# Multiple Genetic Rare Variants in Autism Spectrum Disorders: A Single-Center Targeted NGS Study
## 2 Materials and Methods  
We recruited 40 patients with Autism Spectrum Disorder (36 males, 4 females), coming from all over the Italian territory, who were referred to the Neurodevelopment Disorders Unit of C. Besta Institute between January 2017 and July 2018. The diagnoses of Autism Spectrum Disorder followed the diagnostic criteria of DSM-5 and were confirmed by clinical examination, consisting of an accurate anamnesis, Autism Diagnostic Interview-Revised, ADI-R [16], and the Autism Diagnostic Observation Schedule--II edition, ADOS2 [17]. The cognitive functioning was assessed using the Wechsler Intelligence Scales [18], according to the age of the patients and the Griffiths Mental Developmental Scale for children with chronological or mental age under 4 years [19; 20], considering the correlation between Wechsler Preschool Scale Full IQ and General quotient obtained by Griffiths Scales [21]. The developmental delay or intellectual disability were defined by a GQ or a QI lower than 70. All the patients underwent an accurate clinical evaluation. All the patients were evaluated by a pediatric neurologist, a clinical geneticist, and a child neuropsychological and underwent the following instrumental screening:  
* Brain magnetic resonance imaging (MRI), 1.5 Tesla, without contrast enhancement (with sagittal, transverse, and coronal sequences, as well as FLAIR sequences of the whole brain and cerebellum);
* Electroencephalogram (EEG);
* A plasma amino acid assay using liquid chromatography-tandem mass spectrometry (ESI/MS/MS) and urinary organic acid assay using gas chromatography-mass spectrometry;
* Standard karyotyping on peripheral blood by means of a lymphocyte culture and QFQ banding, with a resolution of 550 bands (number of metaphases analyzed: 16);
* Molecular analysis for fragile X syndrome, using Southern blotting;
* Array-comparative genomic hybridization. We excluded patients with (1) preterm birth, known pregnancy complications or a history of perinatal injuries; (2) major neuroradiologic alterations (i.e., cortical dysplasia);(3) epileptic syndromes (e.g., West syndrome); (4) known infectious or metabolic diseases; (5) major facial and somatic peculiar characteristics; (6) major limb or visceral malformations; (7) the presence of undergrowth or overgrowth syndromes; and (8) genetic diseases (high-resolution karyotype and DNA analysis of Fragile-X syndrome) or positive genetic testing, including the presence of any copy number variants in CGH testing. All the examinations were performed with written informed consent from the subjects' parents. The study was approved by the Ethics Committee of Fondazione IRCCS Istituto Neurologico C. Besta. The genomic DNA was extracted from the whole blood of all the patients enrolled in the study and, when possible, of their parents. The DNA samples were analyzed by targeted resequencing using customized gene panels (Nextera Rapid Capture Custom Enrichment, Illumina) based on a systematic literature review. In detail, we examined (i) the targeted gene panels available for sale (TruSight Autism Target Genes and TruSight Inherited Disease Target Genes, Illumina), (ii) the main genes implicated in ID and ASD reported in the literature [22, 23, 24, 25], and (iii) the SFARI gene database (the most important and complete Autism variants database) (Table 1). In total, 120 genes were selected, and custom probes were designed, focusing on gene coding sequences (CDS), with a +/ - 20 bp intronic flanking region to include splicing regulatory elements. Enrichment of the libraries was performed according to the Nextera Rapid Capture Enrichment protocol (Illumina), and the generated libraries were then loaded and sequenced on the MiSeq platform (Illumina). The generated reads were aligned to the human genome assembly, hg19 (Human February 2009 GRCh37), using BWA (v0.7.9a), and a variant call was performed using GATK (v1.6-23). The variants identified were annotated and filtered (Variant-Studio3.0, Illumina).