Introduction: Hemophilia A is an inherited bleeding disorder caused by mutations in the factor VIII (FVIII) gene. These mutations result in either reduced FVIII synthesis (null variants) or loss of FVIII function (non-null variants). Null variants are typically associated with more severe FVIII deficiency and recurrent joint bleeding, which may adversely affect musculoskeletal health. Objective: To evaluate the relationship between musculoskeletal status and genetic mutations in patients with hemophilia A. Methods: A cross-sectional study was conducted at the Faculty of Medicine Universitas Indonesia-Dr. Cipto Mangunkusumo Hospital from June 2024 to March 2025. Genetic analysis was performed at the Human Genetic Research Center using inverse-shifting PCR and Sanger sequencing. Mutations were classified as null variants (intron-22 inversion, intron-1 inversion, large deletion, and nonsense mutations) and non-null variants (missense and non-conserved splice mutation). Musculoskeletal status was assessed by the presence of target joints and the Hemophilia Joint Health Score (HJHS), which evaluates global gait and joint function of the elbows, knees, and ankles. Higher HJHS scores indicate worse joint health. Results: Sixty patients were included in this study, of which 39 had severe, 15 had moderate, and the remaining 6 had mild hemophilia A. The median age was 9.5 years (range 2-18). Null variants were identified in 45/60 patients and non-null variants in 15/60 patients. The most common target joints were the knees in patients with null variants and the ankles in those with non-null variants. The median HJHS was 4 (Q1-Q3: 2-13.5) in the null variant group and 2 (Q1-Q3: 1-11) in the non-null variant group. No significant association was observed between the target joint and the HJHS and genetic mutations. Further subgroup analysis showed no difference in HJHS between mutation groups among patients receiving prophylaxis (p=0.366) or on-demand treatment (p=0.458). Conclusion: No association was found between genetic mutation type and musculoskeletal status in patients with Hemophilia A. HJHS did not differ between mutation groups regardless of treatment regimens.

Mutant p53(Mtp53) not only loses its canonical tumor-suppressive functions but also acquires oncogenic gain-of-function properties, positioning it as a central orchestrator in reshaping the tumor immune microenvironment. This review systematically delineates how Mtp53 actively establishes and sustains an immunosuppressive niche through multiple interconnected mechanisms, including chronic inflammation, immune cell dysfunction, reprogramming of cancer-associated fibroblasts, metabolic dysregulation, epigenetic hijacking, and potentially aberrant liquid–liquid phase separation, thereby promoting immune evasion and therapeutic resistance. We integrate current evidence to propose a conceptual “metabolism–epigenetics–immunity” axis: Mtp53-driven metabolic reprogramming—such as accumulation of lactate or α-ketoglutarate—can modulate chromatin modifications and immune gene expression. Notably, the full in vivo causal chain of this axis remains unestablished; existing support derives primarily from stepwise experimental data and strong correlations. The immunological impact of Mtp53 is highly context-dependent, shaped by co-mutations and tissue origin. In TP53/KRAS co-mutant non-small cell lung cancer (NSCLC), Mtp53 enhances tumor immunogenicity and improves response to immune checkpoint inhibitors (ICIs); conversely, in immunologically “cold” tumors—such as triple-negative breast cancer, pancreatic ductal adenocarcinoma, and colorectal cancer—it promotes T-cell exhaustion or myeloid suppression, reflecting marked cancer-type heterogeneity. Therapeutic approaches include Mtp53 reactivators (e.g., APR-246, PC14586), degraders, synthetic lethal strategies, and neoantigen vaccines. Although APR-246 showed efficacy in a phase II trial (NCT03072043), it failed to improve survival in phase III (NCT03745716) due to lack of TP53 mutation stratification. Its combination with pembrolizumab (NCT04383938) demonstrated acceptable safety (immune-related adverse events in ∼12%) but limited efficacy, underscoring the need for biomarker-guided, precision-based combinations. Thus, a multidimensional biomarker platform is urgently needed—one integrating TP53 mutation subtypes (e.g., R175H vs . nonsense mutations), dynamic ctDNA monitoring (VAF ≥ 0.01%), tumor immune microenvironment (TIME) features (e.g., TILs, MDSCs), and spatial multi-omics—to enable precise molecular stratification and personalized intervention in Mtp53-driven cancers.

