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Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease causing. This creates a new bottleneck: determining the functional impact of each variant—typically a painstaking, customized process undertaken one or a few genes and variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,448 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of uncertain significance. Our publicly available resource extends our understanding of coding variation in human diseases. [Display omitted] • Screen for missense variant mislocalization for 3,448 variants across 1,269 genes • 16% of pathogenic or likely pathogenic variants are mislocalized • Mislocalization is mainly caused by disruption of protein stability • Distinct localization patterns are associated with pleiotropy and disease severity Pathogenic missense variation can affect protein function in many ways, but the role of protein mislocalization has not been systematically assessed. By characterizing the localization of 3,400 missense variants of over 1,000 genes, we discovered that mislocalization is an unexpectedly common molecular phenotype of coding variation. [ABSTRACT FROM AUTHOR]

Background and Purpose: The ATP‐dependent biliary efflux transporter ABCC2, also known as multidrug resistance protein 2 (MRP2), is essential for the cellular disposition and detoxification of various xenobiotics including drugs as well as endogenous metabolites. Common functionally relevant ABCC2 genetic variants significantly alter drug responses and contribute to side effects. The aim of this study was to determine functional consequences of rare variants identified in subjects with European ancestry using in silico tools and in vitro analyses. Experimental Approach: Targeted next‐generation sequencing of the ABCC2 gene was used to identify novel variants in European subjects (n = 143). Twenty‐six in silico tools were used to predict functional consequences. For biological validation, transport assays were carried out with membrane vesicles prepared from cell lines overexpressing the newly identified ABCC2 variants and estradiol β‐glucuronide and carboxydichlorofluorescein as the substrates. Key Results: Three novel rare ABCC2 missense variants were identified (W227R, K402T, V489F). Twenty‐five in silico tools predicted W227R as damaging and one as potentially damaging. Prediction of functional consequences was not possible for K402T and V489F and for the common linked variants V1188E/C1515Y. Characterisation in vitro showed increased function of W227R, V489F and V1188E/C1515Y for both substrates, whereas K402T function was only increased for carboxydichlorofluorescein. Conclusion and Implications: In silico tools were unable to accurately predict the substrate‐dependent increase in function of ABCC2 missense variants. In vitro biological studies are required to accurately determine functional activity to avoid misleading consequences for drug therapy. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by gradual cognitive decline. Early-onset Alzheimer's disease (EOAD) is defined as AD occurring before age 65. The main pathogenic gene variants associated with EOAD include PSEN1 , PSEN2 , and APP. IDE gene has been identified as a risk factor in the pathogenesis of AD. In this study, we report a 33-year-old male with mutations in the PSEN2 gene (c.640G > T, p.V214L) and IDE gene (c.782G > A, p.R261Q). PSEN2 V214L has been reported in five previous cases, and no reported cases have carried IDE R261Q. He had progressive memory decline, his sister carried the same gene mutations but had no clinical manifestations. Neuroimaging revealed mild cortical atrophy. The concentration of Aβ42 in cerebrospinal fluid (CSF) was obviously decreased. In silico predictive models suggested that these mutations are damaging. Our findings indicate that mutations in the PSEN2 and IDE genes may disrupt the normal functioning of their respective proteins, contributing to the pathogenesis of AD. [ABSTRACT FROM AUTHOR]

Background: Interferon-induced helicase C domain-containing protein 1 (IFIH1) is one of the main pattern recognition receptors that sense viral RNA and activate host cells to mount an effective antiviral immunity. Methods: A case–control study (90 patients with mild/moderate COVID-19 and 90 matched controls) was performed to explore the association of two variants of the IFIH1 gene with COVID-19 risk using the tetra-primer amplification refractory mutation system-polymerase-chain-reaction method. The first is a missense variant, rs1990760 C/T, and the second is an intergenic variant, rs2111485 A/G. In addition, serum IFIH1 levels were assessed using an ELISA kit. Results: Mutant alleles (T and G, respectively) and corresponding homozygous genotypes (TT and GG, respectively) of both variants were significantly associated with increased risk of COVID-19. IFIH1 levels were significantly higher in patients compared to controls and were favorably affected by the rs1990760 and rs2111485 mutant-type genotypes. Conclusion: IFIH1 protein showed up-regulated levels in the serum of patients with mild/moderate COVID-19. In addition, the IFIH1 gene variants rs1990760 C/T and rs2111485 A/G were associated with susceptibility to COVID-19, and the study suggests that their mutant-type genotypes are not only associated with increased risk of COVID-19 but also contributed to higher serum IFIH1 levels. [ABSTRACT FROM AUTHOR]

Background: The PKD2, encoding polycystin-2 (PC2) protein, is second major genetic determinant of autosomal dominant polycystic kidney disease (ADPKD) after PKD1. However, the structural and functional consequences of genetic variants in PKD2 remain poorly understood. Given the complexity and heterogeneous nature of ADPKD, understanding its pathogenesis at cellular and molecular levels is vital for deciphering genotype–phenotype correlations and disease severity, thus informing patient-centered treatments. We analyzed missense variants of PKD2 to assess their impact on RNA structure using computational tools and explored associated protein structure dynamics through MD simulation. Results: Our findings reveal distinct structural alterations and dynamic behaviors associated with specific missense variants. The variants such as c.1789C > A (p.L597M), c.1109G > A (p.S370N), c.1849C > A (p.L617I), and c.646 T > C (p.Y216H) induced major changes not only in RNA structure and accessibility profile but also in protein structure dynamics. In contrast, variants such as c.915C > A (p.N305K), c.1354A > G (p.I452V), and c.568G > A (p.A190T) resulted in minor alterations in RNA structure but exhibited noticeable effects on certain parameters of protein structure dynamics. Conclusion: This study suggests the multifaceted impact of these missense variants on both RNA and protein levels. It lays the groundwork in identifying high-impact variants in terms of pathogenicity and prioritizing these for further implications in understanding disease heterogeneity and eventually contributing to the development of targeted therapeutic interventions for ADPKD. Study Highlights: PKD2 missense variants analyzed for impact on RNA and protein Structures Alterations in RNA Structures and protein dynamics observed Computational integration of analyses aids in prioritizing variants for further study Provide insights into disease heterogeneity and potential therapeutic targets [ABSTRACT FROM AUTHOR]

Autism is a developmental disability. Research demonstrated that children with autism benefit from early diagnosis and early intervention. Genetic factors are considered major contributors to the development of autism. Machine learning (ML), including deep learning (DL), has been evaluated in phenotype prediction, but this method has been limited in its application to autism. We developed a DL model, the Separate Translated Autism Research Neural Network (STAR-NN) model to predict autism status. The model was trained and tested using whole exome sequencing data from 43,203 individuals (16,809 individuals with autism and 26,394 non-autistic controls). Polygenic scores from common variants and the aggregated count of rare variants on genes were used as input. In STAR-NN, protein truncating variants, possibly damaging missense variants and mild effect missense variants on the same gene were separated at the input level and merged to one gene node. In this way, rare variants with different level of pathogenic effects were treated separately. We further validated the performance of STAR-NN using an independent dataset, including 13,827 individuals with autism and 14,052 non-autistic controls. STAR-NN achieved a modest ROC-AUC of 0.7319 on the testing dataset and 0.7302 on the independent dataset. STAR-NN outperformed other traditional ML models. Gene Ontology analysis on the selected gene features showed an enrichment for potentially informative pathways including potassium ion transport. Author summary: Autism is a developmental disability. Genetic factors are considered as major contributors to the development of autism. Here, we present a deep neural network model, Separate Translated Autism Research Neural Network (STAR-NN), to predict autism status using WES data. STAR-NN showed a modest performance and was validated using an independent dataset. However, ML based models may have advantages in autism status prediction using genomic data and should be further studied. [ABSTRACT FROM AUTHOR]

