Dissecting the genetic bases of Amyotrophic lateral sclerosis and Late-onset Pompe disease through Next-generation sequencing

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder that affects motor neurons. ALS genetic landscape continues to shift as the number of genes associated with the disease keeps on growing. Moreover, ALS clinical manifestation is complicated by genetic and phenotypic overlapping with Frontotemporal dementia (FTD), Hereditary spastic paraplegia (HSP), Parkinson’s disease (PD) and other neuromuscular diseases. ALS is now considered an oligogenic disease in which the sum of several variants with small effects in different genes may lead to disease onset. Late-onset Pompe disease (LOPD) is an autosomal recessive disorder, caused by mutations in the GAA gene. The molecular genetic testing for LOPD is based on Sanger sequencing, however, large deletions/duplications are poorly investigated. LOPD is characterized by highly variable clinical presentation, hence genetic factors other than pathogenic GAA mutations may play a role in determining it. The aims of the two Ph.D. projects were 1) the study of the genetic architecture of 248 ALS patients through Next-generation sequencing (NGS); the investigation of the correlation of known ALS-implicated with our ALS cohort; the study of oligogenic bases for ALS onset in our cohort of patients. 2) The genetic investigation through MLPA of LOPD patients which carried only one pathogenic mutation in the GAA gene; the study of the impact of three single nucleotide polymorphisms (SNPs) within the GAA gene on protein stability; the identification of LOPD genetic modifiers through the WES analysis on 30 LOPD patients. All the genetic analyses have been performed upon genomic DNA extraction. Through NGS, we identified 38 pathogenic variants and 31 likely pathogenic variants (16%) out of 423 variants retrieved in the ALS cohort of patients. Among the pathogenic and likely pathogenic variants, 25 were in ALS causative genes, followed by 5 variants in the HSP and PD associated genes respectively and the remaining three variants were in genes linked to other NDs. We established a high degree of genetic heterogeneity among our patients, with 26 different ALS-implicated variants from 14 ALS genes identified among 54,4% of cases. Among the 26 variants, two were significantly associated with our cohort of ALS patients, an in-frame deletion (p.Gly173_Gly174del) within FUS and a missense variant (p.Arg208Trp) within SIGMAR1. Additionally, 7 patients (3%) of our cohort presented more than one of the listed ALS-implicated variants, suggesting oligogenic bases for these patients. Through MLPA analysis we identified two heterozygous deletions in 2 LOPD patients, one in exon 17 and one in exon 1 of the GAA gene. Through NGS we found a total of 176 variants in 103 (34%) out of 302 genes analyzed across six virtual gene panels. Through the statistical analysis, 73 variants out of 176 (43%) resulted significantly associated with our cohort of patients, however, we lack to found genetic modifiers for the clinical presentation of the LOPD pathology. This experimental thesis has reaffirmed the heterogeneity of the genetic of ALS, which takes into consideration not only ALS-causative genes and risk factors in genes notably associated with ALS, but consider also genes associated with other NDs such as FTD, HSP and PD, as well as gene-related to other motor neuron diseases, highlighting the importance of genetic comorbidity studies. Furthermore, here we confirmed the new vision of ALS as a polygenic disease characterized by oligogenic inheritance. Additionally, here we underline also the importance of the utilization of NGS technology in genetically less complex diseases, such as LOPD, to find genetic variants associated with the disease that may at the basis of the clinical variability observed among LOPD patients. Eventually, MLPA technology should be used when only one pathogenic mutation is identified.