Introduction ARHGAP32 gene (Rho GTPase Activating Protein 32) encodes a Rho GTPase activating protein, which is vital for the regulation of synaptic plasticity and cytoskeletal dynamics. ARHGAP32 (11q24.3) has been implicated as a candidate gene for Autism Spectrum Disorder (ASD) in Jacobsen syndrome, where a 243-kb terminal deletion encompasses its locus. A unique patient with de novo (DN) likely gene-disruptive mutation of ARHGAP32 has been reported so far in the medical literature. The present study was undertaken to understand clinical, molecular, and neurobehavioral characteristics of ASD associated with a novel DN nonsense mutation in ARHGAP32 . Methods Clinical characterization included basal and follow-up assessment with standardized measures and comorbidities diagnosis. Trio exome sequencing analyses (WES) and variants annotation were performed. Results WES analyses of a 6-year-old female patient with idiopathic ASD revealed DN heterozygous nonsense variant in ARHGAP32 (NM_001378024.1: c.610C>T; NP_001364953.1: p.(Arg204Ter). The variant is predicted to introduce a premature stop codon, resulting in either a truncated protein or activation of nonsense-mediated mRNA decay, ultimately leading to loss of function. The patient presented with normative growth parameters and cranial measurements, with no congenital morphological anomalies. A diagnosis of idiopathic ASD was made at age 2. Developmental delays were observed, notably language regression beginning at 18 months, mild intellectual disability, and restricted interests accompanied by repetitive motor and verbal behaviors. Significant hyperactivity and attentional difficulties were observed. Over time, she exhibited borderline non-verbal cognitive functioning, persistent speech impairment, and was subsequently diagnosed with comorbid Attention Deficit Hyperactivity Disorder. Discussion This study identifies shared neurobehavioral features of idiopathic Autism Spectrum Disorder (ASD) associated with de novo LoF mutations in ARHGAP32 and reinforces the involvement of RhoGAP family proteins in neurodevelopmental disorders. Taken together with previous evidence, our data support the role of ARHGAP32 as a candidate gene for ASD, expanding the genetic spectrum.

Maltose is one of the most abundant sugars in brewer's wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F2 offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), and MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18's own MAL33ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p result in differences in maltose consumption between strains. We hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of genetic variation, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

Female reproductive aging is characterized by progressive deterioration of ovarian function, yet the molecular mechanisms driving these changes remain incompletely understood. Here, we used long-read direct RNA-sequencing to map transcript isoform changes in mouse ovaries across reproductive age. Comparing young and aged mice after controlled gonadotropin stimulation, we identified widespread alternative splicing changes, including shifts in exon usage, splice site selection, and transcript boundaries. Aged ovaries exhibited increased isoform diversity, favoring distal start and end sites, and a significant rise in exon skipping and intron retention events. Many of these age-biased splicing events altered open reading frames, introduced premature stop codons, or disrupted conserved protein domains. Notably, several mitochondrial genes involved in the respiratory chain were affected. We highlight Ndufs4, a mitochondrial Complex I subunit, as a case in which aging promotes the alternative splicing of a short isoform lacking the canonical Pfam domain. Structural modeling suggests this splice variant could impair Complex I function, resulting in increased ROS production. Our data suggest a mechanistic link between splicing and mitochondrial dysfunction in the aging ovary. These findings support the model of the splicing-energy-aging axis in ovarian physiology, wherein declining mitochondrial function and adaptive or maladaptive splicing changes are intertwined. Our study reveals that alternative splicing is not merely a byproduct of aging but a dynamic, transcriptome-wide regulatory layer that may influence ovarian longevity. These insights open new avenues for investigating post-transcriptional mechanisms in reproductive aging and underscore the need to consider isoform-level regulation in models of ovarian decline.

The potential use of anti-codon engineered tRNAs (ACE-tRNAs) to treat nonsense variants causing inherited retinal diseases.

A novel BRCA mutation classification system reveals differential responses to PARP inhibition and prognostic outcomes in epithelial ovarian cancer: a multicenter study.

The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein’s N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders.