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe hepatic disorder characterized by cholestasis. Elucidating the genotype-phenotype correlations and expanding the mutational spectrum of the ABCB4 gene are crucial for enhancing diagnostic accuracy and therapeutic strategies.Clinical and genetic data from 2 original PFIC3 patients from our institution, along with 118 additional cases identified through a comprehensive literature review, were integrated for a comprehensive analysis. The study included statistical analysis of clinical information, genetic analysis, multi-species sequence alignment, protein structure modeling, and pathogenicity assessment. Machine learning techniques were applied to identify genotype-phenotype relationships. We identified three novel ABCB4 mutations: two missense mutations (c.904G > T and c.2493G > C) and one splicing mutation (c.1230 + 1G > A). Homozygous mutations were associated with significantly earlier disease onset compared to compound heterozygous mutations (p < 0.0001). Missense mutations were predominant (76.9%), with Exon 7 being the most frequently affected region. A random forest model indicated that Exon 10 had the highest feature importance score (9.9%). Liver transplantation remains the most effective treatment modality for PFIC3. This investigation broadens the known mutation spectrum of the ABCB4 gene and identifies key variant sites associated with clinical manifestations. These insights lay a foundation for early diagnosis, optimal treatment selection, and further research into PFIC3. [ABSTRACT FROM AUTHOR]

Aims Methods Results Conclusions Neonatal diabetes is a monogenic condition which can be the presenting feature of complex syndromes. The aim of this study was to identify novel genetic causes of neonatal diabetes with neurological features including developmental delay and epilepsy.We performed genome sequencing in 27 individuals with neonatal diabetes plus epilepsy and/or developmental delay of unknown genetic cause. Replication studies were performed in 123 individuals with diabetes diagnosed aged ≤1 year without a known genetic cause using targeted next‐generation sequencing.Three individuals, all diagnosed with diabetes in the first week of life, shared a rare homozygous missense variant, p.(Arg327Gln), in TARS2. Replication studies identified the same homozygous variant in a fourth individual diagnosed with diabetes at 1 year. One proband had epilepsy, one had development delay and two had both.Biallelic TARS2 variants cause a mitochondrial encephalopathy (COXPD‐21) characterised by severe hypotonia, epilepsy and developmental delay. Diabetes is not a known feature of COXPD‐21. Current evidence suggests that the p.(Arg327Gln) variant disrupts TARS2's regulation of the mTORC1 pathway which is essential for β‐cells.Our findings establish the homozygous p.(Arg327Gln) TARS2 variant as a novel cause of syndromic neonatal diabetes and uncover a role for TARS2 in pancreatic β‐cells. [ABSTRACT FROM AUTHOR]

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies. Dysferlin is essential for plasma membrane repair, and mutations in this protein cause various muscle diseases. Here the authors report the structure of dysferlin, offering insights into its molecular mechanisms and how mutations impair its function. [ABSTRACT FROM AUTHOR]

Background and objectives: Progressive myoclonic epilepsy (PME) is a group of neurological disorders characterized by recurrent myoclonic seizures with progressive neurological deterioration. We investigated the genetics of three unrelated patients with PME from Mali, a country in sub-Saharan Africa highly underrepresented in genetic and genomic research. Methods: Participants were carefully examined and phenotyped. DNA was obtained for genetic analysis including whole exome sequencing (WES). In silico prediction tools and ACMG criteria were used to assess the deleteriousness of putative candidate variants. Results: Pedigree analysis suggests autosomal recessive inheritance patterns for one family and sporadic forms of PME for the two other cases. WES identified novel homozygous missense variants in all the three patients, one each for NHLRC1 , EPM2A , and NEU1. The sequence variants segregated with PME in each family and in silico studies including protein 3D structures, CADD scores and ACMG criteria suggested that they were damaging. Discussion: PME is a group of clinically heterogeneous neurological disorders. Most reported cases in the literature are from European background with only a few cases described in North Africa. We report here novel pathogenic variants in three different genes causing PME phenotypes in three unrelated Malian patients, suggesting that genetic studies of underrepresented populations may expand the genetic epidemiology of PME. These findings also emphasize the need for inclusive genetic research to ensure a more targeted diagnostic and therapeutic approaches for diverse patient populations. [ABSTRACT FROM AUTHOR]

Background: ATP7B (ATPase copper transporting beta gene) is constituted of 21 exons, and codes for a 1465 amino acid. The protein of ATP7B plays an key role of copper metabolism. Many previous reports indicated that mutations in ATP7B are well known to cause defective copper transporting copper-transporting ATPase 2 protein leading to the accumulation of copper, resulting the Wilson disease. Objectives: The meta-analysis was performed to comprehensive gain a thorough grasp of the spectrum of genetic variations. Design: A meta-analysis was conducted according to the guiding of PRISMA. aiming to assess the diversity and frequency of mutations in the ATP7B gene, with an emphasis on mutations located within specific exons. Data sources and methods: The dataset of detected mutations within their positions, types as well as nomenclature, were recorded from previous studies (spanning the year from 2013 to 2023). The analysis focused on exon-specific variations and their prevalence across different populations. Results: A total of 40 studies were enrolled into current data analysis. Our comprehensive study revealed a variety of mutations, most notably over 50% of single nucleotide changes described, distributed over the 21 exons of the gene. Focusing on the exon 8, itisplayed the most diversity of mutations, with 18 studies identifying 53 unique variants, the majority of which were missense mutations (81.13%). Additionally, the variations c.2333G>A/T (p.R778Q/L), c.2305A>G (p.M769V), c.2336G>A (p.W779*), and c.2304dupC (p.M769HfsX26) are reported in many populations. The weighted mean of variants' proportion was used to calculate the pooled estimate of these percentages, which were 14.19% for c.2333G>A/T (p.R778Q/L), 2.70% for c.2305A>G (p.M769V), 1.42% for c.2336G>A (p.W779*), and 2.33% for c.2304dupC (p.M769HfsX26). Conclusion: This design demonstrate to identify the spectrum of ATP7B gene's mutations, especially exon 8, offering important insights into the prevalence and significance of exon 8 mutations. Understanding the mutation in the ATP7B gene offers insights into the mechanisms behind WD and guides strategies for diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Alpha-tubulin 4A encoding gene (TUBA4A) has been associated with familial amyotrophic lateral sclerosis and frontotemporal dementia, based on identification of likely pathogenic variants in patients from distinct amyotrophic lateral sclerosis and frontotemporal dementia cohorts. By screening a multicentric French cohort of 448 unrelated probands presenting with cerebellar ataxia, we identified ultra-rare TUBA4A missense variants, all being absent from public databases and predicted pathogenic by multiple in silico tools. In addition, gene burden analyses in the 100 000 Genomes project (100KGP) showed enrichment of TUBA4A rare variants in the inherited ataxia group compared to controls [odds ratio: 57.0847 (10.2−576.7); P = 4.02 ×10−7]. Taken together, we report 12 patients presenting with spasticity and/or cerebellar ataxia and harbouring a predicted pathogenic TUBA4A missense mutation, including five confirmed de novo cases and a mutation previously reported in a large family presenting with spastic ataxia. Cultured fibroblasts from three patients harbouring distinct TUBA4A missense showed significant alterations in microtubule organization and dynamics, providing insight of TUBA4A variants pathogenicity. Our data confirm the identification of a hereditary spastic ataxia disease gene with variable age of onset, expanding the clinical spectrum of TUBA4A associated phenotypes. [ABSTRACT FROM AUTHOR]

Background and Objectives: Missense variants in exon 7 of the ABO gene can lead to the formation of cisAB alleles. These alleles encode glycosyltransferases (GTs) capable of synthesizing both A and B antigens. In this study, we report the discovery of a novel cisAB allele and characterize it at molecular, protein and serological levels. Materials and Methods: Blood and DNA samples from the proband and seven relatives were examined using standard and modified ABO phenotyping, polymerase chain reaction‐restriction fragment length polymorphism and ABO gene sequencing. We assessed the impact of the p.Leu324Ser variant on the protein structure of the mutant GT using bioinformatics tools. Results: Molecular tests revealed a c.971T>C (p.Leu324Ser) variant in the ABO gene in five of the eight individuals. This variant results in a GT that produces more A antigens and fewer B antigens. Bioinformatics analysis suggests that the amino acid substitution (p.Leu324Ser) could potentially affect enzymatic activity and specificity of the GT. Conclusion: We identified a novel cisAB allele resulting from a c.971T>C variant in the ABO gene. This variant led to the expression of an ABweak phenotype. [ABSTRACT FROM AUTHOR]

