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Pathogenic heterozygous variants in CHD4 cause Sifrim-Hitz-Weiss syndrome, a neurodevelopmental disorder associated with brain anomalies, heart defects, macrocephaly, hypogonadism, and additional features with variable expressivity. Most individuals have non-recurrent missense variants, complicating variant interpretation. A few were reported with truncating variants, and their role in disease is unclear. DNA methylation episignatures have emerged as highly accurate diagnostic biomarkers in a growing number of rare diseases. We aimed to study evidence for the existence of a CHD4-related DNA methylation episignature. We collected blood DNA samples and/or clinical information from 39 individuals with CHD4 variants, including missense and truncating variants. Genomic DNA methylation analysis was performed on 28 samples. We identified a sensitive and specific DNA methylation episignature in samples with pathogenic missense variants within the ATPase/helicase domain. The same episignature was observed in a family with variable expressivity, a de novo variant near the PHD domain, variants of uncertain significance within the ATPase/helicase domain, and a sample with compound heterozygous variants. DNA methylation data revealed higher percentages of shared probes with BAFopathies, CHD8 , and the terminal ADNP variants encoding a protein known to form the ChAHP complex with CHD4. Truncating variants, as well as a sample with a recurrent pathogenic missense variant, exhibited DNA methylation profiles distinct from the ATPase/helicase domain episignature. These DNA methylation differences, together with the distinct clinical features observed in those individuals, provide preliminary evidence for clinical and molecular sub-types in the CHD4-related disorder. In this study, we performed DNA methylation studies on a cohort of individuals with Sifrim-Hitz-Weiss syndrome. We found a recognizable episignature in individuals with pathogenic missense variants within the ATPase helicase domain. Individuals with truncating variants exhibited a different episignature and phenotype with a higher rate of autism spectrum disorder. [ABSTRACT FROM AUTHOR]

BCL11B is a Cys2-His2 zinc-finger (C2H2-ZnF) domain-containing, DNA-binding, transcription factor with established roles in the development of various organs and tissues, primarily the immune and nervous systems. BCL11B germline variants have been associated with a variety of developmental syndromes. However, genotype-phenotype correlations along with pathophysiologic mechanisms of selected variants mostly remain elusive. To dissect these, we performed genotype-phenotype correlations of 92 affected individuals harboring a pathogenic or likely pathogenic BCL11B variant, followed by immune phenotyping, analysis of chromatin immunoprecipitation DNA-sequencing data, dual-luciferase reporter assays, and molecular modeling. These integrative analyses enabled us to define three clinical subtypes of BCL11B -related disorders. It is likely that gene-disruptive BCL11B variants and missense variants affecting zinc-binding cysteine and histidine residues cause mild to moderate neurodevelopmental delay with increased propensity for behavioral and dental anomalies, allergies and asthma, and reduced type 2 innate lymphoid cells. Missense variants within C2H2-ZnF DNA-contacting α helices cause highly variable clinical presentations ranging from multisystem anomalies with demise in the first years of life to late-onset, hyperkinetic movement disorder with poor fine motor skills. Those not in direct DNA contact cause a milder phenotype through reduced, target-specific transcriptional activity. However, missense variants affecting C2H2-ZnFs, DNA binding, and "specificity residues" impair BCL11B transcriptional activity in a target-specific, dominant-negative manner along with aberrant regulation of alternative DNA targets, resulting in more severe and unpredictable clinical outcomes. Taken together, we suggest that the phenotypic severity and variability is largely dependent on the DNA-binding affinity and specificity of altered BCL11B proteins. [Display omitted] Combining genotype-phenotype data of 92 individuals harboring a pathogenic BCL11B variant, with immune phenotyping, analysis of chromatin immunoprecipitation DNA-sequencing data, dual-luciferase reporter assays, and molecular modeling, the authors show here that nearby missense variants and different missense variants affecting the identical amino acid do not necessarily cause similar clinical outcomes. [ABSTRACT FROM AUTHOR]

Dystrophic epidermolysis bullosa is a rare subtype of inherited epidermolysis bullosa, caused by variants in the collagen type VII alpha 1 chain (COL7A1) gene (MIM120120). Both autosomal dominant and recessive inheritance has been reported with variable phenotype. We investigated a Pakistani family with dystrophic epidermolysis bullosa via exome sequencing and identified a pathogenic nonsense variant in COL7A1 NM_000094 c.1573 C > T:p.(Arg525*). The inheritance pattern observed was consistent with a semi-dominant model, where heterozygous parents exhibited a mild phenotype, and homozygous children were more severely affected. For dystrophic epidermolysis bullosa, loss-of-function variants are typically associated with the autosomal recessive form, while missense variants are linked to the autosomal dominant form. A review of the literature suggests a semi-dominance pattern for some missense variants, particularly glycine substitutions, but this concept had not been formally recognized. This study highlights the importance of considering semi-dominant inheritance models for dystrophic epidermolysis bullosa and other Mendelian diseases with an autosomal recessive mode of inheritance, as it can significantly impact diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

Recently, SASH1 has emerged as a novel protein interactor of a few Eph tyrosine kinase receptors like EphA2. These interactions involve the first N-terminal Sam (sterile alpha motif) domain of SASH1 (SASH1-Sam1) and the Sam domain of Eph receptors. Currently, the functional meaning of the SASH1-Sam1/EphA2-Sam complex is unknown, but EphA2 is a well-established and crucial player in cancer onset and progression. Thus, herein, to investigate a possible correlation between the formation of the SASH1-Sam1/EphA2-Sam complex and EphA2 activity in cancer, cancer-linked mutations in SASH1-Sam1 were deeply analyzed. Our research plan relied first on searching the COSMIC database for cancer-related SASH1 variants carrying missense mutations in the Sam1 domain and then, through a variety of bioinformatic tools and molecular dynamic simulations, studying how these mutations could affect the stability of SASH1-Sam1 alone, leading eventually to a defective fold. Next, through docking studies, with the support of AlphaFold2 structure predictions, we investigated if/how mutations in SASH1-Sam1 could affect binding to EphA2-Sam. Our study, apart from presenting a solid multistep research protocol to analyze structural consequences related to cancer-associated protein variants with the support of cutting-edge artificial intelligence tools, suggests a few mutations that could more likely modulate the interaction between SASH1-Sam1 and EphA2-Sam. [ABSTRACT FROM AUTHOR]

Black Americans are 3–4 times more likely to develop nondiabetic kidney disease than other populations. Exclusively found in people of recent African (AFR) ancestry, risk variants in Apolipoprotein L1 (APOL1) termed G1 and G2 contribute significantly to this increased susceptibility. Our group and others showed that a missense variant in APOL1 , rs73885316 (p.N264K, "M1"), is remarkably protective against APOL1 kidney disease when co-inherited with the G2 risk allele. Since the distance between the M1 and G2 variants is only 367 base pairs, we initially suspected that 2 independent mutation events occurred to create non-risk M1-G0 and M1-G2 haplotypes. Here, we examined APOL1 haplotypes in individuals of AFR ancestry from the 1000 Genomes Project, the Nephrotic Syndrome Study Network (NEPTUNE), and an ancient individual from the Allen Ancient Genome Diversity Project to determine how the M1-G2 haplotype arose. We demonstrate that M1 most likely first appeared on a non-risk G0 haplotype, and that a subsequent recombination event bypassed strong recombination hotspots flanking APOL1 and occurred between p.N388Y389del on a G2 haplotype and M1 on a G0 haplotype to create the M1-G2 haplotype. Observing a recombination event within a small region between clinically relevant loci emphasizes the importance of studying the entire haplotype repertoire of a disease gene and the impact of haplotype backgrounds in disease susceptibility. [ABSTRACT FROM AUTHOR]

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Bronchial papillomas are benign tumors, which can be divided into different subtypes based on the cellular features. So far, no bronchial papilloma with basaloid cell features has been reported. We report a bronchial basaloid papillary tumor in a 67-year-old woman. Tumor recurrence and malignant transformation were observed after a long-term follow-up. The clinical, histological, immunohistochemical, and genetic features were reappraised. The primary tumor was characterized by papillary growth pattern and basaloid neoplastic cells, only a small amount of neoplastic cells showed mature characteristics. The tumor originated from respiratory epithelium and had a low proliferation index by Ki-67. Keratin (KRT) 5/6 and KRT7 showed patchy or partial positivity. Myoepithelial markers were negative. P63 was diffusely positive, but it was negative in the small amount of tumor cells with mature characteristics. The common genetic alterations (EGFR, KRAS, BRAF V600E, HER2, and ALK) of lung cancers were not detected. However, tumor recurrence was observed in the mediastinum and esophagus 12 years after surgery. The recurrent tumor had a morphology overlapping with that of the primary tumor; however, it displayed significantly malignant characteristics. The recurrent tumor was not related to high-risk HPV. A high variant allele frequency was observed in tumor suppressor gene BRCA1, TP53, oncogene GNA11, and KIT, which were all missense mutations. Considering the bland features of the primary tumor and the fact of tumor recurrence and undisputed malignant transformation, the basaloid papillary tumor was considered a tumor with uncertain malignant potential. [ABSTRACT FROM AUTHOR]