Unravelling the role of Neurotrimin (NTM), a member of the IgLON family, in mild intellectual disability and anxiety-like behaviors.

This schematic illustrates the genetic, molecular, and clinical consequences of ESAM loss-of-function variants identified in two unrelated Turkish families. Family 1 harbors a splice-site variant (c.451 + 5G>C) predicted to cause aberrant splicing between exons 3 and 4, leading to defective mRNA processing and loss of functional ESAM protein. Family 2 carries a nonsense variant (c.605 T>G; p.Leu202Ter) that introduces a premature stop codon within the second immunoglobulin-like domain, resulting in a truncated protein and expected nonsense-mediated mRNA decay (NMD). Both variants abolish ESAM's membrane anchoring and disrupt endothelial junctional cohesion. Clinically, affected individuals present with a distinct neurovascular phenotype characterized by intrauterine or perinatal intracranial hemorrhage, ventriculomegaly, microcephaly, spastic quadriparesis, and congenital cataracts. The lower panel summarizes the pathophysiological cascade: (1) hemorrhagic stroke resulting from vascular fragility, (2) blood-brain barrier (BBB) disruption due to tight junction dysfunction and increased transcytosis, and (3) impaired vascular network formation caused by defective endothelial tubulogenesis. Collectively, these findings establish ESAM deficiency as a congenital tight-junctionopathy linking molecular endothelial defects to severe neurodevelopmental impairment.

To explore the genetic variants and phenotypic characteristics of patients with Neurofibromatosis type I (NF1). Twenty two NF1 patients who presented at Enze Medical (Center) Group in Taizhou between 2018 and 2024 were selected as the study subjects. Clinical phenotype and family history were collected for the patients. Whole exome sequencing (WES) was carried out for the 22 probands to screen the variants of NF1 gene. Candidate variants were verified by Sanger sequencing of their family members. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: K20230902). The 22 probands were diagnosed between the age of 5 months to 47 years old, and have all shown cafe au lait spots on their skin. Seventeen patients exhibited the phenotype at birth, and 11 had various degrees of neurofibromatosis. Among them, probands 1 and 13 underwent surgical resection of the tumor but had recurred, while proband 12 had amputation due to the huge size and serious impact of the neurofibroma and had no recurrence. Five patients had various degrees of scoliosis. In total 22 germline mutations and one somatic mutation were identified among the 22 families, with 5 variants unreported previously, including 1 nonsense mutation c.1603C>T (Q535*), 3 frameshift mutations [c.7268_7269delCA (Thr2423fs), c.2293del (Arg765Alafs*26), and c.5433_5438delinsGC (Phe1812ArgfsTer50)], and 1 deletion involving exons 41-44 of the NF1 gene and adjacent introns. Proband 13 was found to harbor germline mutation c.6796C>T (Gln2266Ter) and somatic mutation c.1019_1020del (Ser340Cysfs Ter12) in the peripheral blood and tumor tissue, respectively. Among the 22 NF1 probands, 6 had received treatment due to severe illness. Proband 1 had tumor resection in the right upper limb, but was found to have malignant lung tumor and died during follow-up. Proband 12 had multiple recurrence of neurofibroma in the left ring finger. Proband 4 underwent spinal correction surgery due to severe scoliosis. Proband 11 had died due to a central nervous system disease. Among the 22 germline mutations, 6 had led to the occurrence of truncated proteins, which may have a more severe impact on the phenotype. This study investigated the genetic variants and clinical phenotypes of 22 NF1 families and identified 5 novel variants of the NF1 gene, which has expanded the genotypic and phenotypic spectra of the NF1. Preliminary studies have identified an association between truncated mutations, young age, and severe phenotypes, which may provide important clues for prognosis evaluation. For the clinical diagnosis and treatment of NF1, it is necessary to consider the phenotypic characteristics and genetic testing in combination with genetic counseling and long-term follow-up.

