Background: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). Methods: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). Results: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. Conclusion: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

Angelman syndrome (AS) is a neurogenetic disorder characterized by severe developmental delay with absence of speech, happy disposition, frequent laughter, hyperactivity, stereotypies, ataxia and seizures with specific EEG abnormalities. There is a 10–15% of patients with an AS phenotype whose genetic cause remains unknown (Angelman-like syndrome, AS-like). Whole-exome sequencing (WES) was performed on a cohort of 14 patients with clinical features of AS and no molecular diagnosis. As a result, we identified 10de novoand 1 X-linked pathogenic/likely pathogenic variants in 10 neurodevelopmental genes (SYNGAP1,VAMP2,TBL1XR1,ASXL3,SATB2,SMARCE1,SPTAN1,KCNQ3,SLC6A1andLAS1L) and one deleteriousde novovariant in a candidate gene (HSF2). Our results highlight the wide genetic heterogeneity in AS-like patients and expands the differential diagnosis.

KIF1A is a microtubule-dependent motor protein responsible for fast anterograde transport of synaptic vesicle precursors in neurons. Pathogenic variants in KIF1A have been associated with a wide spectrum of neurological disorders. Here, we report a patient presenting a severe neurodevelopmental disorder carrying a novel de novo missense variant p.Arg169Thr (R169T) in the KIF1A motor domain. The clinical features present in our patient match with those reported for NESCAV syndrome including severe developmental delay, spastic paraparesis, motor sensory neuropathy, bilateral optic nerve atrophy, progressive cerebellar atrophy, epilepsy, ataxia, and hypotonia. Here, we demonstrate that the microtubule-stimulated ATPase activity of the KIF1A is strongly reduced in the motor domain of the R169T variant. Supporting this, in silico structural modeling suggests that this variant impairs the interaction of the KIF1A motor domain with microtubules. The characterization of the molecular effect of the R169T variant on the KIF1A protein together with the presence of the typical clinical features indicates its causal pathogenic effect.

High throughput methods such as next generation sequencing are increasingly used in molecular diagnosis. The aim of this study was to develop a workflow for the detection ofBRCA1andBRCA2mutations using massive parallel sequencing in a 454 GS Junior bench top sequencer. Our approach was first validated in a panel of 23 patients containing 62 unique variants that had been previously Sanger sequenced. Subsequently, 101 patients with familial breast and ovarian cancer were studied.BRCA1andBRCA2exon enrichment has been performed by PCR amplification using the BRCA MASTR kit (Multiplicom). Bioinformatic analysis of reads is performed with the AVA software v2.7 (Roche). In total, all 62 variants were detected resulting in a sensitivity of 100%. 71 false positives were called resulting in a specificity of 97.35%. All of them correspond to deletions located in homopolymeric stretches. The analysis of the homopolymers stretches of 6 bp or longer using the BRCA HP kit (Multiplicom) increased the specificity of the detection ofBRCA1andBRCA2mutations to 99.99%. We show here that massive parallel pyrosequencing can be used as a diagnostic strategy to test forBRCA1andBRCA2mutations meeting very stringent sensitivity and specificity parameters replacing traditional Sanger sequencing with a lower cost.

Neurodevelopmental disorders (NDDs) affect 2–5% of the population and approximately 50% of cases are due to genetic factors. Since de novo pathogenic variants account for the majority of cases, a gene panel including 460 dominant and X-linked genes was designed and applied to 398 patients affected by intellectual disability (ID)/global developmental delay (GDD) and/or autism (ASD). Pathogenic variants were identified in 83 different genes showing the high genetic heterogeneity of NDDs. A molecular diagnosis was established in 28.6% of patients after high-depth sequencing and stringent variant filtering. Compared to other available gene panel solutions for NDD molecular diagnosis, our panel has a higher diagnostic yield for both ID/GDD and ASD. As reported previously, a significantly higher diagnostic yield was observed: (i) in patients affected by ID/GDD compared to those affected only by ASD, and (ii) in females despite the higher proportion of males among our patients. No differences in diagnostic rates were found between patients affected by different levels of ID severity. Interestingly, patients harboring pathogenic variants presented different phenotypic features, suggesting that deep phenotypic profiling may help in predicting the presence of a pathogenic variant. Despite the high performance of our panel, whole exome-sequencing (WES) approaches may represent a more robust solution. For this reason, we propose the list of genes included in our customized gene panel and the variant filtering procedure presented here as a first-tier approach for the molecular diagnosis of NDDs in WES studies.