Background: Congenital disorders of glycosylation are a rare group of disorders characterized by impaired glycosylation, wherein STT3A encodes the catalytic subunit of the oligosaccharyltransferase complex, which is crucial for protein N-glycosylation. Previous studies have reported that STT3A-CDG is caused by autosomal recessive inheritance. However, in this study, we propose that STT3A-CDG can be pathogenic through autosomal dominant inheritance. Methods: The variant was identified via trio whole-exome sequencing. We constructed wild-type and variant plasmids, transfected them into HEK293T cells and detected the expression levels of the STT3A protein. We performed CRISPR-Cas9 to establish heterozygous knockdown zebrafish to validate the functional implications of autosomal dominant inheritance of STT3A in pathogenesis. Results: The patient presented with developmental delay, distinctive facial features, short stature, and abnormal discharges. The heterozygous pathogenic missense variant (NM_001278503.2: c.499G > T, NP_001265432.1:p. Asp167Tyr) was identified, and the Western blot results revealed a significant decrease in protein levels. Heterozygous knockdown zebrafish exhibit phenotypes similar to those of patients, including craniofacial dysmorphology (increased eye distance, increased Basihyal's length, increased Ceratohyal's angle), skeletal abnormalities (reduced number of mineralized bones), developmental delay (reduced adaptability under light‒dark stimuli suggesting abnormal locomotion, orientation, and social behavior), and electrophysiological abnormalities. Conclusion: We report a proband with a dominant congenital glycosylation disorder caused by heterozygous pathogenic STT3A variation, which is a new inheritance pattern of STT3A. Our report expands the known phenotype of dominant STT3A-CDGs. Furthermore, we provide in vivo validation through the establishment of a heterozygous knockdown zebrafish model for stt3a and strengthened the compelling evidence for dominant STT3A-related pathogenesis. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to report the spectrum of genetic variations and clinical phenotype in a Vietnamese cohort with confirmed Brugada syndrome (BrS) using the whole exome sequencing (WES). Methods: Fifty patients with confirmed BrS were included in this study. Genomic DNA samples were extracted from peripheral blood and conducted for WES. The variants were annotated using ANNOVAR. The variants in the 13 reported genes associated with BrS were filtered, predicted the functional impact using eight computational tools, and classified according to the 2015 ACMG guidelines. Results: Arrhythmic events were documented in one-fifth of the participants. Twenty-four probands were identified to carry 36 variants in 13 genes. Majority of the variants in our study was SCN5A variants (9/36 variants, 25%), followed by KCNH2 variants (5/36 variants, 14%). The prevalence of SCN5A carriers was 16%; while the prevalence of minor gene carriers was less than 10%. Nine novel missense variants were identified, including four missense SCN5A variants (p.E901D, p.F853L, p.L377F, and p.H184R), two missense ANK2 variants (p.S2845L and V1497L), one missense CACNA1C variant (M1126V), one missense DSP variant (p.K478N), and one intron splicing JUP variant (c.1498-5G>C). Conclusion: Our study underscores the primary significance of the SCN5A gene in BrS, as indicated by variant prevalence, carrier rates, pathogenicity per ACMG classification, in silico predictions, and its correlation with clinical phenotypes. Longitudinal study with larger sample size, pedigree, Sanger sequence confirmation, and functional analysis is recommended. [ABSTRACT FROM AUTHOR]

• Reporting co-occurrence of Retinitis Pigmentosa in a patient with Parkinson's disease. • Whole exome sequencing shows c.190A > T (p.Thr64Ser) variation in SNCAIP gene. • SNCAIP co-occurred with RP associated CNGA-1 gene variation c.1525G > A (p.Gly509Arg). • In-silico analysis shows interaction of SNCAIP and CNGA-1 through SNCA and LGALS4. • Future research of genotypic and phenotypic correlation in PD and RP is warranted. Parkinson's disease (PD) is primarily driven by the protein Alpha Synuclein (A-Syn) accumulation. Synphilin-1 protein, encoded by the SNCAIP gene, which co-localizes with A-Syn is a known risk factor for PD. Retinitis pigmentosa (RP), is a cluster of retinal degenerative disorders, and Cyclic Nucleotide Gated channel subunit Alpha 1 (CNGA1) is one of the initial genes associated with RP. Patients with PD can have various kinds of visual dysfunction as a non-motor manifestation, but to date, CNGA1 mutation and RP as a PD associated visual symptom has not been reported. We report a mutation in the SNCAIP gene in a PD patient, not reported earlier, and its co-occurrence with RP-associated CNGA1 gene mutation. Whole exome sequencing (WES) of the patient DNA sample and in-silico protein–protein interaction (PPI) analysis performed to find out proteins interacting with SNCAIP relevant concerning reported mutation of SNCAIP and further, CNGA1 interaction with SNCAIP. We are reporting, a missense mutation (p.Thr64Ser) at the SNCAIP gene, co-occurring with a missense variation (p.Gly509Arg) in the CNGA1 gene. In silico PPI analysis suggests SIAH1 as an important protein affected by SNCAIP mutation. LGALS4 and SNCA (gene encoding A-Syn) are common interactors between SNCAIP and CNGA1. The current study has determined the co-occurrence of RP and PD, whole exome sequencing ascertains the mutations in SNCAIP and CNGA1 genes, which could be the cause of PD and RP co-occurrence. [ABSTRACT FROM AUTHOR]

The ARID1A gene, frequently mutated in cancer, encodes the AT-rich interactive domain-containing protein 1 A, a key component of the chromatin remodeling SWI/SNF complex. The ARID1A protein features a conserved DNA-binding domain (ARID domain) of approximately 100 residues crucial for its function. Despite the frequency of mutations, the impact on ARID1A's stability and contribution to cancer progression remains unclear. We analyzed five frequent missense mutations R1020S, M1022K, K1047Q, G1063V, and A1089T identified in The Cancer Genome Atlas (TCGA) to assess their effects on the stability of the ARID domain using a hybrid experimental and computational approach. By combining computational stability from web server tools, the structural dynamics from replica exchange discrete molecular simulation (rexDMD), and thermal and chemical denaturation experiments, we found that the R1020S mutation severely decreases structural stability, making it the most impactful, while M1022K has minimal effect, and others lie in between. These findings enhance our understanding of the structural-functional relationship of ARID1A missense mutations at the molecular levels and their role in cancer pathogenesis. This research paves the way for identifying and categorizing which ARID1A mutations are most pathogenic, potentially guiding the development of targeted therapies tailored to specific mutation profiles in cancer treatment. [ABSTRACT FROM AUTHOR]

The cytotoxic T-lymphocyte antigen-4 (CTLA4) is essential in controlling T cell activity within the immune system. Thus, uncovering the molecular dynamics of single nucleotide polymorphisms (SNPs) within the CTLA4 gene is critical. We identified the non-synonymous SNPs (nsSNPs), examined their impact on protein stability, and identified the protein sequences associated with them in the human CTLA4 gene. There were 3134 SNPs (rsIDs) in our study. Out of these, 186 missense variants (5.93%), 1491 intron variants (47.57%), and 91 synonymous variants (2.90%), while the remaining SNPs were unspecified. We utilized SIFT, PolyPhen-2, PROVEAN, and SNAP for identifying deleterious nsSNPs, and SNPs&GO, PhD SNP, and PANTHER for verifying risk nsSNPs in the CTLA4 gene. Following SIFT analysis, six nsSNPs were identified as deleterious and reporting second and third nsSNPs as probably damaging and one as benign, respectively. From upstream analysis, rs138279736, rs201778935, rs369567630, and rs376038796 were found to be deleterious, probably damaging, and disease associated. ConSurf predicted conservation scores for four nsSNPs, and Project Hope suggested that all mutations could disrupt protein interactions. Furthermore, mCSM and DynaMut2 analyses indicated a decrease in ΔΔG stability for the mutants. GeneMANIA and STRING networks highlighted correlations with CD86 and CD80 genes. Finally, MD simulation revealed consistent fluctuation in RMSD and RMSF, consequently Rg, hydrogen bonds, and PCA in the mutant proteins compared with wild-type, which might alter the functional and structural stability of CTLA4 protein. The current comprehensive study shows how various nsSNPs in the CTLA4 gene can modify the structural and functional characteristics of the protein, potentially influencing the pathogenesis of diseases in humans. Further, experimental studies are needed to analyze the effect of these nsSNPs on the susceptibility of pathological phenotype populations. [ABSTRACT FROM AUTHOR]

