# Multiple Genetic Rare Variants in Autism Spectrum Disorders: A Single-Center Targeted NGS Study
## 4 Discussion  
The diagnostic yield of targeted gene panels in the evaluation of individuals with ASD has not been well defined, but it settles at around 12-14% [34,35]. In this study, our purpose is to evaluate the potential contribution of every single rare variant and not to search for a single variant causing the onset of ASD. Interestingly, we found only 4 de novo variants in _ANKRD11_, _NF1_, _SLIT3_, and _PTEN_ in 3 patients (4/105 variants). These data are slightly lower than the ratio published in the literature (according to [PERSON] et al. [36], de novo loss-of-function mutations are in over 5% of ASD patients), and this is probably due to the small number of samples and genes analyzed in our cohort. Another explanation could derive from the recruitment criteria, namely, the exclusion of patients with syndromic characteristics, which are more easily caused by variants in genes already associated with other syndromes and not inherited (De novo). All the four genes are reported in the literature as autism-risk genes or genes causing syndromes, in which subgroups of patients may develop autism [37, 38, 39]. Furthermore, in our cohort, the presence of known variants does not seem to contribute to the pathogenic phenotype more than those variants that have never been associated with ASD. In fact, all the variants belong to the (f) group, except two that fall into the (*) group, and only two variants have two deleterious in silico predictions (SIFT and Polyphen). Thus, although they were already associated with ASD, it is difficult to attribute to them a highly pathogenic role, even though there is always more than one variant in other genes. However, it is certain that they contribute to ASD onset. Only the SLIT3 variant seems to play a determinant role in ASD onset in the L14/95 patient, given its de novo origin, unlike the other 4 variants transmitted by the mother. It would be interesting, in this specific case, to widen the search for variants to other autism-related genes in order to clarify their pathogenic role. Moreover, we noted significantly increased frequencies of rare variants in 9 genes in our study population, with more than or at least 5 variants each. In this regard, we must specify that the analyzed population is composed of patients from all over the Italian territory and that 9 patients have foreign origins: a heterogeneous group comparable to GnomAD population. The results indicate that the mutational frequency in all the studied genes is higher than expected. Our study highlights the importance of using gene panels to better understand the contribution of the different genes already associated with ASD in the onset of the disease. _FAT1_ and _VPS13B_ resulted in the most mutated genes in our cohort, with 12 variants each. The _FAT1_ gene encodes a member of a small family of vertebrate cadherin-like genes, whose gene products play a role in cell migration, lamellipodia dynamics, cell polarity, and cell-cell adhesions (summary by [PERSON] et al. (2016) [40]). Rare de novo missense variants in _FAT1_ have been identified in ASD probands by WES in two reports [37,41], while inherited damaging missense variants in FAT1 have been observed in affected individuals from 3 extended multiplex ASD families [23]. [PERSON] et al. (2015) [42] identified heterozygous missense variants in FAT1 in 10 out of 49 unrelated Japanese patients with a neuromuscular phenotype similar to facioscapulohumeral muscular dystrophy. [PERSON] et al. chose FAT1 as a candidate gene based on the findings of [PERSON] et al. (2013) [43], which demonstrated how hypomorphic Fat1 mice show an FSHD-like phenotype. [PERSON] et al. (2013) [44] reported recurrent somatic mutations in FAT1 in glioblastoma, colorectal cancer, and head and neck cancer. In the SFARI database, FAT1 is classified as a gene, with evidence suggesting its implication in ASD (score 3). The _VPS13B_ gene has been associated with syndromic autism, where a subpopulation of individuals with a given syndrome develop autism.