# Multiple Genetic Rare Variants in Autism Spectrum Disorders: A Single-Center Targeted NGS Study
## 4 Discussion
In particular, rare mutations of the _VPS13B_ gene were associated with [PERSON] syndrome [45]. This gene encodes a potential transmembrane protein that may be involved in the vesicle-mediated transport and sorting of proteins within the cell. This protein may play a role in the development and function of the eye, hematological system, and central nervous system. Multiple splice variantsencoding distinct isoforms were identified for this gene, and when we designed the NGS panel, VPS13B was part of score category S (syndromic). Now, VPS13B belongs to gene category 1; hence, several variants in VPS13B have been associated with ASD. Very interestingly, two patients share 2 variants in 2 different genes (_RAI1_ and _ZEB2_). These variants are not extremely rare in the general population, making it necessary to screen a larger cohort of patients to verify the hypothesis of a potential additive effect of these variants on the development of the clinical phenotype, even though _RAI1_ and _ZEB2_ are reported to be functionally linked [46]. As for the other repeated variants, they are not very rare in the general population, and for this reason, it is necessary to enlarge our cohort to evaluate their role in autism onset. In testing the hypothesis that variants can be considered as susceptibility factors in a family genetic background, we should not expect to find variants on both alleles of the same gene. Nonetheless, in our cohort, we found one homozygous variant in the _MET_ gene, which was inherited from both parents, supporting a potential direct pathogenic role of this mutation. Indeed, the _MET_ gene is classified as a strong candidate on the SFARI database (score 2), and positive associations in the Caucasian, Japanese, and Italian populations were found in multiple studies. In addition, biochemical assays showed a reduction in the MET protein levels and a general disruption of MET signaling in ASD patients. We analyzed the coding regions of 120 ASD candidate genes in 40 autism patients to identify novel mutations, risk genes, and new genotype-phenotype associations. Among the 40 samples screened, only three resulted in carriers of a unique candidate variant, while samples from 36 patients resulted in more than one carrier. This result is very interesting, especially because the panel used allows for the analysis of only 120 genes, despite the approximately one thousand genes currently associated with ASD. The analysis of the 120 genes included in the panel allowed us to confirm the role of specific genes, such as _MET_ and _SLIT3_, as genetic factors strongly associated with ASD. It also highlighted the potentially stronger involvement of other genes, such as _FAT1_, which is classified in the SFARI database with score category 3, and _VPS13B_, which was classified in the SFARI database with score category S, when we designed the gene panel, and only recently moved to gene category 1, showing a frequent mutation in our cohort. In addition to these data, we also evidenced, through the analysis of the variant segregations, a generally equal distribution of parenally and maternally transmitted variants. Here, we find that the situation is the same, although we consider only extremely rare variants or variants with at least a deleterious pathogenicity prediction in silico, as if the extremely rare or deleterious variants, which are potentially more harmful, were causative of disease onset only if present together. This can further support the hypothesis of a threshold effect model. According to this hypothesis, rare and/or deleterious inherited mutations are not sufficient alone to cause the phenotype but need to co-exist with other factors (genetic, immunologic, or environmental), whose contribution to the pathogenesis of the disease remains unknown. The fact that these variants are inherited from a couple of unaffected parents could suggest the presence of a familial mutational burden in these patients.