Background: Glycerol-3-phosphate dehydrogenase 1 (GPD1) gene defect can cause hypertriglyceridemia (HTG), which usually occurs in infants. The gene defect has rarely been reported in adult HTG patients. In the present study, we described the clinical and functional analyses of a novel GPD1 missense variant in a Chinese adult patient with recurrent hypertriglyceridemia‑related acute pancreatitis (HTG-AP), consuming a high-fat diet and smoking heavily. Methods: Exome sequencing was used to analyze the DNA of the adult patient's blood sample. It was found that there was a new variant of GPD1 gene-p.K327N, which was verified by gold standard-sanger sequencing method. In vitro, the corresponding plasmid was constructed and transfected into human renal HEK-293T cells, and GPD1 protein levels were detected. A biogenic analysis was performed to study the population frequency, conservation, and electric potential diagram of the new variant p.K327N. Finally, the previously reported GPD1 variants were sorted and their phenotypic relationships were compared. Results: A novel heterozygous variant of GPD1, p.K327N (c.981G > C), was found in the proband. Furthermore, the patient's daughter carried this variant, whereas his wife did not carry the variant. The proband with obesity suffered eight episodes of HTG-AP from the age of 36 years, and each onset of AP was correlated to high-fat diet consumption and heavy smoking. In vitro, this variant exerted a relatively mild effect on GPD1 functions, which were associated with its effect upon secretion (~ 25% of secretion decreased compared with that of the wild-type); thus, eventually impairing protein synthesis. Additionally, 36 patients with GPD1 variants found in previous studies showed significant transient HTG in infancy. The proband carrying the GDP1 variant was the first reported adult with recurrent HTG-AP. Conclusion: We identified a novel GPD1 variant, p.K327N, in a Chinese adult male patient with recurrent HTG-AP. The variant probably exerted a mild effect on GPD1 functions. The heterozygosity of this GPD1 variant, in addition to high-fat diet consumption and heavy smoking, probably triggered HTG-AP in the patient. [ABSTRACT FROM AUTHOR]

De novo mutations in the RNA binding protein DDX3X cause neurodevelopmental disorders including DDX3X syndrome and autism spectrum disorder. Amongst ~200 mutations identified to date, half are missense. While DDX3X loss of function is known to impair neural cell fate, how the landscape of missense mutations impacts neurodevelopment is almost entirely unknown. Here, we integrate transcriptomics, proteomics, and live imaging to demonstrate clinically diverse DDX3X missense mutations perturb neural development via distinct cellular and molecular mechanisms. Using mouse primary neural progenitors, we investigate four recurrently mutated DDX3X missense variants, spanning clinically severe (2) to mild (2). While clinically severe mutations impair neurogenesis, mild mutations have only a modest impact on cell fate. Moreover, expression of severe mutations leads to profound neuronal death. Using a proximity labeling screen in neural progenitors, we discover DDX3X missense variants have unique protein interactors. We observe notable overlap amongst severe mutations, suggesting common mechanisms underlying altered cell fate and survival. Transcriptomic analysis and subsequent cellular investigation highlights new pathways associated with DDX3X missense variants, including upregulated DNA Damage Response. Notably, clinically severe mutations exhibit excessive DNA damage in neurons, associated with increased cytoplasmic DNA:RNA hybrids and formation of stress granules. These findings highlight aberrant RNA metabolism and DNA damage in DDX3X-mediated neuronal cell death. In sum our findings reveal new mechanisms by which clinically distinct DDX3X missense mutations differentially impair neurodevelopment. Author summary: DDX3X mutations are associated with neurodevelopmental disorders, including DDX3X syndrome and autism spectrum disorder. DDX3X is an RNA binding protein whose loss of function impairs brain development and generation of neurons. In this study, we focus on disease-associated missense mutations containing single amino acid changes in the DDX3X gene. We functionally probe two clinically severe mutations, associated with severe brain anatomical defects and intellectual disability, and two clinically mild mutations, characterized by modest anatomical disruptions and behavioral phenotypes. Using transcriptomics, proteomics, and live imaging, we identify distinct mechanisms by which clinically diverse DDX3X missense mutations disrupt neurodevelopment. Our findings reveal severe variants impair neuronal production and survival, by altering RNA metabolism and DNA damage response. [ABSTRACT FROM AUTHOR]

The eye primordium of vertebrates initially forms exactly at the side of the head. Later, the eyeball architecture is tuned to see ahead with better visual acuity, but its molecular basis is unknown. The position of both eyes in the face alters in patients with holoprosencephaly due to Sonic hedgehog (Shh) mutations that disturb the development of the ventral midline of the neural tube. However, patient phenotypes vary extensively, and microforms without a brain anomaly relate instead to alternation of gene expression of the Shh signaling center in the facial primordia. We identified novel missense mutations of the Shh gene in two patients with a dislocated fovea, where the photoreceptor cells are condensed. Functional assays showed that Shh upregulates Patched and Gli and downregulates Pax6, and that Shh mutations alter these activities. Gain of function of Shh in a chick embryo retards retinal development and eyeball growth depending on the location of Shh expression, while loss of function of Shh promotes these features. We postulate that a signaling molecule like Shh that emanates from the face controls the extent of differentiation of the neural retina in a position-specific manner and that this may result in the formation of the fovea at the correct location. [ABSTRACT FROM AUTHOR]

Achondroplasia, the most prevalent short-stature disorder, is caused by missense variants overactivating the fibroblast growth factor receptor 3 (FGFR3). As current surgical and pharmaceutical treatments only partially improve some disease features, we sought to explore a genetic approach. We show that an enhancer located 29 kb upstream of mouse Fgfr3 (-29E) is sufficient to confer a transgenic mouse reporter with a domain of expression in cartilage matching that of Fgfr3. Its CRISPR/Cas9-mediated deletion in otherwise WT mice reduced Fgfr3 expression in this domain by half without causing adverse phenotypes. Importantly, its deletion in mice harboring the ortholog of the most common human achondroplasia variant largely normalized long bone and vertebral body growth, markedly reduced spinal canal and foramen magnum stenosis, and improved craniofacial defects. Consequently, mouse achondroplasia is no longer lethal, and adults are overall healthy. These findings, together with high conservation of -29E in humans, open a path to develop genetic therapies for people with achondroplasia. [ABSTRACT FROM AUTHOR]

Autosomal dominant CDK13-related disease is characterized by congenital heart defects, dysmorphic facial features, and intellectual developmental disorder (CHDFIDD). Heterozygous pathogenic variants, particularly missense variants in the kinase domain, have previously been described as disease causing. Using the determination of a methylation pattern and comparison with an established episignature, we reveal the first hypomorphic variant in the kinase domain of CDK13, leading to a never before described autosomal recessive form of CHDFIDD in a boy with characteristic features. This highlights the utility of episignatures in variant interpretation, as well as a potential novel diagnostic approach in unsolved cases or for disease prognosis. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder with both genetic and environmental factors contributing to its pathogenesis. While early-onset AD has well-established genetic determinants, the genetic basis for late-onset AD remains less clear. This study investigates a large Italian family with late-onset autosomal dominant AD, identifying a novel rare missense variant in GRIN2C gene associated with the disease, and evaluates the functional impact of this variant. Methods: Affected and unaffected members from a Northern Italian family were included. Genomic DNA from family members was extracted and initially screened for pathogenic mutations in APP, PSEN1, and PSEN2, and screened for 77 genes associated with neurodegenerative conditions using NeuroX array assay. Exome sequencing was performed on three affected individuals and two healthy relatives. Bioinformatics analyses were conducted. Functional analysis was performed using primary neuronal cultures, and the impact of the variant was assessed through immunocytochemistry and electrophysiology. Results: Pathogenic variants were not identified in APP, PSEN1, or PSEN2, nor in the 77 genes in NeuroX array assay. Exome Sequencing revealed the c.3215C > T p.(A1072V) variant in GRIN2C gene (NM 000835.6), encoding for the glutamate ionotropic receptor N-methyl-D-aspartate receptor (NMDA) type subunit 2C (GluN2C). This variant segregated in 6 available AD patients in the family and was absent in 9 healthy relatives. Primary rat hippocampal neurons overexpressing GluN2CA1072V showed an increase in NMDAR-induced currents, suggesting altered glutamatergic transmission. Surface expression assays demonstrated an elevated surface/total ratio of the mutant GluN2C, correlating with the increased NMDAR current. Additionally, immunocytochemistry revealed in neurons expressing the mutant variant a reduced colocalization between the GluN2C subunit and 14-3-3 proteins, which are known to facilitate membrane trafficking of NMDARs. Discussion: We identified a rare missense variant in GRIN2C associated with late-onset autosomal dominant Alzheimer's disease. These findings highlight the role of GluN2C-containing NMDARs in glutamatergic signaling and their potential contribution to AD pathogenesis. [ABSTRACT FROM AUTHOR]