ABSTRACTFucosidosis is an ultra-rare and fatal lysosomal storage disease caused by the impaired lysosomal degradation of fucosylated glycoconjugates due to a deficiency in the lysosomal tissue α-L-fucosidase (FUCA1). The accumulation of fucosylated metabolites within lysosomes leads to a range of severe, primarily neurological, symptoms, including cognitive impairment and progressive motor dysfunction. In this study, we explored a therapeutic approach using translational readthrough (TR) for patients with premature termination codons resulting from nonsense mutations in the FUCA1 gene. We ectopically expressed several clinically identified FUCA1 nonsense variants in a cell line with low endogenous FUCA1 expression. Treatment with the aminoglycoside G418 induced TR, leading to partial recovery of the full-length enzyme and FUCA1 activity. Moreover, combining aminoglycoside treatment with CC-885-induced degradation of the eukaryotic release factor subunit eRF3a further enhanced FUCA1 restoration in two variants (p.Q82X and p.W188X). This study lays the groundwork for individualized TR therapy for patients with fucosidosis with FUCA1 nonsense variants.

Familial adenomatous polyposis (FAP) is a disorder of autosomal dominant inheritance that is responsible for around 1% of colorectal cancer (CRC) cases. To determine the mutation profile of FAP-specific to the Hungarian population. This prospective single-center study enrolled patients with clinically suspected FAP or attenuated FAP (aFAP). Whole-exome next-generation sequencing was performed to detect variants of 50 FAP priority genes and 173 CRC predisposing genes or other CRC disease-associated genes. To identify larger deletions and insertions, a multiplex amplifiable probe hybridization technique was used. The identified genes were then classified according to the American College of Medical Genetics and Genomics guidelines. A total of 26 index patients with clinically suspected FAP (n = 21) and aFAP (n = 5) were enrolled. APC gene alterations were confirmed in 92.31% of the cases (region 1B deletion, n = 2; whole-gene deletion, n = 4; frameshift mutation, n = 2; nonsense mutation, n = 5, and splice mutation, n = 1), with the remaining two cases having CHEK2 and MSH3 gene alterations. According to pathogenicity, 21 cases had pathogenic mutations, 6 cases had likely pathogenic mutations, and 16 cases had variants of unknown significance (VUS). The most frequent of the latter were the POLE (n = 5) and PIEZO1 (n = 4) gene variants. Germline mutations in the APC gene were confirmed in more than 90% of Hungarian patients with clinically suspected FAP. Although the role of VUS genes is unclear, they are highly likely to play a role in the development of CRC.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms defined by co-expression of melanocytic and smooth muscle markers, with fibroma-like PEComa representing a novel and recently described subtype strongly associated with tuberous sclerosis complex (TSC). This article reports the eighth documented example of fibroma-like PEComa, occurring in the subcutaneous wrist tissue of a 6-year-old girl with no prior clinical or family history of TSC. Histopathological examination revealed a hypocellular to moderately cellular proliferation of bland oval, spindle to stellate cells within dense collagenous stroma, accompanied by focal myxoid change and scattered mature adipose tissue. Immunohistochemically, the tumor exhibited diffuse strong glycoprotein nonmetastatic melanoma protein B (GPNMB) expression and focal positivity for HMB45, melan-A, and desmin. DNA-based target next-generation sequencing identified a germline TSC2 nonsense mutation (c.268C > T, p.Q90*) with a variant allele frequency of 83%, confirmed via peripheral blood testing, leading to a definitive diagnosis of TSC. A review of all 8 reported tumors confirms that fibroma-like PEComa occurs predominantly in children and young adults, shows a female predilection (5:3), and favors extremity locations. These tumors typically demonstrate benign behavior with no recurrences or metastases reported. Our report highlights that fibroma-like PEComa can serve as an early indicator of occult TSC and underscores the diagnostic utility of GPNMB immunohistochemistry as a surrogate marker of TSC1/2/MTOR pathway activation. Comprehensive clinical and genetic evaluation for TSC is recommended upon diagnosis of this rare tumor.

