Andrea Accogli

The endoplasmic reticulum (ER) orchestrates the secretion of extracellular matrix (ECM) proteins, many of which exceed the size of conventional transport vesicles and therefore require specialized export machinery. TANGO1, encoded by MIA3 , organizes ER exit sites for bulky cargo export, yet the molecular basis of cargo recognition and its relationship to the collagen-specific chaperone HSP47 remain unclear. Through quantitative secretome profiling of TANGO1- and HSP47-deficient fibroblasts, structural modeling, and binding analyses, we show that TANGO1 directly and selectively recognizes a defined subset of ECM proteins, including specific collagen and fibrillin isoforms, through a conserved tyrosine residue in its luminal MOTH domain, independently of HSP47. Novel MIA3 variants identified in individuals with previously undescribed skeletal dysplasia disrupt this cargo-binding interface, leading to selective intracellular retention of ECM proteins. These findings identify direct cargo recognition, rather than ER exit-site assembly, as the primary molecular defect underlying MIA3 /TANGO1-associated skeletal dysplasia. More broadly, our work establishes TANGO1 as a selective ECM cargo receptor that functions independently of HSP47, providing a new framework for understanding bulky cargo selection at the ER.

Neuronal wiring in the developing nervous system relies on axon guidance, a process which enables proper axon pathfinding to target cells. Disruption of axon guidance results in a wide spectrum of defects including congenital mirror movements (CMM) and corpus callosum agenesis (CCA). Axon guidance signaling pathways, such as Netrin-1/DCC, act through guanine nucleotide exchange factors (GEFs) to activate Rho GTPases, which regulate axon outgrowth and pathfinding. MCF2 is an X-linked gene that encodes a GEF which activates the GTPases Rac1 and Cdc42. We identify a recurrent hemizygous MCF2 variant [NM_001171876.2: c.31C > T p.(R11W)] in two unrelated cases displaying axon guidance and corticospinal defects: an adult male with CMM and an unrelated male fetus with CCA and abnormal corticospinal tract decussation on autopsy. Through biochemical and cellular studies, we demonstrate that MCF2 interacts physically with DCC and is able to induce DCC subcellular relocalization. The MCF2 p.(R11W) variant disrupts the interaction with DCC, is less efficient at relocalizing DCC, and has decreased GEF activity. Together, our results link an MCF2 variant to axon guidance defects in humans. Furthermore, our data suggests that this MCF2 variant may impair axon guidance by impacting DCC signaling, a key regulator of commissural and corticospinal axon guidance.

Background and Objectives Somatic and germline pathogenic variants in genes of the mammalian target of rapamycin (mTOR) signaling pathway are a common mechanism underlying a subset of focal malformations of cortical development (FMCDs) referred to as mTORopathies, which include focal cortical dysplasia (FCD) type II, subtypes of polymicrogyria, and hemimegalencephaly. Our objective is to screen resected FMCD specimens with mTORopathy features on histology for causal somatic variants in mTOR pathway genes, describe novel pathogenic variants, and examine the variant distribution in relation to neuroimaging, histopathologic classification, and clinical outcomes. Methods We performed ultra-deep sequencing using a custom HaloPlex^HS Target Enrichment kit in DNA from 21 resected fresh-frozen histologically confirmed FCD type II, tuberous sclerosis complex, or hemimegalencephaly specimens. We mapped the variant alternative allele frequency (AAF) across the resected brain using targeted ultra-deep sequencing in multiple formalin-fixed paraffin-embedded tissue blocks. We also functionally validated 2 candidate somatic MTOR variants and performed targeted RNA sequencing to validate a splicing defect associated with a novel DEPDC5 variant. Results We identified causal mTOR pathway gene variants in 66.7% (14/21) of patients, of which 13 were somatic with AAF ranging between 0.6% and 12.0%. Moreover, the AAF did not predict balloon cell presence. Favorable seizure outcomes were associated with genetically clear resection borders. Individuals in whom a causal somatic variant was undetected had excellent postsurgical outcomes. In addition, we demonstrate pathogenicity of the novel c.4373_4375dupATG and candidate c.7499T>A MTOR variants in vitro. We also identified a novel germline aberrant splice site variant in DEPDC5 (c.2802-1G>C). Discussion The AAF of somatic pathogenic variants correlated with the topographic distribution, histopathology, and postsurgical outcomes. Moreover, cortical regions with absent histologic FCD features had negligible or undetectable pathogenic variant loads. By contrast, specimens with frank histologic abnormalities had detectable pathogenic variant loads, which raises important questions as to whether there is a tolerable variant threshold and whether surgical margins should be clean, as performed in tumor resections. In addition, we describe 2 novel pathogenic variants, expanding the mTORopathy genetic spectrum. Although most pathogenic somatic variants are located at mutation hotspots, screening the full-coding gene sequence remains necessary in a subset of patients.