AbstractInherited prion diseases (IPD) secondary to mutations of the prion protein gene, PRNP, exhibit diverse clinical phenotypes, capable of mimicking numerous primary neurodegenerative conditions. We describe the clinical phenotype and neuropathological findings in a family from County Limerick in Ireland presenting with Alzheimer’s disease-like cognitive decline and motor symptoms caused by a novel missense mutation of PRNP. This mutation occurs in the PRNP central lysine cluster (CLC; codon 101-110), resulting in substitution of threonine with isoleucine at codon 107 (T107I). This case series highlights that IPD can be hard to distinguish from overlapping clinical syndromes seen in other neurodegenerative diseases. We also discuss similarities and differences of the novel mutation T107I to other pathogenic mutations of the CLC of PRNP. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC), half of which are lung adenocarcinoma (LUAD), is one of the most widely spread cancers in the world. Telomerase, which maintains telomere length and chromosomal integrity, enables cancer cells to avoid replicative senescence. When telomerase is inhibited, cancer cells' senescence began, preventing them from growing indefinitely. Cellular senescence and telomeres are intrinsically linked. As of yet, still laking a systematic study of the involvement of telomere-senescence related genes in lung adenocarcinoma development. In this study, myeloid cells were identified as the cell type which are most correlated with cellular senescence based on its highest telomere-related gene activity. GO, KEGG, GSEA and GSVA analyses were used to explore the biological function of telomere-senescence related genes in LUAD. The combined analysis of single-cell RNA-sequencing and bulk-RNA sequencing identified a gene signature composed of 14 genes which can accurately predict the prognosis of patients with LUAD. In one training and four validation sets, patients with higher Telomere Related Gene Signature (TRGS) had a worse prognosis than those with lower TRGS. Different TRGS patient groups showed varying degrees of immune cell infiltration, frequency of gene missense mutation, sensitivity to different drugs, and tumor mutation burden (TMB). Collectively, we developed a brand new signature composed of telomere-senescence related genes that can accurately predicts patients' prognosis in LUAD, which provides new insights for future research into the role of cellular senescence in LUAD. [ABSTRACT FROM AUTHOR]

Recurrent missense mutations in the human epidermal growth factor receptor 2 (HER2) have been identified across various human cancers. Among these mutations, the active S310F mutation in the HER2 extracellular domain stands out as not only oncogenic but also confers resistance to pertuzumab, an antibody drug widely used in clinical cancer therapy, by impeding its binding. In this study, we have successfully employed computational-aided rational design to undertake directed evolution of pertuzumab, resulting in the creation of an evolved pertuzumab variant named Ptz-SA. This variant, with only two mutations (T30S/D31A) located on its heavy chain, effectively reinstates binding to the mutated antigen, at the expense of a 35-fold reduction in binding affinity to HER2 (S310F) compared to the wild-type pair. Subsequently, Ptz-SA demonstrates potent killing capacity through antigen-dependent cytotoxicity. Moreover, upon engineering Ptz-SA into antibody-drug conjugates, such as Ptz-SA-MMAE, it manifests notable in vitro and in vivo antitumor efficacy by efficiently delivering cytotoxic payload into tumor cells expressing HER2 (S310F). Cryoelectron microscopy studies elucidate the molecular mechanism underlying the restored binding ability of Ptz-SA toward the S310F mutation. The steric hindrance induced by the S310F mutation is efficiently circumvented by the T30S and D31A mutations, which provides adequate space to accommodate the larger phenylalanine. Additionally, Ptz-SA also exhibits binding capacity to HER2 (S310Y), another mutation occurring at the S310 site of HER2 with high frequency. The computational-aided evolution of pertuzumab provides an alternative strategy for overcoming point mutation-mediated resistance to therapeutic antibodies. [ABSTRACT FROM AUTHOR]

Purpose: To explore possible pathogenic genes for concomitant exotropia using whole-exome sequencing. Methods: In this study, 47 individuals from 10 concomitant exotropia (including intermittent exotropia and constant exotropia) pedigrees were enrolled. Whole-exome sequencing was used to screen mutational profiles in 25 affected individuals and 10 unaffected individuals. Sanger sequencing and in silico analysis were performed for all participants. Two target genes were used to capture the sequences of 220 sporadic samples. Results: All 10 concomitant exotropia pedigrees presented autosomal dominant inheritance with childhood onset (3.35 ± 1.51 years old). Eleven different missense variants were identified among seven potential pathogenic genes (COL4A2, SYNE1, LOXHD1, AUTS2, GTDC2, HERC2 and CDH3) that cosegregated with pedigree members. All variants were predicted to be deleterious and had low frequencies in the general population. Distinct variants of COL4A2 were present in three pedigrees, and distinct variants of SYNE1 were present in two pedigrees. Fifteen variants in AUTS2 and four variants in GTDC2 were identified in 220 patients with sporadic concomitant exotropia using a target-capture sequencing approach. Conclusion: This is the first study to explore the genetic mechanism of concomitant exotropia and identify seven associated genes (COL4A2, SYNE1, LOXHD1, AUTS2, GTDC2, HERC2 and CDH3) that may be candidate genes causing concomitant exotropia. More samples and in-depth studies are needed to verify these findings. [ABSTRACT FROM AUTHOR]

Background: Pancreatic adenocarcinoma (PAAD) has a very poor prognosis, and there are few treatments for PAAD. Therefore, it is important to find some biomarkers for the diagnosis and treatment of PAAD. Although some members of Armadillo repeat containing proteins (ARMCs) have been implicated in the development of certain cancers, their relationship with PAAD remains unknown. In this study, we aimed to explore the expression and prognostic value of ARMCs in PAAD. Methods: We used the The Cancer Genome Atlas (TCGA) database for survival analysis. Then, Gene Expression Profiling Interactive Analysis (GEPIA), the cBioPortal database, the Human Protein Atlas (HPA), Kaplan-Meier Plotter, LinkedOmics Database, Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG), Cytoscape and Timer were used to analyze the relationship between ARMCs and PAAD. In addition, we established a prognostic model of ARMCs for PAAD. Immunohistochemistry (IHC) was also performed. Then Image-J was used to analyze all images obtained from the experiment, and GraphPad-Prism (9.5.1) was used for statistical analysis to verify the expression of ARMCs in PAAD. Results: In the TCGA database, the expressions of ARMC1, 2, 3, 5, 6, 7, 8, 9 and 10 in PAAD tissues were significantly higher than those in normal tissues. And higher expressions of ARMC1 and 10 were associated with lower survival rate of PAAD patients. In addition, ARMC2, 5, 6, and 10 were positively associated with advanced stages of PAAD. ARMCs mutations occur in 11% of PAAD patients, and missense mutations and amplification of ARMCs account for most of them. In addition, ARMC5 and ARMC10 were independent prognostic factors in univariate and multivariate Cox regression analyses. Finally, through our confirmation experiment, it was found that the expression of ARMC1 and 10 in PAAD tissues was significantly increased compared with those in paracancer tissue. Conclusion: This study suggests that ARMCs may be able to play important roles in PAAD, and they can act as biomarkers, providing valuable clues for the treatment and diagnosis of PAAD. [ABSTRACT FROM AUTHOR]

Innovative and easy-to-implement strategies are needed to improve the pathogenicity assessment of rare germline missense variants. Somatic cancer driver mutations identified through large-scale tumor sequencing studies often impact genes that are also associated with rare Mendelian disorders. The use of cancer mutation data to aid in the interpretation of germline missense variants, regardless of whether the gene is associated with a hereditary cancer predisposition syndrome or a non-cancer-related developmental disorder, has not been systematically assessed. We extracted putative cancer driver missense mutations from the Cancer Hotspots database and annotated them as germline variants, including presence/absence and classification in ClinVar. We trained two supervised learning models (logistic regression and random forest) to predict variant classifications of germline missense variants in ClinVar using Cancer Hotspot data (training dataset). The performance of each model was evaluated with an independent test dataset generated in part from searching public and private genome-wide sequencing datasets from ~1.5 million individuals. Of the 2,447 cancer mutations, 691 corresponding germline variants had been previously classified in ClinVar: 426 (61.6%) as likely pathogenic/pathogenic, 261 (37.8%) as uncertain significance, and 4 (0.6%) as likely benign/benign. The odds ratio for a likely pathogenic/pathogenic classification in ClinVar was 28.3 (95% confidence interval: 24.2–33.1, p < 0.001), compared with all other germline missense variants in the same 216 genes. Both supervised learning models showed high correlation with pathogenicity assessments in the training dataset. There was high area under precision-recall curve values (0.847 and 0.829) and area under the receiver-operating characteristic curve values (0.821 and 0.774) for logistic regression and random forest models, respectively, when applied to the test dataset. With the use of cancer and germline datasets and supervised learning techniques, our study shows that cancer mutation data can be leveraged to improve the interpretation of germline missense variation potentially causing rare Mendelian disorders. Author summary: Our study introduces an approach to improve the interpretation of rare genetic variation, specifically missense variants that can alter proteins and cause disease. We found that published evidence from somatic cancer sequencing studies may be relevant to understanding the impact of the same variant in the context of rare inherited (Mendelian) disorders. By using widely available datasets, we noted that many cancer driver mutations have also been observed as rare germline variants associated with inherited disorders. This intersection led us to employ machine learning techniques to assess how cancer mutation data can predict the pathogenicity of germline variants. We trained machine learning models and tested them on a separate dataset curated by searching public and private genome-wide sequencing data from over a million participants. Our models were able to successfully identify pathogenic genetic changes, demonstrating strong performance in predicting disease-causing variants. This study highlights that cancer mutation data can enhance the interpretation of rare missense variants, aiding in the diagnosis and understanding of rare diseases. Integrating this approach into current genetic classification frameworks could be beneficial, and opens new avenues for leveraging existing cancer research to benefit broader genetic research and diagnostics for rare genetic conditions. [ABSTRACT FROM AUTHOR]