Bipolar disorder's etiology involves genetics, environmental factors, and gene–environment interactions, underlying its heterogeneous nature and treatment complexity. In 2020, Forstner and colleagues catalogued 378 sequence variants co-segregating with familial bipolar disorder. A notable candidate was an R59Q missense mutation in the PDZ (PSD-95/Dlg1/ZO-1) domain of RGS12. We previously demonstrated that RGS12 loss removes negative regulation on the kappa opioid receptor, disrupting basal ganglia dopamine homeostasis and dampening responses to dopamine-eliciting psychostimulants. Here, we investigated the R59Q variation in the context of potential PDZ domain functional alterations. We first validated a new target for the wildtype RGS12 PDZ domain—the SAPAP3 C-terminus—by molecular docking, surface plasmon resonance (SPR), and co-immunoprecipitation. While initial molecular dynamics (MD) studies predicted negligible effects of the R59Q variation on ligand binding, SPR showed a significant reduction in binding affinity for the three peptide targets tested. AlphaFold2-generated models predicted a modest reduction in protein–peptide interactions, which is consistent with the reduced binding affinity observed by SPR, suggesting that the substituted glutamine side chain may weaken the affinity of RGS12 for its in vivo binding targets, likely through allosteric changes. This difference may adversely affect the CNS signaling related to dynorphin and dopamine in individuals with this R59Q variation, potentially impacting bipolar disorder pathophysiology. [ABSTRACT FROM AUTHOR]

Treacher Collins syndrome (TCS) is a rare congenital craniofacial disorder with variable penetrance and high genetic and phenotypic heterogeneity. It is caused by pathogenic variants in the TCOF1, POLR1D, POLR1C, and POLR1B genes, and its major characteristic features are malar and mandibular hypoplasia, downward slanting of the palpebral fissures, and conductive hearing loss. In this study, five patients (two males and three females, age range from 2 to 29 years) with TCS were tested by Next-Generation Sequencing (NGS)-based sequencing and clinically characterized. Genetic analyses detected two deletions and one insertion in the TCOF1 gene and one missense variant in the POLR1D gene. Two novel mutations, c.1371_1372insT (p.Lys458*) in the TCOF1 gene and c.295 G>C (p.Gly99Arg) in the POLR1D gene, were identified. Moreover, two already known mutations, c.4369_4373del (p.Lys1457Glufs*12) and c.2103_2106del (p.Ser701Argfs*9) in the TCOF1 gene, were detected. The novel TCOF1 c.1371_1372insT mutation was associated with mild craniofacial manifestations and very rare symptoms of TCS, i.e., developmental delay and moderate intellectual disability. Although incomplete penetrance is a known phenomenon in TCS, surprisingly, the majority of our patients inherited the disease-causing variants from an asymptomatic mother. The unique feature of our study is the observation of causative mutation transmission between asymptomatic family members. Our results expanded the clinical and mutational spectrum of TCS and further confirmed the inter- and intra-familial variability of this disorder. [ABSTRACT FROM AUTHOR]

The kiang (Equus kiang) can only be observed in the Qinghai–Tibet Plateau (QTP). The kiang displayed excellent athletic performance in the high‐altitude environment, which attracted wide interest in the investigation of the potential adaptive mechanisms to the extreme environment. Here, we assembled a chromosome‐level genome of the kiang based on Hi‐C sequencing technology. A total of 324.14 Gb clean data were generated, and the chromosome‐level genome with 26 chromosomes (25 + X) and scaffold N50 of 101.77 Mb was obtained for the kiang. The genomic synteny analysis revealed large‐scale chromosomal rearrangement during the evolution process of Equus species. Phylogenetic and divergence analyses revealed that the kiang was the sister branch to the ass and diverged from a common ancestor at approximately 13.5 Mya. The expanded gene families were mainly related to the hypoxia response, metabolism, and immunity. The kiang suffered a significant loss of olfaction‐related genes, which might indicate decreased olfactory sensibility. Positively selected genes (PSGs) detected in the kiang were mainly associated with hypoxia response. Especially, there were two species‐specific missense amino acid mutations in the PSG STAT3 annotated in the hypoxia‐inducible factor 1 signal pathway, which may play an important role in the high‐altitude adaptation of the kiang. Moreover, structure variations in the kiang genome were also identified, which possibly contributed to the high‐altitude adaptation of the kiang. Comparative analysis revealed a lot of species‐specific insertions and deletions in the kiang genome, such as PIK3CB and AKT with 3258 and 189 bp insertions in the intron region, respectively, possibly affecting the expression and regulation of hypoxia‐related downstream pathways. This study provided valuable genomic resources, and our findings help a better understanding of the underlying adaptive strategies to the high‐altitude environment in the kiang. [ABSTRACT FROM AUTHOR]

MEDNIK syndrome is a rare autosomal recessive disease characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma, and caused by variants in the adaptor-related protein complex 1 subunit sigma 1 (AP1S1) gene. This gene encodes the σ1A protein, which is a subunit of the adaptor protein complex 1 (AP-1), a key component of the intracellular protein trafficking machinery. Previous work identified three AP1S1 nonsense, frameshift and splice-site variants in MEDNIK patients predicted to encode truncated σ1A proteins, with consequent AP-1 dysfunction. However, two AP1S1 missense variants (c.269 T > C and c.346G > A) were recently reported in patients who presented with severe enteropathy but no additional symptoms of MEDNIK. This condition was described as a novel non-syndromic form of congenital diarrhea caused specifically by the AP1S1 missense variants. In this study, we report two patients with the same c.269 T > C variant, who, contrary to the previous cases, presented as complete MEDNIK syndrome. These data substantially revise the presentation of disorders associated with AP1S1 gene variants and indicate that all the identified pathogenic AP1S1 variants result in MEDNIK syndrome. We also provide a series of functional analyses that elucidate the impact of the c.269 T > C variant on σ1A function, contributing to a better understanding of the molecular pathogenesis of MEDNIK syndrome. Key messages: A missense AP1S1 c.269 T > C (σ1A L90P) variant causes full MEDNIK syndrome. The σ1A L90P variant is largely unable to assemble into the AP-1 complex. The σ1A L90P variant fails to bind [DE]XXXL[LI] sorting motifs. The σ1A L90P variant results in loss-of-function of the protein. [ABSTRACT FROM AUTHOR]

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/ threonine kinase (CIT) missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CITKI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly. [ABSTRACT FROM AUTHOR]

Background & Objective: Autosomal recessive cerebellar ataxias (ARCA) are rare heterogenous neurodegenerative disorders characterized by degeneration of the cerebellum and spinal cord with an early onset before the age of 20 years. PMPCA (MIM: 613036), is a key enzyme in mitochondrial protein processing which is critical for cell survival and growth. Our objective was to investigate Peptidase, Mitochondrial Processing Subunit Alpha (PMPCA) mutations linked with Spinocerebellar ataxia, autosomal recessive 2 (SCAR2). Method: In the current study, Whole Exome Sequencing (WES) was done followed by Sanger sequencing for the validation of the WES results. Results: WES results identified a novel homozygous variant, NM_015160.2: c.802C>T p.(Arg268Trp) in PMPCA gene. Mutation in this gene leads to progressive cerebellar ataxia with fine motor skills difficulties, intentional tremors, slow slurred speech and learning difficulties in a 12-year-old Saudi patient. WES results were further validated by Sanger sequencing technique. Conclusions: Identified phenotype in our case was similar as previously described for SCAR2 related conditions. To our knowledge, this is the first reported mutation in PMPCA gene leading to SCAR2 in Saudi Arabia. These findings will enrich the scarce literature, further provide a new insight on the role of PMPCA gene-related disorders leading to SCAR2 and expand the disease concept. In addition, this will help to establish a database for the disease and its causative factors will further help in controlling diseases resulting from consanguinity in Saudi population. [ABSTRACT FROM AUTHOR]

