MOLECULAR AND GENETIC CHARACTERIZATION OF ALS PATIENTS

Amyotrophic lateral sclerosis (ALS) is an adult‑onset, rapidly progressive and ultimately fatal neurodegenerative disorder characterised by degeneration of upper and lower motor neurons. This leads to weakness, muscular atrophy and spasticity, which relentlessly progress to complete paralysis with a low survival rate beyond 5 years from symptom onset. In 10% of cases the disease is considered to be genetically transmitted (FALS) while in the remaining cases it occurs sporadically in the population (SALS). To date cases of hereditary ALS have been attributed to mutations in more than 16 different genes, the most common being SOD1, FUS, TARDBP and C9ORF72; mutations in other genes are rare. The above genes explain 60% of the cases of familial ALS and 15% of sporadic cases. The disease can be subdivided into bulbar (25%) and spinal‑onset (75‑80%) forms. Nevertheless, it is currently recognized that pathological changes are not limited to the motor system: patients with ALS may exhibit cognitive abnormalities ranging from impaired frontal executive dysfunction to overt frontotemporal dementia (FTD). In spite of the above evidence, ALS is regarded as a complex disease in which multiple environmental and genetic risk factors contribute to disease susceptibility. Furthermore it is possible that the phenotypic variability, which is frequently detected within families, could be due to multiple genetic factors as devised in the oligogenic disease model. In the present study we analysed 285 SALS and 17 FALS cases. Globally, our molecular analysis explained 10.3% of all ALS cases (31/302). The genes screened were SOD1, TARDBP, FUS and C9ORF72. Genomic DNA was extracted from peripheral blood through a salting out procedure; coding regions and exon‑intron boundaries of SOD1 (5 exons), TARDBP (1 exon), and FUS (5 exons) genes were amplified from genomic DNA and sequenced. Otherwise the repeat‑primed PCR assay, used for C9ORF72 gene, was performed in order to screen for the presence of the GGGGCC hexanucleotide repeats expansion in ALS patients. The repeat unit of 6 nucleotides expands up to more than several hundred times in the affected individuals. Fewer than 10 repeats are not associated with a pathological phenotype, while more than 30 repeats are associated. However we still do not know the meaning of intermediate repeat sizes (11‑29). This fact complicates the attribution of the size expansion to the pathological phenotype observed. According to the oligogenic disease model, all patients were screened for the most common ALS‑associated genes and all mutated subjects were tested also for ANG. The affected/unaffected family members, when available for the study, were screened for SOD1, TARDBP, FUS and C9ORF72 in order to identify their genetic difference from the proband and to evaluate if this difference could explain an heterogeneous phenotypic expression of the disease. Following these analyses we selected and described in detail 5 FALS and 2 SALS cases and one SETX case, with different intrafamilial phenotypic expression. In one of our SOD1 mutation carriers, the proband manifested the disease after the delivery; together with a specific angiogenin genetic variant this condition seems to have anticipated the age of disease onset and contributed to the aggressive clinical course observed in the proband compared to other family members. We also found one case in which we observed the phenomenon of anticipation, which could be due to hormonal treatments together with the simultaneous presence of 2 mutations (C9ORF72/ TARDBP), as suggested in the oligogenic disease model. Indeed, the neuroprotective effects of estrogens could account for the later age at onset in women as we have tested in another family harbouring the R521C mutation. In this case a male subject developed the disease, whereas his older sister didn’t show any neurological signs. In another family we observed a wide spectrum of clinical symptoms associated with A382T TARDBP mutation. Some family members showed cognitive impairment without signs of ALS, conversely, other relatives showed a typical ALS without any signs of dementia. In addition we have evidence of TARDBP mutation carriers without a neurological phenotype. Surprisingly, two subjects harbouring the mutation in homozygous state display different spectra of clinical symptoms. The screening of SOD1, FUS, C9ORF72 and ANG in the family members, to identify other mutations that could explain this intrafamilial phenotypic heterogeneity, resulted negative; indicating that other genes/conditions could play a role in ALS disease. These data support the hypothesis that phenotypic variability seen in patients carrying the same mutation, could be attributed to additional genetic and/or environmental factors, which could modify the penetrance of the disease. Furthermore we identified a clinical subgroup of patients that develop the disease during pregnancy (child-bearing age), thus indicating an additional modifier conditions linked to hormonal changes. For all these reasons predictive or presymptomatic testing should be undertaken with caution, especially in unaffected family members. Indeed, it is impossible for the genetic test to predict clinical outcomes based on the genotypic results only. A number of uncertainties remain, due to variability in penetrance, expressivity, and modifying factors, such as gene‑gene interactions and environmental influences. This adds to the debate for the ethical and psychological dilemmas about genetic testing, since still more has to be discovered related to this devastating disease.