About 50% of all cancers carry a mutation in p53 that impairs its tumor suppressor function. The p53 missense mutation p53R175H (p53R172H in mice) is a hotspot mutation in various cancer types. Therefore, monoclonal antibodies selectively targeting clinically relevant mutations like p53R175H could prove immensely value. We aimed to evaluate the in vitro and in vivo binding properties of two novel anti-p53R175H monoclonal antibodies and to assess their performance as agents for molecular imaging. In vitro, 125I-4H5 and 125I-7B9 demonstrated long shelf life and antigen-specific binding. Our in vivo study design allowed head-to-head comparison of the antibodies in a double tumor model using repeated SPECT/CT imaging, followed by biodistribution and autoradiography. Both tracers performed similarly, with marginally faster blood clearance for 125I-7B9. Repeated molecular imaging demonstrated suitable imaging characteristics for both antibodies, with the best contrast images occurring at 48 h post-injection. Significantly higher uptake was detected in the mut-p53-expressing tumors, confirmed by ex vivo autoradiography. We conclude that molecular imaging with an anti-p53R175H tracer could be a promising approach for cancer diagnostics and could be further applied for patient stratification and treatment response monitoring of mutant p53-targeted therapeutics. [ABSTRACT FROM AUTHOR]

Mutations can disrupt the native function of protein by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms for misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours - acting to stabilize a wide array of cytosolic proteins, including wild-type variants, and to specifically 'buffer' some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein. [ABSTRACT FROM AUTHOR]

Background/Objectives: The failure of physiological left-right (LR) patterning, a critical embryological process responsible for establishing the asymmetric positioning of internal organs, leads to a spectrum of congenital abnormalities characterized by laterality defects, collectively known as "heterotaxy". MMP21 biallelic variants have recently been associated with heterotaxy syndrome and congenital heart defects (CHD). However, the genotype–phenotype correlations and the underlying pathogenic mechanisms remain poorly understood. Methods: Patients harboring biallelic MMP21 missense variants who underwent diagnostic genetic testing for CHD or heterotaxy were recruited at the Institute for Maternal and Child Health—I.R.C.C.S. "Burlo Garofolo". Additionally, a literature review on MMP21 missense variants was conducted, and clinical data from reported patients, along with molecular data from in silico and modeling tools, were collected. Results: A total of 18 MMP21 missense variants were reported in 26 patients, with the majority exhibiting CHD (94%) and variable extra-cardiac manifestations (64%). In our cohort, through Whole-Exome Sequencing (WES) analysis, the missense p.(Met301Ile) variant was identified in two unrelated patients, who both presented with heterotaxy syndrome. Conclusions: Our comprehensive analysis of MMP21 missense variants supports the pathogenic role of the p.(Met301Ile) variant and provides significant insights into the disease pathogenesis. Specifically, missense variants are distributed throughout the gene without clustering in specific regions, and phenotype comparisons between patients carrying missense variants in compound heterozygosity or homozygosity do not reveal significant differences. These findings may suggest a potential loss-of-function mechanism for MMP21 missense variants, especially those located in the catalytic domain, and highlight their critical role in the pathogenesis of heterotaxy syndrome. [ABSTRACT FROM AUTHOR]

Background/Objectives: The ectodysplasin A (EDA) gene, a member of the tumor necrosis factor ligand superfamily, is involved in the early epithelial–mesenchymal interaction that regulates ectoderm-derived appendage formation. Numerous studies have shown that mutations in the EDA gene can cause X-linked ectodermal dysplasia (ED) and non-syndromic oligodontia (NSO). Accordingly, this study aimed to identify the causative genetic mutations of the EDA gene. Methods: We investigated EDA gene mutations in two X-linked oligodontia families using candidate gene sequencing and whole-exome sequencing, with a single proband identified and studied for each family. The first family included a patient with NSO, while the second family had a patient exhibiting variable expression of ED. Results: Mutational analysis identified two missense mutations in the EDA gene (NM_001399.5): one novel mutation, c.787A>C p.(Lys263Gln), in family 2; and one previously reported mutation, c.457C>T p.(Arg153Cys), in family 1. All mutated residues are evolutionarily highly conserved amino acids. The p.(Arg153Cys) mutation would destroy the furin recognition site and affect the cleavage of EDA. The p.(Lys263Gln) mutation in a TNF homology domain would interfere with the binding of the EDA receptor. The p.(Lys263Gln) mutation was associated with NSO, while the other mutation demonstrated ED. Conclusions: This study helps to better understand the nature of EDA-related ED and NSO and their pathogenesis, and it expands the mutational spectrum of EDA mutations. [ABSTRACT FROM AUTHOR]

A couple presented to the office with an apparently healthy infant for a thorough clinical assessment, as they had previously lost two male children to a neurodegenerative disorder. They also reported the death of a male cousin abroad with a comparable condition. We aimed to evaluate a novel coding pathogenic variant c.1097T>A, PLA2G6, within the affected family, previously identified in a deceased cousin, but its clinical significance remained undetermined. A 200 bp PCR product of target genome (including codon 366 of PLA2G6) was amplified followed by enzymatic digestion (MboI) and sequencing. Structural pathogenic variant analysis was performed using PyMOL 2.5.4. In RFLP analysis, the mutant-type allele produced a single band of 200 bp, and the wild-type allele manifested as two bands of 112 bp and 88 bp. The pathogenic variant was identified in nine family members, including two heterozygous couples with consanguineous marriages resulting in affected children. It was predicted to be deleterious by multiple bioinformatic tools. The substitution of nonpolar isoleucine with polar asparagine of iPLA2 (Ile366Asn) resulted in a eense pathogenic variant (ATC>AAC). A missense variant (p. Ile366Asn) in the PLA2G6 gene is associated with clinically evident infantile neuroaxonal dystrophy, which is transmitted in an autosomal recessive pattern, and is also predicted to be dysfunctional by bioinformatic analyses. [ABSTRACT FROM AUTHOR]

Background Alterations in forkhead box A1 (FOXA1), a pioneer transcription factor, are associated with poor prognosis in breast cancer and prostate cancer. We characterized FOXA1 genomic alterations and their clinical impacts in a large pan-cancer cohort from the American Association for Cancer Research Genomics, Evidence, Neoplasia, Information, Exchange database. Methods FOXA1 alterations were characterized across more than 87 000 samples from more than 30 cancer types for primary and metastatic tumors alongside patient characteristics and clinical outcomes. FOXA1 alterations were queried in the Memorial Sloan Kettering - Metastatic Events and Tropisms (MSK-MET) cohort (a GENIE subset), allowing definition of hazard ratios (HRs) and survival estimates based on Cox proportional hazard models. Results FOXA1 was altered in 1869 (2.1%) samples, with distinct patterns across different cancers: prostate cancer enriched with indel-inframe alterations, breast cancer with missense mutations, and lung cancers with copy number amplifications. Of 74 715 samples with FOXA1 copy number profiles, amplification was detected in 834 (1.1%). Amplification was most common in non–small cell lung cancer (NSCLC; 3% in primary; 6% in metastatic) and small cell lung cancer (4.1% primary; 3.5% metastatic), followed by breast cancer (2% primary; 1.6% metastatic) and prostate cancer (2.2% primary; 1.6% metastatic). Copy number amplifications were associated with decreased overall survival in NSCLC (HR = 1.45, 95% confidence interval [CI] = 1.06 to 1.99; P  = .02), breast cancer (HR = 3.04, 95% CI = 1.89 to 4.89; P  = 4e−6), and prostate cancer (HR = 1.94, 95% CI = 1.03 to 3.68; P  = .04). Amplifications were associated with widespread metastases in NSCLC, breast cancer, and prostate cancer. Conclusions FOXA1 demonstrates distinct alteration profiles across cancer sites. Our findings suggest an association between FOXA1 amplification and enhanced metastatic potential and decreased survival, highlighting prognostic and therapeutic potential in breast cancer, prostate cancer, and NSCLC. [ABSTRACT FROM AUTHOR]

