NTNG2 encodes netrin-G2, a membrane-anchored protein implicated in the molecular organization of neuronal circuitry and synaptic organization and diversification in vertebrates. In this study, through a combination of exome sequencing and autozygosity mapping, we have identified 16 individuals (from seven unrelated families) with ultra-rare homozygous missense variants in NTNG2; these individuals present with shared features of a neurodevelopmental disorder consisting of global developmental delay, severe to profound intellectual disability, muscle weakness and abnormal tone, autistic features, behavioral abnormalities, and variable dysmorphisms. The variants disrupt highly conserved residues across the protein. Functional experiments, including in silico analysis of the protein structure, in vitro assessment of cell surface expression, and in vitro knockdown, revealed potential mechanisms of pathogenicity of the variants, including loss of protein function and decreased neurite outgrowth. Our data indicate that appropriate expression of NTNG2 plays an important role in neurotypical development.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Major Facilitator Superfamily Domain containing 2a (MFSD2A) is an essential endothelial lipid transporter at the blood-brain barrier. Biallelic variants affecting function in MFSD2A cause autosomal recessive primary microcephaly 15 (MCPH15, OMIM# 616486). We sought to expand our knowledge of the phenotypic spectrum of MCPH15 and demonstrate the underlying mechanism of inactivation of the MFSD2A transporter. We carried out detailed analysis of the clinical and neuroradiological features of a series of 27 MCPH15 cases, including eight new individuals from seven unrelated families. Genetic investigation was performed through exome sequencing (ES). Structural insights on the human Mfsd2a model and in-vitro biochemical assays were used to investigate the functional impact of the identified variants. All patients had primary microcephaly and severe developmental delay. Brain MRI showed variable degrees of white matter reduction, ventricular enlargement, callosal hypodysgenesis, and pontine and vermian hypoplasia. ES led to the identification of six novel biallelic MFSD2A variants (NG_053084.1, NM_032793.5: c.556+1G>A, c.748G>T; p.(Val250Phe), c.750_753del; p.(Cys251SerfsTer3), c.977G>A; p.(Arg326His), c.1386_1435del; p.(Gln462HisfsTer17), and c.1478C>T; p.(Pro493Leu)) and two recurrent variants (NM_032793.5: c.593C>T; p.(Thr198Met) and c.476C>T; p.(Thr159Met)). All these variants and the previously reported NM_032793.5: c.490C>A; p.(Pro164Thr) resulted in either reduced MFSD2A expression and/or transport activity. Our study further delineates the phenotypic spectrum of MCPH15, refining its clinical and neuroradiological characterization and supporting that MFSD2A deficiency causes early prenatal brain developmental disruption. We also show that poor MFSD2A expression despite normal transporter activity is a relevant pathomechanism in MCPH15.

To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype–phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.

Synaptotagmins are integral synaptic vesicle membrane proteins that function as calcium sensors and regulate neurotransmitter release at the presynaptic nerve terminal. Synaptotagmin‐2 (SYT2), is the major isoform expressed at the neuromuscular junction. Recently, dominant missense variants in SYT2 have been reported as a rare cause of distal motor neuropathy and myasthenic syndrome, manifesting with stable or slowly progressive distal weakness of variable severity along with presynaptic NMJ impairment. These variants are thought to have a dominant‐negative effect on synaptic vesicle exocytosis, although the precise pathomechanism remains to be elucidated. Here we report seven patients of five families, with biallelic loss of function variants in SYT2, clinically manifesting with a remarkably consistent phenotype of severe congenital onset hypotonia and weakness, with variable degrees of respiratory involvement. Electrodiagnostic findings were consistent with a presynaptic congenital myasthenic syndrome (CMS) in some. Treatment with an acetylcholinesterase inhibitor pursued in three patients showed clinical improvement with increased strength and function. This series further establishes SYT2 as a CMS‐disease gene and expands its clinical and genetic spectrum to include recessive loss‐of‐function variants, manifesting as a severe congenital onset presynaptic CMS with potential treatment implications.

Bi‐allelic TECPR2 variants have been associated with a complex syndrome with features of both a neurodevelopmental and neurodegenerative disorder. Here, we provide a comprehensive clinical description and variant interpretation framework for this genetic locus. Through an international collaboration, we identified 17 individuals from 15 families with bi‐allelic TECPR2‐variants. We systemically reviewed clinical and molecular data from this cohort and 11 cases previously reported. Phenotypes were standardized using Human Phenotype Ontology terms. A cross‐sectional analysis revealed global developmental delay/intellectual disability, muscular hypotonia, ataxia, hyporeflexia, respiratory infections and central/nocturnal hypopnea as core manifestations. A review of brain MRI scans demonstrated a thin corpus callosum in 52%. We evaluated 17 distinct variants. Missense variants in TECPR2 are predominantly located in the N‐ and C‐terminal regions containing β‐propeller repeats. Despite constituting nearly half of disease associated TECPR2 variants, classifying missense variants as (likely) pathogenic according to ACMG criteria remains challenging. We estimate a pathogenic variant carrier frequency of 1/1,221 in the general and 1/155 in the Jewish Ashkenazi populations. Based on clinical, neuroimaging and genetic data, we provide recommendations for variant reporting, clinical assessment, and surveillance/treatment of individuals with TECPR2‐associated disorder. This sets the stage for future prospective natural history studies.