PURPOSE: Coffin-Siris and Nicolaides-Baraitser syndromes, are recognisable neurodevelopmental disorders caused by germline variants in BAF complex subunits. The SMARCC2 BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort. METHODS: Clinical symptoms for 41 novel and 24 previously published cases were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlation, molecular data were standardized and grouped into non-truncating and likely gene-disrupting variants (LGD). Missense variant protein expression and BAF subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays. RESULTS: Neurodevelopmental delay with intellectual disability, muscular hypotonia and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, while non-truncating variants were mostly de novo and presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms. In vitro testing showed decreased protein expression for N-terminal missense variants similar to LGD. CONCLUSION: This study improved SMARCC2 variant classification and identified discernible SMARCC2 -associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

Objective: Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies.Methods: Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two‐electrode voltage clamp.Results: We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore‐lining S6 α‐helix of K_V1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co‐expressed with wild‐type K_V1.6 or K_V1.1 subunits.Significance: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain‐of‐function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.

Context The etiology of central precocious puberty (CPP) includes a spectrum of conditions. Girls younger than age 6 years with CPP should undergo cranial magnetic resonance imaging (MRI), but it remains controversial whether all girls who develop CPP between the ages of 6 and 8 years require neuroimaging examination. Objective To investigate the frequency of brain MRI abnormalities in girls diagnosed with CPP and the relationship between maternal factors, their age at presentation, clinical signs and symptoms, hormonal profiles, and neuroimaging findings. Methods Data were collected between January 2005 and September 2019 from 112 girls who showed clinical pubertal progression before 8 years of age who underwent brain MRI. Results MRI was normal in 47 (42%) idiopathic (I) scans, 54 (48%) patients had hypothalamic-pituitary anomalies (HPA) and/or extra-HP anomalies (EHPA), and 11 (10%) had brain tumors or tumor-like conditions (BT/TL), including 3 with neurological signs. Associated preexisting disorders were documented in 16. Girls with BT/TL had a higher LH peak after GnRH test (P = 0.01) than I, and those older than age 6 years had a higher craniocaudal diameter of the pituitary gland (P = 0.01); their baseline FSH and LH (P = 0.004) and peak FSH (P = 0.01) and LH (P = 0.05) values were higher than I. Logistic regression showed maternal age at menarche (P = 0.02) and peak FSH (P = 0.02) as BT/TL risk factors. Conclusions MRI provides valuable information in girls with CPP by demonstrating that fewer than half have a normal brain MRI and that few can have significant intracranial lesions after the age of 6, despite the absence of suggestive neurological signs.

Purpose To explore the relationships between clinical-radiological features and surgical outcomes in subjects with interhemispheric cysts (IHC) and corpus callosum anomalies. Methods We reviewed the clinico-radiological and neurosurgical data of 38 patients surgically treated with endoscopic fenestration, shunting, or combined approaches from 2000 to 2018 (24 males, median age 9 years). Pre- and postoperative changes in IHC volume were calculated. Outcome assessment was based on clinico-radiological data. Group comparisons were performed using χ², Fisher exact, Mann–Whitney U, and Kruskal–Wallis tests. Results Median age at first surgery was 4 months (mean follow-up 8.3 years). Eighteen individuals (47.3%) required > 1 intervention due to IHC regrowth and/or shunt malfunction. Larger preoperative IHC volume (P = .008) and younger age at surgery (P = .016) were associated with cyst regrowth. At last follow-up, mean cystic volume was 307.8 cm³, with IHC volume reduction > 66% in 19/38 (50%) subjects. The neurological outcome was good in 14/38 subjects (36.8%), fair in 18/38 (47.3%), and poor in 6/38 (15.7%). There were no differences in the postoperative cyst volume with respect to either the type of first surgery or overall surgery type. Higher absolute postoperative IHC reduction was observed in subjects who underwent both IHC fenestration and shunting procedures (P < .0001). No differences in neurological outcome were found according to patient age at surgery or degree of IHC reduction. Conclusion Endoscopic fenestration and shunting approaches are both effective but often require multiple procedures especially in younger patients. Larger IHC are more frequently complicated by cyst regrowth after surgery.