Next‐generation sequencing is advancing in low‐ and middle‐income countries, but accessibility remains limited. In Pakistan, many members of the Pashtun population practice familial marriage and maintain distinct socio‐cultural traditions, isolating them from other ethnic groups. As a result, they may harbor genetic variants that could unveil new gene‐disease associations. To investigate the genetic basis of epilepsy in the Pashtun community we recently established a collaboration between Bannu University and the University of Tuebingen. Here we report our first results of exome sequencing of four families with presumed monogenetic epilepsy and Mendelian inheritance pattern. In Family #201, we identified distinct disease‐causing variants. One had a homozygous pathogenic missense variant in TSEN54 (c.919G > T, p.(Ala307Ser)), linked to Pontocerebellar Hypoplasia Type 2A. The second individual had a homozygous class IV missense variant in MOCS2 (c.226G > A, p.(Gly76Arg)) which is associated with Molybdenum cofactor deficiency. In family EP02, one affected individual carried a heterozygous class III variant in OPHN1 (c.1490G > A, p.(Arg497Gln)), related to syndromic X‐linked intellectual disability with epilepsy. Our small study demonstrates the promise of next‐generation sequencing in genetic epilepsies among the Pashtun population. Diagnostic next‐generation sequencing should be established in Pakistan as soon as possible, and if not feasible, genetic research projects may pioneer this path. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2 regulatory subunit B56δ related neurodevelopmental disorder (PPP2R5D‐related NDD) is largely caused by de novo heterozygous missense PPP2R5D variants. We report medical characteristics, longitudinal adaptive functioning, and in‐person neurological, motor, cognitive, and electroencephalogram (EEG) activity for PPP2R5D‐related NDD. Forty‐two individuals (median age 6 years, range = 0.8–25.3) with pathogenic/likely pathogenic PPP2R5D variants were assessed, and almost all variants were missense (97.6%) and de novo (85.7%). Common clinical symptoms were developmental delay, hypotonia, macrocephaly, seizures, autism, behavioral challenges, and sleep problems. The mean Gross motor functional measure‐66 was 60.2 ± 17.3% and the mean Revised upper limb module score was 25.9 ± 8.8. The Vineland‐3 adaptive behavior composite score (VABS‐3 ABC) at baseline was low (M = 61.7 ± 16.8). VABS‐3 growth scale value scores increased from baseline in all subdomains (range = 0.6–5.9) after a mean follow‐up of 1.3 ± 0.3 years. EEG beta and gamma power were negatively correlated with VABS‐3 score; p < 0.05. Individuals had a mean Quality‐of‐life inventory‐disability score of 74.7 ± 11.4. Twenty caregivers (80%) had a risk of burnout based on the Caregiver burden inventory. Overall, the most common clinical manifestations of PPP2R5D‐related NDD were impaired cognitive, adaptive function, and motor skills; and EEG activity was associated with adaptive functioning. This clinical characterization describes the natural history in preparation for clinical trials. [ABSTRACT FROM AUTHOR]

Aim: To expand the phenotypic spectrum of ADGRL1 and explore the correlation between epilepsy and the ADGRL1 genotype. Method: We performed whole‐exome sequencing in a cohort of 115 families (including 195 males and 150 females) with familial febrile seizure or epilepsy with unexplained aetiology. The damaging effects of variants was predicted using protein modelling and multiple in silico tools. All reported patients with ADGRL1 pathogenic variants were analysed. Results: One new ADGRL1 variant (p.Pro753Leu) was identified in one family with genetic epilepsy with febrile seizures. Further analysis of 12 ADGRL1 variants in 16 patients revealed that six patients had epilepsy. Epilepsy types ranged from early‐onset epileptic encephalopathy to genetic epilepsy with febrile seizures plus (GEFS+). All four variants associated with epilepsy were located in the non‐helix or sheet region of ADGRL1. Three of the four epilepsy‐associated variants were missense variants. Thus, all three variants located in the G‐protein‐coupled receptor autoproteolytic‐inducing domain exhibited epilepsy. Interpretation: We found one new missense variant of ADGRL1 in one family with GEFS+. ADGRL1 may be a potential candidate or susceptibility gene for epilepsy. ADGRL1‐associated epilepsy ranged from benign GEFS+ to severe epileptic encephalopathy; the genotypes and variant locations may help explain the phenotypic heterogeneity of patients with the ADGRL1 variant. [ABSTRACT FROM AUTHOR]

Objective: De novo variants in cullin‐3 ubiquitin ligase (CUL3) have been strongly associated with neurodevelopmental disorders (NDDs), but no large case series have been reported so far. Here, we aimed to collect sporadic cases carrying rare variants in CUL3, describe the genotype–phenotype correlation, and investigate the underlying pathogenic mechanism. Methods: Genetic data and detailed clinical records were collected via multicenter collaboration. Dysmorphic facial features were analyzed using GestaltMatcher. Variant effects on CUL3 protein stability were assessed using patient‐derived T‐cells. Results: We assembled a cohort of 37 individuals with heterozygous CUL3 variants presenting a syndromic NDD characterized by intellectual disability with or without autistic features. Of these, 35 have loss‐of‐function (LoF) and 2 have missense variants. CUL3 LoF variants in patients may affect protein stability leading to perturbations in protein homeostasis, as evidenced by decreased ubiquitin‐protein conjugates in vitro. Notably, we show that 4E‐BP1 (EIF4EBP1), a prominent substrate of CUL3, fails to be targeted for proteasomal degradation in patient‐derived cells. Interpretation: Our study further refines the clinical and mutational spectrum of CUL3‐associated NDDs, expands the spectrum of cullin RING E3 ligase‐associated neuropsychiatric disorders, and suggests haploinsufficiency via LoF variants is the predominant pathogenic mechanism. ANN NEUROL 2025;97:76–89 [ABSTRACT FROM AUTHOR]

Background and Purpose: Pathogenic gain‐of‐function mutations in Cav1.3 L‐type voltage‐gated Ca2+‐channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain‐of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca2+‐channel blocker isradipine, a potential therapeutic for affected patients. Experimental Approach: Wildtype and mutant channels were expressed in tsA‐201 cells and their gating analysed using whole‐cell and single‐channel patch‐clamp recordings. The voltage‐dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations. Key Results: Both mutations were confirmed pathogenic due to characteristic shifts of voltage‐dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30‐ to 40‐fold). The IC50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated. Conclusions and Implications: Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage‐dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Cav1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels. [ABSTRACT FROM AUTHOR]

SCN2A gene-related early-infantile developmental and epileptic encephalopathy (EI-DEE) is a rare and severe disorder that manifests in early infancy. SCN2A mutations affecting the fast inactivation gating mechanism can result in altered voltage dependence and incomplete inactivation of the encoded neuronal Nav1.2 channel and lead to abnormal neuronal excitability. In this study, we evaluated clinical data of seven missense Nav1.2 variants associated with DEE and performed molecular dynamics simulations, patch-clamp electrophysiology and dynamic clamp real-time neuronal modelling to elucidate the molecular and neuron-scale phenotypic consequences of the mutations. The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q149K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics that were similar to those of N1662D, whereas the M1501V, M1501T, F1651C, P1658S and A1659V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. Our results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A -related EI-DEE. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a distinct cellular mechanism in the pathogenesis of SCN2A -related DEE. [ABSTRACT FROM AUTHOR]

Each human genome has approximately 5 million DNA variants. Even for complete loss-of-function variants causing inherited, monogenic diseases, current understanding based on gene-specific molecular function does not adequately predict variability observed between people with identical mutations or fluctuating disease trajectories. We present a parallel paradigm for loss-of-function variants based on broader consequences to the cell when aberrant polypeptide chains of amino acids are translated from mutant RNA to generate mutated proteins. Missense variants that modify primary amino acid sequence, and nonsense/frameshift variants that generate premature termination codons (PTCs), are placed in context alongside emergent themes of chaperone binding, protein quality control capacity, and cellular adaptation to stress. Relatively stable proteostasis burdens are contrasted with rapid changes after induction of gene expression, or stress responses that suppress nonsense mediated decay (NMD) leading to higher PTC transcript levels where mutant proteins can augment cellular stress. For known disease-causal mutations, an adjunctive variant categorization system enhances clinical predictive power and precision therapeutic opportunities. Additionally, with typically more than 100 nonsense and frameshift variants, and ∼10,000 missense variants per human DNA, the paradigm focuses attention on all protein-coding DNA variants, and their potential contributions to multimorbid states beyond classically designated inherited diseases. Experimental testing in clinically relevant systems is encouraged to augment current atlases of protein expression at single-cell resolution, and high-throughput experimental data and deep-learning models that predict which amino acid substitutions generate enhanced degradative burdens. Incorporating additional dimensions such as pan-proteome competition for chaperones, and age-related loss of proteostasis capacity, should further accelerate health impacts. A parallel paradigm for loss-of-function DNA variants is presented, based on cellular consequences when aberrant polypeptide chains of amino acids are translated. Missense variants and nonsense/frameshift variants that generate premature termination codons (PTCs) are considered alongside emergent themes of chaperone binding, protein quality control capacity, and cellular adaptation to stress. [ABSTRACT FROM AUTHOR]

Developmental language disorder (DLD) is a neurodevelopmental disorder involving impaired language abilities. Its genetic etiology is heterogeneous, involving rare variations in multiple susceptibility loci. However, family-based studies on gene mutations are scarce. We performed whole-exome sequencing (WES) of a first-time-described Tunisian-family with DLD. Analyses of segregation patterns with stringent filtering of the exome data identified disease-causing compound heterozygous variants. In the MRNIP gene, two variants were detected including a synonymous low-frequency variant c.345G > C and a nonsense rare variant c.112G > A predicted pathogenic. In the ABHD14A gene, four variants were identified including a rare missense variant c.689T > G and three splice-site variants c.70-8C > T, c.282-25A > T and c.282-10G > C with low-frequency MAF < 5%. Complementary analyses showed that these variants are predicted pathogenic and the missense variant Leu230Arg significantly affects the stability and structure modelling of the ABHD14A protein. Biological functions and interconnections analyses predicted the potential roles of ABHD14A and MRNIP in neuronal development pathways. These results suggest ABHD14A and MRNIP, as putative candidate genes for DLD susceptibility. Our findings reveal the involvement of novel candidate genes in the genetic etiology of DLD and explore the potential future utility of WES in the diagnosis of such complex disorders. [ABSTRACT FROM AUTHOR]