The paper presents in silico mutational research on the energetics of the most resolved protein structure of HIV-1 NNRTIs, the 4G1Q HIV-1 reverse transcriptase protein. Twenty nearby residues that are less than 6 Å from the center of the embedded ligand are subjected to in silico alterations. For the current study, 380 unique proteins are generated in silico using a set of 380 surrounding residues that have been altered. Analyses and comparison with the parent protein have been done to determine the impact of mutation on the change in folding-unfolding free energy (G), protein stability, and solvation energy. To evaluate the impact of mutation (i) by and (ii) on a particular amino acid residue, a two-fold investigation is conducted. The findings imply that in 12 designed proteins (G − 3.0), folding-unfolding is significantly favored, resulting in the creation of a highly stable conformation. The 11 designed proteins are unstable because the positive values of G > 0.5 in these proteins point to unfavorable mutations. However, the G value in 171 designed proteins is − 1.0, which suggests that mutations cause designed proteins to adopt a stable conformation. According to the findings, out of the 380 created proteins, 11 had extremely unfavorable, 69 less-favorable, and 270 favorable folding-unfolding changes. [ABSTRACT FROM AUTHOR]

Objective: Developmental and epileptic encephalopathies (DEEs) can result from dominant, gain of function variants of neuronal ion channels. More than 450 de novo missense variants of the sodium channel gene SCN8A have been identified in individuals with DEE. Methods: We studied a mouse model carrying the patient Scn8a variant p.Asn1768Asp. An AAV‐PHP.eB virus carrying an allele‐specific single guide RNA (sgRNA) was administered by intracerebroventricular injection. Cas9 was provided by an inherited transgene. Results: Allele‐specific disruption of the reading frame of the pathogenic transcript generated out‐of‐frame indels in 1/4 to 1/3 of transcripts throughout the brain. This editing efficiency was sufficient to rescue lethality and seizures. Neuronal hyperexcitability was reduced in cells expressing the virus. Interpretation: The data demonstrate efficient allele‐specific editing of a dominant missense variant and support the feasibility of allele‐specific therapy for DEE epilepsy. ANN NEUROL 2024;96:958–969 [ABSTRACT FROM AUTHOR]

Background: KCNQ is a voltage-gated K + channel that controls neuronal excitability and is mutated in epilepsy and autism spectrum disorder (ASD). We focus on the KV7.2 voltage-gated potassium channel gene (KCNQ2), which is known for its association with developmental delay and various seizures (including self-limited benign familial neonatal epilepsy and epileptic encephalopathy). But the pathogenicity of many variants remains unproven, potentially leading to misinterpretation of their functional consequences. Methods: In this study, we studied a patient who visited Nanhua Hospital. Targeted next-generation sequencing and Sanger sequencing were used to identify the pathogenic variants. Meanwhile, computational models, including hydrogen bonding and docking analyses, suggest that variants cause functional impairment. In addition, functional validation was performed in the drosophila to further evaluate the missense variant in the KCNQ2 gene as the cause of this patient. Results: A new missense variant in the KCNQ2 gene was identified: NM_172107.4:c.1007C > A(p.ALa336Glu), which resulted in the change from alanine to glutamate at amino acid position 336 in the KCNQ2 gene. After computational modeling, including hydrogen bond analysis and docking analysis, it is indicated that the variants cause functional impairment. Furthermore, RNAi-mediated KCNQ knockout in flies led to the onset of epileptic behavior, lifespan and climbing capacity were affected, expression of the normal human KCNQ2 rescues the in flies RNAi-mediated KCNQ knockout behavioral abnormalities. Conclusion: Our findings expands the genetic profile of KCNQ2 and enhances the genotype – phenotype link. [ABSTRACT FROM AUTHOR]

Intellectual disability (ID) is a kind of nervous developmental disorder and affects more than 1% of people worldwide. SLC45A1 as a transmembrane protein is implicated in the regulation of glucose homoeostasis. Through trio‐based exome sequencing, the missense mutations of SLC45A1 c.103G>A (p.V35M) and c.1211T>G (p.F404C) were identified in the proband with syndromic ID. The distribution, expression and activity of SLC45A1 wild‐type (WT) and variants were assayed in transfected COS7 cells. In SLC45A1 variants, the hydrogen bonds surrounding the 35th and 404th amino acid were changed, location on the cytomembrane was failed, their activity to transport glucose was also significantly decreased to contrast with SLC45A1‐WT. No difference was observed at the mRNA and protein level. In conclusion, the compound heterozygous variants of SLC45A1 might be the genetic etiology for syndromic ID. These novel mutations probably attenuated its activity to transport glucose by the alteration of tertiary structure and failure of intracellular location. [ABSTRACT FROM AUTHOR]

GTPases of the Rab family are important orchestrators of membrane trafficking, and their dysregulation has been linked to a variety of neuropathologies. In 2017, we established a causal link between RAB11A variants and developmental and epileptic encephalopathy. In this study, we expand the phenotype of RAB11A -associated neurodevelopmental disorder and explore genotype-phenotype correlations. We assessed 16 patients with pathogenic or likely pathogenic RAB11A variants, generally de novo , heterozygous missense variants. One individual had a homozygous nonsense variant, although concomitant with a pathogenic LAMA2 variant, which made their respective contributions to the phenotype difficult to discriminate. We reinforce the finding that certain RAB11A missense variants lead to intellectual disability and developmental delays. Other clinical features might include gait disturbances, hypotonia, magnetic resonance imaging abnormalities, visual anomalies, dysmorphisms, early adrenarche, and obesity. Epilepsy seems to be less common and linked to variants outside the binding sites. Individuals with variants in the binding sites seem to have a more multisystemic, nonepileptic phenotype. Similar to other Rab-related disorders, RAB11A -associated neurodevelopmental disorder can also impact gait, tonus, brain anatomy and physiology, vision, adrenarche, and body weight and structure. Epilepsy seems to affect the minority of patients with variants outside the binding sites. [ABSTRACT FROM AUTHOR]

Background: There are 49 B alleles in the ISBT ABO blood group list. This study will describe a new missense mutation, c.784G>T, in exon 7 of the ABO in a Chinese individual. Methods: A weak B was analyzed by serologic techniques. Exons 6 and 7 were sequenced directly through polymerase chain reaction-based typing (PCR-SBT). Subsequently, the heterozygous mutation sites in exon 7 were determined through cloning and sequencing. The mutated GTB proteins were expressed in HEK293F cells after being subcloned into a pCAG vector with a Strep-tag. The potential impact of the mutations on GTB stability was predicted using mCSM software, while UCSF Chimera X software was utilized for visualization of the mutation. Results: The ABO blood typing of serologic characteristics showed weak B phenotype, and the heterozygous site ABO*B.01 (c.784G>T) in Exon 7 was identified by PCR-SBT analysis after TA cloning, which led to an alteration of Asp to Tyr at residue 262 in B glycosyltransferase. Like the ABO*BW.17 (D262Y), D262N also significantly decreased ABO*B.01 expression and lead to GTB destabilizing. Conclusions: The novel B allele with 784G>T caused an alteration of Asp to Tyr at residue 262 in B glycosyltransferase, affecting the expression of GTB protein and influencing GTB structural instability. [ABSTRACT FROM AUTHOR]

