radiographic, and genetic analysis. A thorough clinical examination and molecular study were conducted utilizing whole exome sequencing and Sanger sequencing. Results: Whole exome sequencing of the proband revealed a novel de novo nonsynonymous/nonsense variant (c.1468A>T; (p.Lys490Ter) in exon 6 of the HCN1 gene. This variant may cause channel dysfunction via nonsynonymous/nonsense-mediated decay or aberrant protein, which may be associated with EIEE phenotypes. Conclusions: This evidence backs the idea that HCN1 has a vital role in brain development and lose of function can cause a range of debilitating conditions. Still, the functional characterization study of the HCN1 variants will be the fundamental tool for a better understanding of EIEE in the near future.

Non-Hotspot PIK3CA Variants Have Higher Variant Allele Frequency and are More Common in Syndromic Vascular Malformations.

PIK3CA variants are known to cause vascular malformations. We were interested in studying the phenotypic spectrum, the location within the PIK3CA gene, and the variant allele frequency (VAF) of somatic PI3KCA variants in vascular malformations. Clinical data of consecutive patients with extracranial/extraspinal vascular malformations were collected in the context of the VASCOM cohort (2008-2022, n = 558). Starting October 2020, biopsy samples were tested with the TSO500 gene panel (Illumina). All consenting patients with PIK3CA variants were included in this study. Eighty-nine patients had available genetic results by June 2022. PIK3CA variants (n = 25) were found in 16 simple/combined (nonsyndromic) vascular malformations and in nine vascular malformations associated with other anomalies (syndromic). Four hotspot variants in exons 9 and 20 (c.1624G>A, c.1633G>A, c.3140A>G, c.3140A>T) were identified in 16/25 patients (VAF 0.9%-9.7%). Six non-hotspot variants (c.328_330del, c.323_337del, c.353G>A, c.1258T>C, c.3132T>A, c.3195_3203delinsT) were detected in nine patients (VAF 3.6%-31.7%). Non-hotspot variants were more frequent in syndromic than nonsyndromic vascular malformations (p = 0.0034) and exhibited a higher VAF than hotspot variants (p = 0.0253). Our study contributes to the growing body of knowledge of the genetic background in vascular malformations. Further studies will enrich the ever-growing list of pathogenic PIK3CA variants associated with vascular malformations.