Missense mutations in the Leucine-rich repeat kinase 2 (LRRK2) protein are strongly linked to the pathogenicity of both sporadic and familial Parkinson's Disease (PD). Considering their prevalence and functional consequence, mutations causing kinase dysfunction have experienced the greatest exploration. However, variants of LRRK2 with unaltered kinase activity have also been found to cause PD. This study focuses on one such case: the novel R1501W mutation in the Roc-COR (GTPase) domain of LRRK2. Given its novelty, there are scarcely any studies inspecting this variant. Thus, we employed molecular dynamics simulations of 200 ns alongside structural analyses such as root-mean-square (RMS) fluctuation, RMS deviation, radius of gyration, solvent-accessible surface-area, principal component analysis, and free energy landscape to characterise the structural changes occurring in the R1501W mutant in comparison to the wild-type. Our results reveal the introduction of a closed hydrophobic nest in the mutated region, as well as an increase in flexibility, instability, and compactness of the tertiary structure. We speculate that these alterations contribute to a kinase-independent mechanism for pathogenesis. Overall, our investigation showcases the structural dynamics of R1501W mutant LRRK2, providing a backbone for the design of future studies to further inspect the pathogenic pathway employed by this novel variant. [ABSTRACT FROM AUTHOR]

Hepatic fibrinogen storage disease is an uncommon autosomal dominant hereditary illness marked by hypofibrinogenemia and the accumulation of variant fibrinogen in the hepatic endoplasmic reticulum. We present an asymptomatic 15-month-old male with elevated liver enzymes. Test results indicate hypofibrinogenemia. The liver biopsy revealed circular eosinophilic inclusion bodies within the hepatocyte cytoplasm. After diastase pretreatment, the inclusion bodies did not stain using the periodic acid – Schiff procedure. Ultrastructural examination revealed the characteristic fibrinogen storage curvilinear inclusions. Sequence analysis using the Blueprint Genetics (BpG) FLEX Bleeding Disorder/Coagulopathy Panel identified a heterozygous missense variant FGG c.1075 G>C, p. (Gly359Arg). Thus, the patient was diagnosed with hepatic fibrinogen storage disease. Our findings suggest that in patients with asymptomatic elevated liver enzymes presenting with unanticipated hypofibrinogenemia, hepatic fibrinogen storage disorder must be included in the differential diagnosis. Furthermore, our results underscore the significance of molecular diagnosis in patients diagnosed with cryptogenic liver disease. [ABSTRACT FROM AUTHOR]

Background: Frontotemporal lobar degeneration (FTLD) is one of the leading causes of early onset dementia. Pathogenic variants in GRN have been reported to cause 5–25% of familial and 5% of sporadic FTLD. Here, we present two novel, likely pathogenic variants in GRN. Methods: Four patients from four different families underwent whole exome sequencing (WES) with additional copy-number variance (CNV) analysis in a clinical setting. TMEM106B rs1990622 and rs3173615 SNPs and 3’UTR insertion were tested in one presymptomatic carrier. In three probands and one presymptomatic carrier, plasma progranulin (PGRN) levels were measured using a specific ELISA kit. In two probands, neuropathological diagnosis was established using current neuropathological criteria. Results: Through CNV analysis on WES data, we identified a partial deletion, NM_002087.2 (GRN):c.1179 + 104_1536delinsCTGA, p.(?), in three patients with primary progressive aphasia and/or corticobasal syndrome. Haplotype analysis revealed a shared haplotype block, suggesting that the deletion represents a founder mutation. Additionally, we found a novel, missense variant, NM_002087.2 (GRN):c.23 T > A, p.(Val8Glu), in one proband with a negative family history. The proband’s unaffected parent—in their 80 s—carried the same variant, yet was homozygous for the TMEM106B risk haplotype. The pathogenicity of both GRN variants was supported by typical neuropathological features and reduced PGRN levels. Conclusion: We recommend a thorough genetic screening, including CNV analysis, for both familial and apparent sporadic FTLD patients. Furthermore, the presymptomatic carrier homozygous for the TMEM106B risk haplotype exemplifies the presence of other protective factors that modify disease onset and urges caution in genetic counselling based on the TMEM106B haplotype. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusion Variants in the GABRA2 gene, which encodes the α2 subunit of the γ‐aminobutyric acid A receptor, have been linked to a rare form of developmental and epileptic encephalopathy (DEE) referred to as DEE78. Only eight patients have been reported globally. This study presents the clinical presentation and genetic analysis of a Chinese family with a child diagnosed with DEE78, due to a novel GABRA2 variant.Genetic diagnosis was performed using trio‐whole exome sequencing, followed by bioinformatics predictions of pathogenicity. Structural modeling assessed the potential impact of the variant. A mutant plasmid was constructed and transfected into 293 T cells. Western blotting (WB) was used to evaluate mutant protein expression, while co‐immunoprecipitation (Co‐IP) analyzed interactions with GABRB3 and GABRG2 proteins. Immunofluorescence (IF) assessed the subcellular localization of the mutant protein.The 6‐year‐old male proband presented with seizures starting at age two, along with global developmental delay and hypotonia. Genetic testing revealed a heterozygous de novo variant in GABRA2 gene (NM_000807: c.923C>T, p.Ala308Val). Structural modeling suggested that this variant is located within the extracellular domain, which may disrupt hydrogen bonding interactions with GABRB3 and GABRG2. WB and Co‐IP showed reduced protein expression and impaired interactions, potentially destabilizing the pentamer receptor complex. If analysis revealed that the variant did not affect subcellular localization.This study identified a novel likely pathogenic GABRA2 extracellular domain variant in a Chinese family causing the DEE phenotype. The results expand the genotypic and phenotypic spectrum of GABRA2‐related DEE. [ABSTRACT FROM AUTHOR]

Objective: A biallelic missense mutation in mitofusin 2 (MFN2) causes multiple symmetric lipomatosis and partial lipodystrophy, implicating disruption of mitochondrial fusion or interaction with other organelles in adipocyte differentiation, growth and/or survival. In this study, we aimed to document the impact of loss of mitofusin 1 (Mfn1) or 2 (Mfn2) on adipogenesis in cultured cells. Methods: We characterised adipocyte differentiation of wildtype (WT), Mfn1-/- and Mfn2-/- mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes in which Mfn1 or 2 levels were reduced using siRNA. Results: Mfn1-/- MEFs displayed striking fragmentation of the mitochondrial network, with surprisingly enhanced propensity to differentiate into adipocytes, as assessed by lipid accumulation, expression of adipocyte markers (Plin1, Fabp4, Glut4, Adipoq), and insulin-stimulated glucose uptake. RNA sequencing revealed a corresponding pro-adipogenic transcriptional profile including Pparg upregulation. Mfn2-/- MEFs also had a disrupted mitochondrial morphology, but in contrast to Mfn1-/- MEFs they showed reduced expression of adipocyte markers. Mfn1 and Mfn2 siRNA mediated knockdown studies in 3T3-L1 adipocytes generally replicated these findings. Conclusions: Loss of Mfn1 but not Mfn2 in cultured pre-adipocyte models is pro-adipogenic. This suggests distinct, non-redundant roles for the two mitofusin orthologues in adipocyte differentiation. [ABSTRACT FROM AUTHOR]

CHAMP1 (chromosome alignment-maintaining phosphoprotein 1) plays a role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The CHAMP1 gene is one of the genes mutated in individuals with intellectual disability. The majority of the mutations are premature termination codon (PTC) mutations, while missense mutations have also been reported. How these mutations affect the functions of CHAMP1 has not been clarified yet. Here we investigated the effects of the CHAMP1 mutations on HR. In Epstein-Barr virus-induced lymphoblastoid cells and fibroblasts derived from individuals with CHAMP1 PTC mutations, truncated CHAMP1 proteins of the expected sizes were detected. When DSBs were induced in fibroblasts with PTC mutations, a defect in HR was detected. U2OS cells expressing the CHAMP1 mutants did not show an HR defect in the presence of endogenous wild-type (WT) CHAMP1, whereas they were unable to restore HR activity when CHAMP1 WT was depleted, suggesting that the PTC mutations are loss-of-function mutations. On the other hand, the CHAMP1 mutants with missense mutations restored HR activity when CHAMP1 WT was depleted. In DLD-1 cells, heterozygous depletion of CHAMP1 resulted in an HR defect, indicating haploinsufficiency. These results suggest that CHAMP1 PTC mutations cause an HR defect through a haploinsufficient mechanism, while CHAMP1 missense mutations do not affect the HR function of CHAMP1. [ABSTRACT FROM AUTHOR]