Rodent management involves the use of anticoagulant rodenticides (ARs). This use has resulted in the selection of numerous resistance alleles in the Vkorc1 gene, encoding the target enzyme of ARs. In Africa, although rodents are a major problem as a consequence of their transport and transmission of zoonotic pathogens, and damage to crops, the use of ARs and the spread of resistance alleles are poorly documented. We attempted to address both issues in Chad which is one of the largest countries in Africa. Owing to its location at the crossroads of central and northern Africa, Chad is representative of many African countries. Methods: Using a sampling of nearly 300 rodents composed of invasive and endemic rodents collected in six of Chad's largest cities, exposure to ARs was analyzed by their quantification in the liver; the spread of AR resistance alleles was analyzed by Vkorc1 sequencing. Results: We demonstrate the use of both ARs generations in Chadian cities and report the total sequencing of the Vkorc1 for 44 Mastomys natalensis with detection of two different haplotypes, the sequencing of the Vkorc1 for two other endemic rodent species, M. kollmannspergeri and Arvicanthis niloticus, and finally the detection of three new missense mutations – V29E, V69E and D127V – in R. rattus, potentially associated with resistance to ARs. Discussion: These results should argue for the implementation of a reasoned management of rodent populations in Africa to avoid the spread of ARs resistance alleles. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Context CHEK2 is a cell cycle checkpoint regulator gene with a long-established role as a clinically relevant, moderate risk breast cancer predisposition gene, with greater risk ascribed to truncating variants than missense variants. Objective To assess the rate and pathogenicity of CHEK2 variants amongst individuals with pituitary adenomas (PAs). Methods We assessed 165 individuals with PAs for CHEK2 variants. The study population comprised a primary cohort of 29 individuals who underwent germline and tumor whole-exome sequencing, and a second, independent cohort of 136 individuals who had a targeted next-generation sequencing panel performed on both germline and tumor DNA (n = 52) or germline DNA alone (n = 84). Results We identified rare, coding, nonsynonymous germline CHEK2 variants amongst 3 of 29 (10.3%) patients in our primary cohort, and in 5 of 165 (3.0%) patients overall, with affected patients having a range of PA types (prolactinoma, thyrotropinoma, somatotropinoma, and nonfunctioning PA). No somatic variants were identified. Two variants were definitive null variants (c.1100delC, c.444 + 1G > A), classified as pathogenic. Two variants were missense variants (p.Asn186His, p.Thr476Met), classified as likely pathogenic. Even when considering the null variants only, the rate of CHEK2 variants was higher in our cohort compared to national control data (1.8% vs 0.5%; P =.049). Conclusion This is the first study to suggest a role for the breast cancer predisposition gene, CHEK2 , in pituitary tumorigenesis, with pathogenic/likely pathogenic variants found in 3% of patients with PAs. As PAs are relatively common and typically lack classic autosomal dominant family histories, risk alleles—such as these variants found in CHEK2 —might be a significant contributor to PA risk in the general population. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is thought to be a key component of neurodevelopmental disorders such as autism, intellectual disability, and attention-deficit hyperactivity disorder (ADHD). However, little is known about the molecular mechanisms that protect against mitochondrial dysfunction during neurodevelopment. Here, we address this question through the investigation of rbm-26, the Caenorhabditis elegans ortholog of the RBM27 autism candidate gene, which encodes an RNA-binding protein whose role in neurons is unknown. We report that RBM-26 (RBM26/27) protects against axonal defects by negatively regulating expression of the MALS-1 (MALSU1) mitoribosomal assembly factor. Autism-associated missense variants in RBM-26 cause a sharp decrease in RBM-26 protein expression along with defects in axon overlap and axon degeneration that occurs during larval development. Using a biochemical screen, we identified the mRNA for the MALS-1 mitoribosomal assembly factor as a binding partner for RBM-26. Loss of RBM-26 function causes a dramatic overexpression of mals-1 mRNA and MALS-1 protein. Moreover, genetic analysis indicates that this overexpression of MALS-1 is responsible for the mitochondrial and axon degeneration defects in rbm-26 mutants. These observations reveal a mechanism that regulates expression of a mitoribosomal assembly factor to protect against axon degeneration during neurodevelopment. Mitochondrial dysfunction contributes to neurodevelopmental disorders, such as autism or ADHD. This study shows that an ortholog of an autism-candidate gene protects against axonal defects by negatively regulating the expression of the mitoribosomal assembly factor MALS-1. [ABSTRACT FROM AUTHOR]

Polydactyly was recorded before 100 BCE and attracted widespread interest because of its relationship to limb health and ancestral traits in horses. However, the underlying reasons for the development of polydactyly remain unclear. To search for polydactyly-related genes, we utilize a paternal half-sib family and screen for variants that match the mode of inheritance. Through this screening process, 77 variants in 65 genes are filtered. A missense variant (EqCab3.0 chr4: <107353368> A > G) (rs1138485164) in the 3rd exon of LMBR1 is identified as a source of amino acid sequence variation. Gene editing confirms that the variant down-regulates LMBR1expression, increases the proliferative viability of mutant cells, and inhibits apoptosis. This study suggests that LMBR1 might play a role in the development of polydactyly and that the variant detected in this study is related to polydactyly in horses. However, further research is needed to determine whether a direct relationship exists. Identification of an LMBR1variant suggests a potential role in equine polydactyly development, revealing its impact on gene expression, cell proliferation, apoptosis and Shh signalling pathway. [ABSTRACT FROM AUTHOR]

Variant effect predictors (VEPs) are computational tools developed to assess the impacts of genetic mutations, often in terms of likely pathogenicity, employing diverse algorithms and training data. Here, we investigate the performance of 35 VEPs in the discrimination between pathogenic and putatively benign missense variants across 963 human protein-coding genes. We observe considerable gene-level heterogeneity as measured by the widely used area under the receiver operating characteristic curve (AUROC) metric. To investigate the origins of this heterogeneity and the extent to which gene-level VEP performance is predictable, for each VEP, we train random forest models to predict the gene-level AUROC. We find that performance as measured by AUROC is related to factors such as gene function, protein structure, and evolutionary conservation. Notably, intrinsic disorder in proteins emerged as a significant factor influencing apparent VEP performance, often leading to inflated AUROC values due to their enrichment in weakly conserved putatively benign variants. Our results suggest that gene-level features may be useful for identifying genes where VEP predictions are likely to be more or less reliable. However, our work also shows that AUROC, despite being independent of class balance, still has crucial limitations when used for comparing VEP performance across different genes. [ABSTRACT FROM AUTHOR]

Protein aggregate myopathies can result from pathogenic variants in genes encoding protein chaperones. DNAJB4 is a cochaperone belonging to the heat shock protein-40 (HSP40) family and plays a vital role in cellular proteostasis. Recessive loss-of-function variants in DNAJB4 cause myopathy with early respiratory failure and spinal rigidity, presenting from infancy to adulthood. This study investigated the broader clinical and genetic spectrum of DNAJB4 myopathy. In this study, we performed whole-exome sequencing on seven patients with early respiratory failure of unknown genetic etiology. We identified five distinct pathogenic variants in DNAJB4 in five unrelated families of diverse ethnic backgrounds: three loss-of-function variants (c.547 C > T, p.R183*; c.775 C > T, p.R259*; an exon 2 deletion) and two missense variants (c.105G > C, p.K35N; c.181 A > G, p.R61G). All patients were homozygous. Most affected individuals exhibited early respiratory failure, and patients from three families had rigid spine syndrome with axial weakness in proportion to appendicular weakness. Additional symptoms included dysphagia, ankle contractures, scoliosis, neck stiffness, and cardiac dysfunction. Notably, J-domain missense variants were associated with a more severe phenotype, including an earlier age of onset and a higher mortality rate, suggesting a strong genotype‒phenotype correlation. Consistent with a loss of function, the nonsense variants presented decreased stability. In contrast, the missense variants exhibited normal or increased stability but behaved as loss-of-function variants in yeast complementation and TDP-43 disaggregation assays. Our findings suggest that DNAJB4 is an emerging cause of myopathy with rigid spine syndrome of variable age of onset and severity. This diagnosis should be considered in individuals presenting with suggestive symptoms, particularly if they exhibit neck stiffness during infancy or experience respiratory failure in adults without significant limb muscle weakness. Missense variants in the J domain may predict a more severe phenotype. [ABSTRACT FROM AUTHOR]