BackgroundShigellacauses severe diarrheal disease, andS. flexneriandS. sonneiare the targets for multivalent vaccine development. Culture-based agglutination has been the gold standard for serotyping, but it is limited by logistics, subjectivity, and the availability of antisera for emerging serotypes. Newer methods, including a qPCR-based approach and whole-genome sequencing offer alternatives, but their performance inShigellaendemic populations are not well documented.MethodsShigellaisolates obtained from the Enterics for Global Health (EFGH) study in Iquitos, Peru were simultaneously serotyped using four methods: culture-based agglutination, isolate-based qPCR serotyping, stool-based qPCR serotyping and WGS using the in-silico tool ShigaPass. The definitive adjudicated serotype was established by an expert analysis of the WGS data, involving the mapping of sequence reads to known O-antigen biosynthesis and modification genes to identify key mutations.ResultsResults from all four serotyping methods were available for 107/114 isolates. Accuracy for vaccine subtypesS. flexneri1b, 2a, 3a, 6, andS. sonnei, ranged from 93.3-100% for all methods. Complete concordance between methods was noted in 83/107 isolates, while 24/107 (22.4%) exhibited at least one discrepancy. Most discrepancies derived fromS. flexneriserotypes Y, Yv and 1a. Agglutination misclassified eight Y/Yv isolates as 4a, and six isolates correctly classified as 1a by agglutination were classified as 1b by the other methods, a discrepancy associated with a nonsense mutation in theoacgene.ConclusionAll four serotyping methods achieved acceptable accuracy forShigellavaccine efficacy evaluation. Although discrepancies are infrequent, WGS provides information of their genomic basis.ImportanceShigella serotyping is critically important for the evaluation of future multivalent vaccines, of which there are several in advanced stages of development, as well as for monitoring of emerging Shigella serotypes. Culture based agglutination is the most widely used serotyping method, yet its successful implementation is associated with key logistical constraints. This study compares culture-based, qPCR-based, and whole-genome sequencing serotyping methods using isolates from aShigella-endemic population in Peru. The study demonstrates that molecular and genomic approaches achieve high accuracy for vaccine-relevant serotypes and identifies the genomic basis of serotyping discrepancies. These methods would also reduce variation and improve data quality for future vaccine trials and epidemiologic surveillance. Ultimately, this work informs clinical microbiology laboratories and public health programs that seek a reliable and scalable alternative to traditional serotyping methods.

Accurate classification of novel globin gene variants is critical for the diagnosis and management of thalassaemia. The adaptation of ACMG/AMP guidelines for globin genes represents an important step toward standardising variant interpretation and enhancing clinical utility in the field. This study reports the haematological and molecular characteristics of a novel α2-globin variant identified in a Malay family. A Malay family from Taiping, Perak, Malaysia, with a history of α-thalassaemia, was recruited for this study. The proband's phenotype was assessed through comprehensive haematological analysis and clinical evaluation. Known α-thalassaemia deletions and non-deletional mutations were screened using gap-PCR and ARMS-PCR. Sanger sequencing of the HBA genes was conducted to characterise the proband's genotype in detail. A novel pathogenic HBA2 variant was identified, expanding the known mutational spectrum of α-thalassaemia. This variant introduces a premature stop codon, occurs in trans with a known pathogenic allele associated with a significant clinical phenotype, segregates with the disease in the family, and is absent from major population databases. Based on haematological data, molecular findings, in silico predictions, and protein modeling, the variant meets the ACMG/AMP criteria for pathogenicity adapted for α-globin genes. We have designated this variant Hb Taiping, named after the location of its discovery. Its accurate classification is vital for carrier screening, genetic counselling, and prenatal diagnosis, thereby supporting improved clinical management. This study identifies and characterises a novel α-globin gene variant, Hb Taiping, in a Malay family with α-thalassaemia. The discovery contributes to the growing body of pathogenic mutations linked to α-thalassaemia and underscores the importance of precise variant classification for effective diagnosis, risk assessment, and genetic counselling.