ABSTRACT Hereditary spastic paraplegias (HSP) are a diverse group of neurodegenerative diseases characterized by lower limb spasticity and weakness. To date, over 80 genes have been associated with HSP, but many families remain without a molecular diagnosis.In this study, linkage analysis and whole‐exome sequencing (WES) were performed to identify the causal gene in a HSP family with autosomal recessive inheritance. Multipoint linkage analysis revealed a maximum significant multipoint LOD score of 4.6 on chromosome 4. WES analysis focused on this region led to the identification of a homozygous missense variant in AIMP1 (c.223G>A). Minigene assays showed that the presumed missense variant in AIMP1 caused loss of the exon 3 donor splice site. Ultimately, this led to the use of an alternative splice site within the intron and the insertion of a premature stop codon.The identification of a novel AIMP1 causal variant contributes to the growing list of HSP genes. Furthermore, it shows that, considering also previous reported cases, disruption of AIMP1 causes a spectrum of disorders ranging from intellectual disability to more complex neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

ABSTRACT Phosphoribosylaminoimidazole carboxylase (PAICS) deficiency, caused by biallelic variants in PAICS gene, is an inborn error of de novo purine synthesis. Only two patients from a consanguineous family have been reported, with multiple congenital malformations, resulting in early neonatal death. Molecular analysis identified a homozygous p.(Lys53Arg) missense variant. We report the third case of PAICS deficiency in a 7 years old boy, presenting with polymalformative syndrome, but normal neurodevelopment. We report malformations not previously described in PAICS deficiency, notably congenital cardiopathy, and support the consistency of skeletal and oesophageal defects. Genome Sequencing identified the homozygous pathogenic variant p.(Lys53Arg), suggesting a recurrent variant in PAICS. A probable recurrence of PAICS deficiency occurred in a sibling, with a similar polymalformative syndrome antenatally diagnosed, but could not be confirmed molecularly. We further delineate the phenotype of PAICS deficiency and provide new insights concerning prognosis, notably in terms of neurodevelopment. [ABSTRACT FROM AUTHOR]

ABSTRACT Objectives Design, Patients and Measurements Results Conclusion Pheochromocytomas and paragangliomas (PPGLs) are manifestations of von Hippel‐Lindau (VHL) disease. This study aims to describe the clinical features of PPGLs in VHL patients and the distinctions between VHL disease‐related PPGLs and sporadic PPGLs.The study included all patients with VHL disease and PPGLs treated in a single centre from 2007 to 2023. A total of 145 patients were included in the sporadic PPGLs group. Their clinical data were retrospectively reviewed. Genetic testing for VHL mutation was conducted using Sanger sequencing. Statistical analysis was performed using SPSS 22.Fifty‐nine (65.6%) of the 90 VHL disease patients had PPGLs (male: female, 38:21; age at diagnosis, 25.0 ± 13.3 years). 42 (71.2%) patients had lesions only in the adrenal gland, and 16 (27.1%) patients had lesions both in and out of the adrenal gland. 45 (76.3%) patients had multiple lesions. Eighteen (45.0%) patients developed recurrence after surgery. Fifty‐eight patients with PPGLs underwent genetic testing and had pathogenic or likely pathogenic mutations in the VHL gene. Fifty‐three (91.4%) patients had missense mutations, 34 of which were located in the Elongin‐C binding domain. The hotspot mutation sites were codon 161 and codon 167. Five novel mutations were identified. The clinical characteristics showed no significant differences between groups with different mutation sites. Compared to sporadic PPGLs, VHL disease‐related PPGLs were more frequently located in the adrenal gland (71.2% vs. 49.0%, p < 0.001), had a higher prevalence of multiple lesions (76.3% vs. 11.0%, p < 0.001), and secreted noradrenaline (80.4% vs. 43.2%, p < 0.001). They were also more likely to relapse after surgery (45.0% vs. 15.3%, p < 0.001).VHL disease‐related PPGLs were often multifocal and noradrenergic, and more likely to relapse compared with sporadic PPGLs. No relationships were identified between the mutation sites and the clinical characteristics of PPGLs. [ABSTRACT FROM AUTHOR]

Allosteric conformational change is an important paradigm in the regulation of protein function, which is typically triggered by the binding of small cofactors, metal ions or protein partners. Here, we found those conformational transitions can be effectively monitored by 19F NMR, facilitated by a site-specific 19F incorporation strategy at the protein C-terminus using asparaginyl endopeptidase (AEP). Three case studies show that C-terminal 19F-nuclei can reveal protein dynamics not only adjacent but also distal to C-terminus, including those occurring in a hemoprotein neuroglobin (Ngb), calmodulin (CaM), and a cobalt metalloregulator (CoaR) responding to both cobalt and tetrapyrrole. In Ngb, the heme orientation disorder is affected by missense mutations that perturb backbone rigidity or surface charges close to the heme axial ligands. In CaM, the C-terminal 19F-nuclei is an ideal probe for detecting the binding states of Ca2+, peptides and inhibitors. Furthermore, multiple 19F-moieties were incorporated into the two domains of CoaR, revealing the intrinsically disordered C-terminal metal binding tail might be an allosteric conformational switch to maintain cobalt homeostasis and balance corrinoid biosynthesis. This study demonstrates that the AEP-based 19F-modification strategy can be applied to various targets to study allosteric regulation, especially for those biological processes modulated by the protein C-terminus. The site-specific 19F labeling at protein C-terminus catalyzed by OaAEP1C247A is an efficient approach to utilize 19F NMR, which is compatible with the genetic code expansion technology. Three cases studies show this approach is suitable for probing allosteric conformational transitions in metalloproteins. [ABSTRACT FROM AUTHOR]

Li–Fraumeni syndrome (LFS) is one of the most common hereditary cancer predisposition syndromes in Brazil. The high frequency of the syndrome is due to a founding variant (R337H) in the country. LFS is characterized by a wide variety of malignant phenotypes. Despite the great epidemiological importance of the R337H variant, the frequency of other types of pathogenic variants is like other populations, with the majority of these being missense variants. There is strong evidence that radiotherapy is associated with secondary sarcomas, including angiosarcomas, and this finding is especially true for LFS patients. Angiosarcoma is not described as overrepresented in individuals with LFS, except in patients submitted to radiotherapy. Germline testing in all breast cancer patients under 65 will reveal many germline mutations in TP53 without a family history of cancers associated with the syndrome. We present a case of a previously undescribed pathogenic variant in TP53 (c788del, pAns263llefs ∗82) in a patient with no family history of cancer, with a previous diagnosis of breast carcinoma treated with radiotherapy, who developed angiosarcoma after a few years leading to germline testing. The presence of angiosarcoma in a radiotherapy bed should raise suspicion for LFS. The recent recommendation of testing breast cancer patients under the age of 65, even without any family history, can be a source of discoveries of new mutations and assist in therapeutic decisions. [ABSTRACT FROM AUTHOR]

Doublecortin (DCX) is a microtubule (MT)-associated protein in immature neurons. DCX is essential for early brain development, 1 and DCX mutations account for nearly a quarter of all cases of lissencephaly-spectrum brain malformations 2,3 that arise from a neuronal migration failure through the developing cortex. 4 By analyzing pathogenic DCX missense mutations in non-neuronal cells, we show that disruption of MT binding is central to DCX pathology. In human-induced pluripotent stem cell (hiPSC)-derived cortical i3Neurons, genome edited to express DCX-mEmerald from the endogenous locus, DCX-MT interactions polarize very early during neuron morphogenesis. DCX interacts with MTs through two conserved DCX domains 5,6 that bind between protofilaments and adjacent tubulin dimers, 7 a site that changes conformation during guanosine triphosphate (GTP) hydrolysis. 8 Consequently and consistent with our previous results, 5 DCX specifically binds straight growth cone MTs and is excluded from the GTP/guanosine diphosphate (GDP)-inorganic phosphate (Pi) cap recognized by end-binding proteins (EBs). Comparing MT-bound DCX fluorescence to mEmerald-tagged nanocage standards, we measure approximately one hundred DCX molecules per micrometer growth cone MT. DCX is required for i3Neuron growth cone advance in soft microenvironments that mimic the viscoelasticity of brain tissue, and using high-resolution traction force microscopy, we find that growth cones produce comparatively small and transient traction forces. Given our finding that DCX stabilizes MTs in the growth cone periphery by inhibiting MT depolymerization, we propose that DCX contributes to growth cone biomechanics and reinforces the growth cone cytoskeleton to counteract actomyosin-generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated traction may be insufficient for productive growth cone advance. [Display omitted] • DCX binds straight microtubules in human cortical neuron growth cones • DCX stabilizes growth cone microtubules by inhibiting depolymerization • Growth cone-generated traction stresses are small and transient • DCX-stabilized microtubules counter contractility to promote growth cone advance Mutations in doublecortin (DCX), a microtubule-associated protein in immature neurons, can cause brain malformations. Dema et al. show that DCX binds and stabilizes straight growth cone microtubules in hiPSC-derived cortical neurons and propose that these microtubules mechanically support growth cone advance in physiologically soft surroundings. [ABSTRACT FROM AUTHOR]