Ethanol stress in Saccharomyces cerevisiae is a well-studied phenomenon, but pinpointing specific genes or polymorphisms governing ethanol tolerance remains a subject of ongoing debate. Naturally found in sugar-rich environments, this yeast has evolved to withstand high ethanol concentrations, primarily produced during fermentation in the presence of suitable oxygen or sugar levels. Originally a defense mechanism against competing microorganisms, yeast-produced ethanol is now a cornerstone of brewing and bioethanol industries, where customized yeasts require high ethanol resistance for economic viability. However, yeast strains exhibit varying degrees of ethanol tolerance, ranging from 8 to 20%, making the genetic architecture of this trait complex and challenging to decipher. In this study, we introduce a novel QTL mapping pipeline to investigate the genetic markers underlying ethanol tolerance in an industrial bioethanol S. cerevisiae strain. By calculating missense mutation frequency in an allele located in a prominent QTL region within a population of 1011 S. cerevisiae strains, we uncovered rare occurrences in gene IRA2. Following molecular validation, we confirmed the significant contribution of this gene to ethanol tolerance, particularly in concentrations exceeding 12% of ethanol. IRA2 pivotal role in stress tolerance due to its participation in the Ras-cAMP pathway was further supported by its involvement in other tolerance responses, including thermotolerance, low pH tolerance, and resistance to acetic acid. Understanding the genetic basis of ethanol stress in S. cerevisiae holds promise for developing robust yeast strains tailored for industrial applications. [ABSTRACT FROM AUTHOR]

Cerebellar vermis hypoplasia refers to a varying degree of incomplete development of the cerebellum and vermis. A Saudi family with four affected individuals with cerebellar vermis hypoplasia, facial dysmorphology, visual impairment, skeletal, and cardiac abnormalities was ascertained in this study. Three out of four patients could not survive longer and had died in early infancy. Genetic analysis of the youngest affected was performed by genome-wide homozygosity mapping coupled with whole exome sequencing (WES), followed by Sanger validation. Genome-wide genotyping analysis mapped the phenotype to chromosome 8q24.3. Using an autosomal recessive model, considering deleterious variants with minor allele frequency of less than 0.001 in WES data, a homozygous missense variant (NM_025251.2; ARHGAP39; c.1301G > T; p.Cys434Phe) was selected as a potential candidate for the phenotype. The variant (c.1301G > T) in the ARHGAP39 is in the region of homozygosity on chromosome 8q24.3. ARHGAP39 is a Rho GTPase-activating protein 39 and has been known to regulate apoptosis, cell migration, neurogenesis, and cerebral and hippocampal dendritic spine morphology. Mice homozygous for arhgap39 knockouts have shown premature embryonic lethality. Our findings present the first ever human phenotype associated with ARHGAP39 alteration. [ABSTRACT FROM AUTHOR]

Olmsted syndrome (OS) is a rare congenital skin disorder, typically characterized by symmetrical, severe palmoplantar and periorificial keratoderma, often accompanied by alopecia, and onychodystrophy, with varying degrees of pruritus and pain. Gain-of-function variants of the transient receptor potential cation channel subfamily V member 3 (TRPV3) were described as a cause of OS. Here, we report an atypical case of OS caused by a novel mutation in the TRPV3 gene that has not been described before in OS. The patient presented with disabling, bilateral palmoplantar keratoderma, and subsequently developed squamous cell carcinoma on the right sole. Genetic analysis identified a novel heterozygous p.Val306Met missense mutation in the exon 8 of TRPV3. Our findings expand the phenotypic spectrum of TRPV3-related OS and underscore the need for vigilant long-term monitoring of these patients. [ABSTRACT FROM AUTHOR]

Anophthalmia, microphthalmia and coloboma (AMC) comprise a spectrum of developmental eye disorders, accounting for approximately 20% of childhood visual impairment. While non-coding regulatory sequences are increasingly recognised as contributing to disease burden, characterising their impact on gene function and phenotype remains challenging. Furthermore, little is known of the nature and extent of their contribution to AMC phenotypes. We report two families with variants in or near MAB21L2, a gene where genetic variants are known to cause AMC in humans and animal models. The first proband, presenting with microphthalmia and coloboma, has a likely pathogenic missense variant (c.338 G > C; p.[Trp113Ser]), segregating within the family. The second individual, presenting with microphthalmia, carries an ~ 113.5 kb homozygous deletion 19.38 kb upstream of MAB21L2. Modelling of the deletion results in transient small lens and coloboma as well as midbrain anomalies in zebrafish, and microphthalmia and coloboma in Xenopus tropicalis. Using conservation analysis, we identify 15 non-coding conserved elements (CEs) within the deleted region, while ChIP-seq data from mouse embryonic stem cells demonstrates that two of these (CE13 and 14) bind Otx2, a protein with an established role in eye development. Targeted disruption of CE14 in Xenopus tropicalis recapitulates an ocular coloboma phenotype, supporting its role in eye development. Together, our data provides insights into regulatory mechanisms underlying eye development and highlights the importance of non-coding sequences as a source of genetic diagnoses in AMC. Coding sequence variants in the MAB21L2 gene can cause human eye disorders. Here the authors show that a deletion upstream of MAB21L2 leads to similar eye anomalies in humans, zebrafish and frogs due to the disruption of evolutionarily conserved regulatory elements. [ABSTRACT FROM AUTHOR]

Cancer genomes are rife with genetic variants; one key outcome of this variation is widespread gain-of-cysteine mutations. These acquired cysteines can be both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain unidentified via chemoproteomics; identification is a critical step to enable functional analysis, including assessment of potential druggability and susceptibility to oxidation. Here, we pair cysteine chemoproteomics—a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues—with genomics to reveal the hidden landscape of cysteine genetic variation. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized two-stage false discovery rate (FDR) error controlled proteomic search, which is further enhanced with a user-friendly FragPipe interface. Chemoproteogenomics analysis reveals that cysteine acquisition is a ubiquitous feature of both healthy and cancer genomes that is further elevated in the context of decreased DNA repair. Reference cysteines proximal to missense variants are also found to be pervasive, supporting heretofore untapped opportunities for variant-specific chemical probe development campaigns. As chemoproteogenomics is further distinguished by sample-matched combinatorial variant databases and is compatible with redox proteomics and small molecule screening, we expect widespread utility in guiding proteoform-specific biology and therapeutic discovery. Cancer genomes are rife with genetic variants, and one key outcome of this variation is widespread gain-of-cysteine mutations. Here, the authors pair cysteine chemoproteomics with genomics to investigate the landscape of cysteine genetic variation. [ABSTRACT FROM AUTHOR]

The Cornelia de Lange syndrome (CdLS) is a rare genetic disease, which is characterized by a cohesinopathy. Mutations of the NIPBL gene are observed in 65% of CdLS patients. A novel iPSC (induced Pluripotent Stem Cell) line was reprogrammed from the leukocytes of a CdLS patient carrying a missense mutation of the NIPBL gene. A mutation-corrected isogenic iPSC-line and two iPSC-lines generated from the healthy parents were used as controls. The iPSC lines were differentiated along the hepatocyte-lineage. Comparative immunofluorescence, RNA-seq and ATAC-seq analyses were performed on undifferentiated and differentiated iPSCs. In addition, chromatin organization was studied by ChIP-Seq analysis on the patient derived iPSCs as well as the respective controls. Relative to the mutation-corrected and the healthy-parents iPSCs, the patient-derived counterparts are defective in terms of differentiation along the hepatocyte-lineage. One-third of the genes selectively up-regulated in CdLS-derived iPSCs and hepatic cells are non-protein-coding genes. By converse, most of the selectively down-regulated genes code for transcription factors and proteins regulating neural differentiation. Some of the transcriptionally silenced loci, such as the DPP6 gene on chromosome 7q36.2 and the ZNF gene cluster on chromosome 19p12, are located in closed-chromatin regions. Relative to the corresponding controls, the global transcriptomic differences observed in CdLS undifferentiated iPSCs are associated with altered chromatin accessibility, which was confirmed by ChIP-Seq analysis. Thus, the deficits in the differentiation along the hepatocyte lineage observed in our CdLS patient is likely to be due to a transcriptional dysregulation resulting from a cohesin-dependent alteration of chromatin accessibility. [ABSTRACT FROM AUTHOR]

