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Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel ( SpastiSure3.0 ) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy.

In humans, the most common genomic disorder is the hemizygous deletion of the chromosome 22q11.2 region, that results in the "22q11.2 deletion syndrome" (22q11.2DS). A peculiarity of 22q11.2DS is its great phenotypic variability that makes this pathology a classic example of a syndrome with variable expressivity and incomplete penetrance. The reasons for this variability have not been elucidated yet, and the molecular substrates underlying the different clinical features of 22q11.2DS are still debated. A cohort of 21 patients has been analyzed by array CGH in order to detect some of the genetic differences that may influence this variability. Two aspects have been investigated: (1) the precise localization of the deletion breakpoints within the low copy repeats (LCRs), (2) the additional Copy Number Variations (CNVs) elsewhere in the genome, by analyzing their gene content. Both protein-coding genes and miRNAs were considered, in order to discover possible epistatic interactions between genes of the 22q11.2 region and the rest of the genome. Eighteen out of twenty-one patients had a deletion of ~3 Mb mediated by LCR22-A and D, whereas 3/21 had a smaller deletion. The breakpoints within the LCR22-A and D do not have a major role in the phenotypic variability since they are rather clustered and the small differences concern genes that are not directly related to clinical signs of 22q11.2DS. A detailed analysis of the gene content of 22q11.2 deleted region indicates that this syndrome could be a bioenergetic disorder or consequence of an altered post-transcriptional gene regulation, due to the presence of DGCR8 , a major player of the microRNA (miRNA) biogenesis. Only four genes with mitochondrial function are harbored in the additional CNVs, whereas 11 miRNA, all related to biological pathways present in the 22q11.2DS, have been detected in 19/21 patients. CNVs and miRNAs are new entities that have changed the order of complexity at the level of gene expression and regulation, thus CNV-miRNAs (miRNA harbored in the CNVs) are potential functional variants that should be considered high priority candidate variants in genotype-phenotype association studies. Deletion of DGCR8 , the main actor in miRNA biogenesis, amplifies this variability. To our knowledge, this is the first report that focus on the miRNA-CNVs in 22q11.2DS, with the aim of trying to better understand their role in the variable expressivity and incomplete penetrance.