Genetic profile of ALS patients in Portugal

Background Mutation frequency of the two main Amyotrophic Lateral Sclerosis (ALS)-related genes, C9orf72 and SOD1, varies considerably across the world. To date, there is no available information about the prevalence of genetic mutations in ALS Portuguese patients or about the phenotypic characteristics of mutation carriers. The combination of genetic information with detailed clinical phenotyping will have implications in future genetic studies, in genetic counseling and in devising future trials targeted to specific mutations. Objectives To assess the mutation frequency of SOD1 and C9orf72 , in a Portuguese ancestry population-based series of cases and compare it with other populations; to analyze the demographic and clinical features of those patients; and to further investigate the genetic cause of FALS and/or concomitant Frontotemporal Dementia (FTD) in patients without mutations in those two genes. Methods We analyzed SOD1 and C9orf72 in a large population of Portuguese ALS patients (n=371) recruited over 12 years. Fragment length analysis and repeat-primed PCR was used to screen for the presence of >30 GGGGCC repeats in the first intron of C9orf72 gene. The coding region and the flanking intron-exon boundaries of SOD1 were analyzed by bidirectional Sanger sequencing. Next generation sequencing was used to further investigate genetic variation in a panel of 25 known ALS-related genes in a cohort of 10 patients with familial ALS and/or concomitant FTD. Results Familial ALS (FALS) was disclosed in 11.6% of patients. Mutations in either SOD1 or C9orf72 were found in 9.2% of patients and accounted for 40% of FALS and 5.2% of apparently sporadic ALS (SALS). Only three (0.83%) SOD1 mutations were found: the very rare G16S (c.49G>A), the D90A (c.272A>C) and a novel likely pathogenic A152P (c.457G>C). The C9orf72 hexanucleotide repeat expansion (HRE) accounted for 4.6% of SALS and 37.5% of FALS; in these patients, FTD was prevalent. Among the additional 25 ALS-related genes studied we identified some previously unreported rare variants, most of which of uncertain clinical significance. Still, the majority of patients with FALS or concomitant FTD did not carry any probable or possible pathogenic variant. Discussion This study adds to our knowledge of the worldwide frequency of mutations in known ALS genes, as well as genotype–phenotype correlations. Of the ALS genes investigated so far in the Portuguese population, C9orf72 is the most often mutated gene. We identified a novel SOD1 missense mutation but the SOD1 mutation frequency was significantly lower than in most countries. Although patients with C9orf72 HRE have a higher prevalence of FTD, the concomitance of ALS and FTD cannot be explained by C9orf72 HRE in 42% of FALS and 83% of SALS patients. Moreover, most FALS did not carry a SOD1 mutation or C9orf72 HRE. Further studies demonstrated widespread absence of likely pathogenic variants in the 25 ALS-related genes investigated and thus, many concurrent FALS and FTD lacking the C9orf72 HRE remain unexplained. The mutation incidence in ALS patients and associated phenotypes suggest that genetic tests should be offered to more patients and other genes should be investigated.