Sorting Rare ALS Genetic Variants by Targeted Re-Sequencing Panel in Italian Patients: OPTN, VCP, and SQSTM1 Variants Account for 3% of Rare Genetic Forms

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease due to motor neuron loss variably associated with frontotemporal dementia (FTD). Next generation sequencing technology revealed an increasing number of rare and novel genetic variants and interpretation of their pathogenicity represents a major challange in the diagnosis of ALS. We selected 213 consecutive patients with sporadic or familial (16%) ALS, tested negative for SOD1, FUS, TARDBP, and C9orf72 mutations. To reveal rare forms of genetic ALS, we performed a comprehensive multi-gene panel screening including 46 genes associated with ALS, hereditary motor neuronopathies, spastic paraplegia, and FTD. Our study allowed the identification of pathogenic or likely pathogenic variants in 4.2% of patients. The genes with the highest percentage of pathogenic variants were OPTN (1%), VCP (1%) SQSTM1(1%), SETX (0.4%), FIG4 (0.4%), and GARS1 (0.4%) genes. We also found 49 novel or rare gene variants of unknown significance in 30 patients (14%), 44 unlikely pathogenic variants (39%), and 48 variants in ALS susceptibility genes. The results of our study suggest the screening of OPTN, VCP, and SQSTM1 genes in routine diagnostic investigations for both sporadic and familial cases, and confirm the importance of diagnosis and couselling for patients and their relative family members.

High-Throughput Genetic Testing in ALS: The Challenging Path of Variant Classification Considering the ACMG Guidelines

“…Comparing our results with those presented in previous reports [ 5 , 6 ], we can confirm that the majority of pathogenic variants identified are C9orf72 repeat expansions (39/59, 66%), followed by SOD1 (10/59, 17%) and TARDBP (8/59, 14%) variants.…”

“…We were probably more conservative than other authors [ 5 ] in variant classification, since we found pathogenic or likely pathogenic variants only in 8 out 39 tested genes (21%). For instance, Pensato and colleagues identified these categories of variants in 12 out of 46 genes (26%) [ 5 ].…”

“…However, according to the ACMG guidelines, the circumstance that a variant is expected to be synonymous only guarantees a supporting evidence of benignity (BP7), and only if computational evidence predicts no impact on RNA splicing and no evolutionary conservation of the nucleotide. Therefore, the integration of splice prediction tools in the variant annotation pipeline would be a good practice for a more comprehensive analysis, as already implemented in some literature studies [ 5 ].…”

“…By applying these criteria, variants should be classified in five different categories: “Pathogenic”, “likely pathogenic”, “uncertain significance”, “likely benign”, and “benign”. Recently, researchers used the ACMG standards to interpret variants found in ALS patients [ 4 , 5 , 6 ]. In this study, we discuss the application of ACMG guidelines to ALS, combining data reported in literature with results obtained from a large ALS cohort, screened with gene panel sequencing over a four-year period.…”

Targeted sequencing panels in Italian ALS patients support different etiologies in the ALS/FTD continuum

“…Four major genes, chromosome 9 open reading frame 72 (C9orf72), superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP), and fused in sarcoma (FUS) cover up to 60% of fALS and 10-13% of sALS cases [9]. Variants in other genes are found in < 1% of patients [10,11].…”