*2.3. Genomic Analysis*

Karyotyping was performed according to standard methods. The commercially available D22S75/N25 probe (Cytocell, Cambridge, UK) was used, according to the manufacturer's instructions, to perform fluorescent in situ hybridization (FISH) analysis.

Genomic DNA of the patients (tests) was isolated from peripheral blood using standard methods; DNA from healthy subjects (reference) was used as controls (Agilent Technologies, Santa Clara, CA, USA); tests and reference DNA were differentially labeled with Cy5-dCTP or with Cy3-dCTP using random primer labeling and applied to 60K arrays, according to the manufacturer's protocol (Agilent, Santa Clara, CA, USA). Quality slide evaluation was performed using the Agilent dedicated software (Feature Extraction, Agilent). We elaborated only on those experiments that met the "excellent" criteria, as determined by the QC report (Cytogenomic software, Agilent). In particular, the derivative log ratio spread (DLRS) was the main value considered for further analysis of the data: when >0.16, the experiment was discarded and repeated. CNVs were identified with Cytogenomics 4.0.3.12 (Agilent) using the ADM-2 (aberration detection method 2) algorithm. The threshold was set to a minimum of 6 with the minimum number of 3 probes required in a region and a minimum absolute log ratio of 0.25. We analyzed all the CNVs with 3 or more contiguous probes for deletions and duplications. The CNVs reported in the Database of Genomic Variants (http://projects.tcag.ca/variation/ (accessed on 25 February 2022)) were classified as benign and not further analyzed. All the other genomic imbalances were compared with those collected in DECIPHER (https://decipher.sanger.ac.uk/ (accessed on 25 February 2022)) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ (accessed on 25 February 2022)). Data about the biological function of genes and their interactions were retrieved from UCSC Genome Browser (http://genome.ucsc.edu/ (accessed on 25 February 2022)), PubMed (https://www.ncbi.nlm.nih.gov/pubmed/ (accessed on 25 February 2022)), and OMIM (https://www.omim.org/ (accessed on 25 February 2022)).

#### **3. Results**