Heart function depends on cardiomyocyte contractile apparatus and proper sarcomere protein expression. Variants in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. Recently, a sarcomere component, myosin-binding protein-H like (MyBP-HL), was identified. MyBP-HL is mainly expressed in cardiac atria and is homologous to the last three C-terminal domains of cardiac myosin-binding protein-C (cMyBP-C). The MYBPHL R255X nonsense variant has been linked to atrial enlargement, dilated cardiomyopathy, and arrhythmias. Similar nonsense mutations in MYBPC3 are linked to hypertrophic cardiomyopathy, with these mutations preventing myofilament incorporation and the degradation of the truncated protein. However, the allele frequency of the MYBPHL R255X variant is too high in the human population to be pathogenic. We sought to determine whether MYBPHL nonsense variants impact on MyBP-HL sarcomere integration and degradation of the truncated protein, and whether the MyBPHL nonsense variants lead to changes in cardiomyocyte calcium dynamics and contractility. We mimicked human MYBPHL nonsense variants in the mouse Mybphl cDNA sequence and tested their sarcomere incorporation. We demonstrated that full-length MyBP-HL overexpression showed the expected C-zone sarcomere incorporation. Nonsense variants showed defective sarcomere incorporation. We demonstrated that full-length MyBP-HL and MyBP-HL nonsense variants were degraded by both proteasome and calpain mechanisms. We did not observe changes in calcium transients. In addition, we observed changes in contraction kinetics, including sarcomere shortening. Together, these data support the hypothesis that MYBPHL nonsense variants are functionally similar.

Clathrin-mediated endocytosis (CME) is essential for maintaining homeostasis in mammalian cells. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. We show by quantitative live cell imaging that siRNA-mediated knockdown of CCDC32, a poorly characterized endocytic accessory protein, leads to the accumulation of unstable flat clathrin assemblies. CCDC32 interacts with the α-appendage domain (AD) of AP2 in vitro and with full-length AP2 complexes in cells. Deletion of aa78-98 in CCDC32, corresponding to a predicted α-helix, abrogates AP2 binding and CCDC32's early function in CME. Furthermore, clinically observed nonsense mutations in CCDC32, which result in C-terminal truncations that lack aa78-98, are linked to the development of cardio-facio-neuro-developmental syndrome (CFNDS). Overall, our data demonstrate the function of a novel endocytic accessory protein, CCDC32, in regulating CCP stabilization and invagination, critical early stages of CME.

Rewriting RNA information to alter function requires controllable tools to edit RNA sequences within a user-defined region. Here we report a single-strand deaminase-assisted platform for adjustable RNA information manipulation (AIM). AIM is composed of a loop-forming guide RNA bound to an RNA-targeting Cas protein and an evolved TadA. AIM induces a loop, flanked by paired regions, in the target RNA; the loop size can be adjusted to allow conversions of single and multiple bases. We evolve TadA to achieve A-to-I, C-to-U or simultaneous A+C editing in coding and noncoding regions. We apply AIM to suppress the ochre nonsense codon (UAA) in disease-relevant cell and animal models, in which the two As must be simultaneously edited to rewrite the coding information. Moreover, we use AIM to manipulate adjacent phosphorylation sites important for protein function. Collectively, AIM is a versatile platform for manipulating RNA information within user-defined regions, opening additional avenues for functional RNA modulation.

Background/Objectives: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by pathogenic variants in the adenomatous polyposis coli (APC) gene. Its attenuated form (AFAP) is characterized by fewer colorectal polyps and later onset of colorectal cancer. We aimed to characterize the molecular effects of a novel APC gene variant (NM_000038.6: c.1620_1624delinsT) identified in a patient with AFAP. Methods: A 56-year-old man with the AFAP phenotype underwent germline testing via a multigene NGS panel, which identified a novel APC gene variant (NM_000038.6: c.1620_1624delinsT). In silico analyses predicted disruption of the canonical donor splice site and a frameshift followed by the introduction of a premature stop codon. The transcriptional impact of the identified APC gene variant was investigated by mRNA analysis. Results: mRNA analysis revealed two distinct APC transcripts: the first transcript led to a truncated protein (p.Leu540PhefsTer8), and the second transcript lacked exon 12, resulting in an in-frame 26 amino acid deletion of APC protein (p.Ala517_Gly542del). The transcript lacking exon 12 was more abundant than the transcript with a premature stop codon, likely due to degradation through nonsense-mediated decay. Conclusions: The APC gene variant (NM_000038.6: c.1620_1624delinsT) exhibits a dual transcriptional effect, revealing its pathogenic role in AFAP. This study highlights the diagnostic value of combined DNA-RNA germline testing for improving the clinical classification of novel APC gene variants and their genotype-phenotype correlations in FAP.