Objective Methods Results Significance Determining the pathogenicity of missense variants in clinical genetic tests for individuals with epilepsy is crucial for guiding personalized treatment. However, achieving a definitive pathogenic classification remains challenging, with most missense variants still classified as variants of uncertain significance (VUS) and with the availability of many computational tools which may provide conflicting predictions. Here, we aim to evaluate the performance of state‐of‐the‐art computational tools in pathogenicity prediction of missense variants in epilepsy‐associated genes. This will assist in selecting the most appropriate tool and critically assess their use in clinical setting.We assessed the performance of nine in silico pathogenicity prediction tools for missense variants in epilepsy‐associated genes on three carefully curated data sets. The first two data sets comprise missense variants in epilepsy associated genes that have been uploaded to ClinVar in the last year and were, therefore, not part of the training set of any of the nine considered tools. These two data sets are based on two different lists of epilepsy‐associated genes and comprise ~700 and ~ 250 missense variants, respectively. The third data set includes ~400 missense variants within epilepsy‐associated genes for which the functional effects have been determined experimentally and are therefore used here to infer pathogenicity. These three data sets represent the best available approximation to blind and independent test sets.Among the nine assessed tools, AlphaMissense (area under the curve [AUC]: .93, .88, and .95) and REVEL (AUC: .93, .88, and .93) showed the best classification performance, also outperforming other tools in the number of classified variants.We show which recently developed prediction tools achieve higher performance in epilepsy‐associated genes and should be integrated, therefore, into the American College of Medical Genetics and Genomics/Association of Molecular Pathology (AGMC/AMP) variant classification process. Periodic reevaluation of genetic test results with newly developed or updated tools should be incorporated into standard clinical practice to improve diagnostic yield and better inform precision medicine. [ABSTRACT FROM AUTHOR]

Introduction: Severe childhood obesity can be caused by pathogenic variants in several genes involved in monogenic and syndromic obesity. Recently, heterozygous variants in pleckstrin homology domain interacting protein (PHIP) have been identified in patients with obesity as part of Chung-Jansen syndrome. Case Presentation: The index patient is a 5-year-old boy with severe obesity since 1 year of age, developmental delay, facial dysmorphism, and behavior problems. Whole-exome sequencing identified a novel missense variant in PHIP (c.3182C>A, p.Ala1061Glu) in the index patient. Further genetic testing in family members revealed segregation of the same PHIP variant in the brother and mother, who both presented with severe childhood obesity and developmental delay or learning difficulties. The PHIP missense variant was predicted pathogenic by multiple in silico tools and affects a highly conserved residue. Conclusion: Early-onset obesity may be monogenic. Our finding expands the spectrum of disease-causing variants in PHIP and demonstrates variable intrafamilial clinical expressivity and severity. Screening for PHIP variants should be included in genetic testing in patients with severe early-onset obesity. [ABSTRACT FROM AUTHOR]

ABSTRACT Disorders of somatic mosaicism (DoSMs) are rare genetic disorders arising from postzygotic alteration leading to segmental/nonsegmental disease. Current professional guidelines for standardized variant interpretation focus on germline and cancer variants, making them suboptimal for DoSM variant interpretation. The Brain Malformations Variant Curation Expert Panel (BMVCEP) modified existing guidelines to account for brain‐specific disorders of somatic mosaicism, but applicability to other DoSM presentations is limited. At Washington University in St. Louis School of Medicine, we have adopted the BMVCEP interpretation framework but suggested alterations that make it more suitable for generalized DoSM variant classification. These modifications include (1) expanding applicability beyond genes associated with brain malformations, (2) introduction of a criterion to interpret truncating variants at the C‐terminus of gain of function genes, (3) establishment of a variant allele fraction (VAF) cutoff for applying de novo criteria, and (4) demonstration that in silico prediction tools are relevant to interpretation of gain of function missense variants. Furthermore, modifications to BMVCEP guidelines reduce the number of variants classified as uncertain. The variant classification considerations that we propose have the potential to improve the accuracy of somatic variant classification, better inform clinical care, and may benefit clinical laboratories also conducting DoSM testing. [ABSTRACT FROM AUTHOR]

Defective renal excretion and increased production of uric acid engender hyperuricemia that predisposes to gout. However, molecular mechanisms underlying defective uric acid excretion remain largely unknown. Here, we report a rare genetic variant of gout-unprecedented NUMB gene within a hereditary human gout family, which was identified by an unbiased genome-wide sequencing approach. This dysfunctional missense variant within the conserved region of the NUMB gene (NUMBR630H) underwent intracellular redistribution and degradation through an autophagy-dependent mechanism. Mechanistically, we identified the uric acid transporter, ATP Binding Cassette Subfamily G Member 2 (ABCG2), as a novel NUMB-binding protein through its intracellular YxNxxF motif. In polarized renal tubular epithelial cells (RTECs), NUMB promoted ABCG2 trafficking towards the apical plasma membrane. Genetic loss-of-function of NUMB resulted in redistribution of ABCG2 in the basolateral domain and ultimately defective excretion of uric acid. To recapitulate the clinical situation in human gout patients, we generated a NUMBR630H knock-in mouse strain, which showed marked increases of serum urate and decreased uric acid excretion. The NUMBR630H knock-in mice exhibited clinically relevant hyperuricemia. In summary, we have uncovered a novel NUMB-mediated mechanism of uric acid excretion and a functional missense variant of NUMB in humans, which causes hyperuricemia and gout. [ABSTRACT FROM AUTHOR]

Over 700 syndromes associated with hearing loss (HL) have been identified. Labyrinthine aplasia, microtia, and microdontia (LAMM syndrome, OMIM: 610706) is a rare HL syndrome characterized by congenital sensorineural HL, labyrinthine aplasia, type I microtia and microdontia, which is caused by biallelic variants in the FGF3 gene. Using Whole-exome sequencing (WES), we identified a novel missense FGF3 variant (c.137G > C, p. Arg46Pro (NM_005247.4) in three unrelated Uyghur ethnic families. This variant is classified as a variant of uncertain significance according to ACMG guidelines, with the applied criteria of PM3, PM2_Supporting, PP3 and PP4. Patients from the three families revealed variable clinical features. We found a novel phenotype, sparse hair, in one of the proband. Our findings expanded the variant and phenotype spectrum of LAMM syndrome and provided new insights to the diagnose and pathogenesis investigation of the disease. [ABSTRACT FROM AUTHOR]

UNC-89 is a giant sarcomeric M-line protein required for sarcomere organization and optimal muscle function. UNC-89 contains two protein kinase domains, PK1 and PK2, separated by an elastic region. Here we show that PK2 is a canonical kinase expected to be catalytically active. C. elegans expressing UNC-89 with a lysine to alanine (KtoA) mutation to inactivate PK2 have normally organized sarcomeres and SR, and normal muscle function. PK2 KtoA mutants have fragmented mitochondria, correlated with more mitochondrially-associated DRP-1. PK2 KtoA mutants have increased ATP levels, increased glycolysis and altered levels of electron transport chain complexes. Muscle mitochondria show increased complex I and decreased complex II basal respiration, each of which cannot be uncoupled. This suggests that mutant mitochondria are already uncoupled, possibly resulting from an increased level of the uncoupling protein, UCP-4. Our results suggest signaling from sarcomeres to mitochondria, to help match energy requirements with energy production. UNC-89, a giant protein localized to muscle sarcomeres, contains 2 protein kinase domains, one of which, PK2 is a canonical kinase. A missense mutation expected to inactivate PK2 results in worms with an unexpected phenotype suggesting signaling from sarcomeres to mitochondria. [ABSTRACT FROM AUTHOR]

ABSTRACT During the past two decades, an emerging group of genes coding for proteins involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis are being implicated in early‐infantile epileptic encephalopathy. Amongst these, a hypomorphic promoter mutation in the mannosyltransferase‐encoding PIGM gene was described in seven patients to date, exhibiting intractable absence epilepsy, portal and cerebral vein thrombosis and intellectual disability (ID). We describe here three siblings exhibiting intractable epilepsy and ID, found to harbor a homozygous c.224G>A p.(Arg75His) missense variant in PIGM, which segregated with the disease in the family. The variant is evolutionary conserved, extremely rare in general population databases and predicted to be deleterious. Structural modeling of the PIGM protein and the p.(Arg75His) variant indicates that it is located in a short luminal region of the protein, predicted to be hydrophilic. Functional prediction suggests that the entire local region is sensitive to mutations, with the p.(Arg75His) variant in particular. This is the first report of a PIGM coding variant, and the second variant altogether to be described affecting this gene. This phenotype differs from that of patients with the shared PIGM promoter mutation by lack of thrombotic events and no decrease in PIGM cDNA levels or CD59 expression on red blood cells. [ABSTRACT FROM AUTHOR]

Deep learning models for predicting variant pathogenicity have not been thoroughly evaluated on real-world clinical phenotypes. Here, we apply state-of-the-art pathogenicity prediction models to hereditary breast cancer gene variants in UK Biobank participants. Model predictions for missense variants in BRCA1, BRCA2 and PALB2, but not ATM and CHEK2, were associated with breast cancer risk. However, deep learning models had limited clinical utility when specifically applied to variants of uncertain significance. [ABSTRACT FROM AUTHOR]

Background: the role of the R202Q (c.605G>A, p.Arg202Gln) missense variant of the MEFV gene has been debated as either a benign polymorphism or a potentially pathogenic mutation. We report and discuss here the case of a young female with corticosteroid-dependent recurrent pericarditis carrying the homozygous R202Q variant, exhibiting distinctive clinical features possibly influenced by this genetic variant. Methods: a 30-year-old woman with a previous diagnosis of cancer and recent respiratory infection presented with severe pleuritic chest pain, hypotension, tachycardia, and fever. Initial diagnostic evaluation indicated cardiac tamponade, and emergent pericardiocentesis was performed. Despite initial treatment with NSAIDs, colchicine, and corticosteroids, the patient experienced multiple recurrences. Genetic testing identified homozygous R202Q variant in the MEFV gene. Given the corticosteroid dependency and recurrent nature of her condition, IL-1 inhibitor anakinra was introduced, leading to significant improvement, although tapering below 150 mg per week failed to prevent recurrences. Results: the introduction of anakinra resulted in rapid symptom relief and resolution of pericardial effusion. However, attempts to taper or discontinue anakinra led to pericarditis recurrences. Ultimately, a maintenance dose of 50 mg every three days was established, which maintained remission for 18 months without recurrence. Despite multiple tapering attempts, further reduction in anakinra dosage was unsuccessful without triggering relapses. Conclusions: the R202Q variant, although typically considered benign, may contribute to an autoinflammatory phenotype resembling familial Mediterranean fever. This case underscores the potential pathogenicity of the homozygous R202Q variant in recurrent pericarditis and its responsiveness to IL-1 inhibition. In patients with corticosteroid-dependent recurrent pericarditis, genetic testing for the R202Q variant should be considered when anti-IL-1 drugs cannot be withdrawn. Further studies are warranted to elucidate the variant's role in pericardial inflammation and guide personalized treatment strategies. [ABSTRACT FROM AUTHOR]

The DP1 family of integral membrane proteins stabilize high membrane curvature in the endoplasmic reticulum and phagophores. Mutations in the human DP1 gene REEP1 are associated with Hereditary Spastic Paraplegia type 31 and distal hereditary motor neuropathy. Four missense mutations map to a putative dimerization interface but the impact of these mutations on DP1 structure and tubule formation are unknown. Combining biophysical measurements, functional assays, and computational modeling in the context of the model protein Yop1, we found that missense mutations have variable effects on DP1 dimer structure and in vitro tubulation activity, and provide mechanistic insights into the role of DP1 oligomerisation on membrane curvature stabilization. Whereas the mutations P71L and S75F decreased dimer homogeneity and led to polydisperse oligomerization and impaired membrane curving activity, A72E introduced new polar interactions between subunits that stabilized the Yop1 dimer and allowed robust tubule formation but prevented formation of more highly-curved lipoprotein particles (LPP). The introduction of a BRIL domain to the cytoplasmic loop of A72E rescued LPP formation, consistent with a requirement for dimer splaying in highly curved membranes. These results suggest that the membrane curving activity of DP1 proteins requires both dimer stability and conformational plasticity at the intermolecular interface. [ABSTRACT FROM AUTHOR]

Background: Pulmonary arterial hypertension is a life-threatening progressive disorder characterised by high blood pressure (hypertension) in the arteries of the lungs (pulmonary artery). Although treatable, there is no known cure for this rare disorder, and its exact cause is unknown. Mutations in the bone morphogenetic protein receptor type-2 (BMPR2) are the most common genetic cause of familial pulmonary arterial hypertension. This study represents the first-ever trial of treatments aimed at directly rescuing the BMPR2 pathway, repurposing two drugs that have shown promise at restoring levels of BMPR2 signalling: hydroxychloroquine and phenylbutyrate. Methods: This three-armed phase II precision medicine study will investigate BMPR2 target engagement and explore the efficacy of two repurposed therapies in pulmonary arterial hypertension patients with BMPR2 mutations. Patients will be stratified based on two BMPR2 mutation classes: missense and haploinsufficient mutations. Eligible subjects will be randomised to one of the three arms (two active therapy arms and a placebo arm, all plus standard of care) following a Bayesian response-adaptive design implemented independently in each stratum and updated in response to a novel panel of primary biomarkers designed to assess biological modification of the disease. Discussion: The results of this trial will provide the first randomised evidence of the efficacy of these therapies to rescue BMPR2 function and will efficiently explore the potential for a differential response of these therapies per mutation class to address causes rather than consequences of this rare disease. Trial registration: The study has been registered with ISRCTN (ISRCTN10304915, 22/09/2023). [ABSTRACT FROM AUTHOR]

ABSTRACT 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]

Introduction: FAS has been implicated in the development of various cancers, but its involvement in lung cancer has not been systematically characterized. In this study, we performed data mining in online tumor databases to investigate the expression, methylation, alterations, protein interactions, co-expression and prognostic significance of FAS in lung cancer. Method: The expression, prognostic significance and molecular interactions of FAS in lung cancer was mined and analyzed using GENT2, GEPIA2, UALCAN, cBioPortal, STRING, GeneMANIA, UCSC Xena, Enrichr, and OSluca databases. FAS expression was subsequently investigated at the protein level in samples from 578 lung cancer patients to understand its protein-level expression. In vitro validation of FAS gene expression was performed on H1299, H1993, A549 and HBE cell lines. Result: We found that the expression of FAS was significantly downregulated in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) compared to normal lung tissue. In addition, we observed a higher level of FAS promoter methylation in LUSC tissue than in normal tissue. FAS alterations were rare (1.9%) in lung cancer samples, with deep deletions being more common than missense mutations, which occurred mainly in the TNFR-like cysteine-rich domain and the death domain. We also identified a list of proteins interacting with FAS and genes co-expressed with FAS, with LUAD having 11 co-expressed genes and LUSC having 90 co-expressed genes. Our results also showed that FAS expression has limited prognostic significance (HR=1.302, 95% CI=0.935-1.139, P=0.530). Protein level investigation revealed that FAS expression varied among individuals, with nTPM values ranging from 5.2 to 67.2. Conclusion: This study provides valuable insights into the involvements and characteristics of FAS in lung cancer. Further studies are needed to investigate the clinical significance of FAS alterations in lung cancer and to explore the potential of targeting FAS for therapeutic intervention. [ABSTRACT FROM AUTHOR]