**2. Cu/Zn Superoxide Dismutase (SOD1 gene)**

Superoxide dismutase [Cu-Zn] also known as superoxide dismutase 1 or SOD1 is a soluble protein acting as a 32 kDa homodimeric enzyme. SOD1 is one of three human superoxide dismutases.

Its main function is the conversion, naturally occurring, but harmful, superoxide radicals to molecular oxygen and hydrogen peroxide.

SOD1 binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. The encoded isozyme is a soluble cytoplasmic and mitochondrial inter-membrane space protein, acting as a homodimer to convert naturally occurring, but harmful, superoxide radicals to molecular oxygen and hydrogen peroxide

### **2.1. Genotype**

**g.** Pure lower motor neuron phenotype is characterised by clinical and electrophysiological evidence of progressive LMN involvement. Patients with family history of inherited spinal muscular atrophy are excluded. It has a low incidence rate and is twice as frequent in men. Patients with this form are younger than those with any other ALS phenotype, with a peak of incidence rate in the seventh decade among men and in the sixth decade

among women. Nobody has FTD and mean survival is the longest (7 years) [4].

(more than 10 years) [4].

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**h.** Patients with pure upper motor neuron have signs of UMN involvement (severe spastic para/tertaparesis associated with Babinski or Hoffmann sign, hyperactive reflexes, clonic jaw jerk, dysarthric speech and pseudobulbar affect). Patients with clinical or EMG signs of LMN involvement or with history of spastic para/tetraparesis in family such as hereditary spastic paraplegia are excluded. It has a low incidence rate with peak in the sixth decade in both genders, Median survival time is the longest among ALS phenotype

**Table 2.** Mean age at onset, mean time delay from onset to diagnosis and frequency of frontotemporal dementia [4].

**Table 3.** Amyotrophic lateral sclerosis phenotypes. Overall and men versus women mean annual crude incidence

raters (/100000 population), 95% CIs and gender incidence rate ratios [4].

The human *SOD1* gene (Entrez Gene ID 6647) is located on chromosome 21q22.11, and it codes for the monomeric SOD1 polypeptide (153 amino acids, molecular weight 16 kDa).

The vast majority of which are missense substitutions distributed throughout the five exons of the gene. Also frame-shift deletions and insertions, all clustered in exons 4 and 5, which lead

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Collectively, SOD1 mutations are found in ~20% of all FALS patients, and in ~3% of

In Italian ALS population, different screening have been performed [2, 18] and both confirmed

About FALS the percentage of SOD1-mutated FALS patients was 14.7% [18]. In the most recent screening of 480 SALS patients in 48 FALS has been found that the percentage of mutations

Novel mutations are continuous discovered, the last one in Italian patient has been described

Patients with SOD1 mutations, FALS and SALS, show a phenotypic heterogeneity even within the same mutation although some sporadic missense mutations carry a consistently worse or better prognosis. A lot of mutations have been described during the time, we have focused in this paragraph on the most interesting in the Italian population for eventually presence of

Patients with FALS and G41S mutations have similar clinical phenotype with early upper and lower motor neuron involvement in one or both lower limbs, rapidly spreading to upper limbs,

In 2010 Battistini et al. [22] described a family with 9 members ALS affected, in which there was evidence of a missense mutation in exon 4 (L106F) in SOD1 gene. In this family there was autosomal dominant inheritance. The clinical presentation was characterized by relatively

to a premature truncation of the protein have been described (Figure 2).

that the percentage of mutation in SOD1 gene in Italian SALS was 4.5%.

**Figure 2.** Distribution of *SOD1* mutations detected in sporadic ALS patients.

correlation between genotype and phenotype, both for FALS and SALS.

appearance of bulbar signs within 1 years and death a few months later [21].

SALS cases [17].

was totally 2.1% [19].

in January 2012 [20].

**2.2. Phenotype**

**Figure 1.** Summarize the percentage of mutations in Italian ALS patients, (a) SALS, (b) FALS [2, 9, 10, 11, 12, 13, 14].

In 1991, Siddique and collaborators [15] identified a linkage between familial ALS and the SOD1 locus on chromosome 21q22 and demonstrated genetic locus heterogeneity in FALS studying 23 ALS families.

In 1993, Rosen and collaborators [16] have reported tight genetic linkage between ALS and *SOD1* gene, establishing *SOD1* as the first causative gene for ALS. More than 150 *SOD1* mutations have been reported in 68 of the 153 codons, spread over all five exons (ALS Online Genetic Database, ALSOD: http://alsod.iop.kcl.ac.uk/).

The vast majority of which are missense substitutions distributed throughout the five exons of the gene. Also frame-shift deletions and insertions, all clustered in exons 4 and 5, which lead to a premature truncation of the protein have been described (Figure 2).

Collectively, SOD1 mutations are found in ~20% of all FALS patients, and in ~3% of SALS cases [17].

In Italian ALS population, different screening have been performed [2, 18] and both confirmed that the percentage of mutation in SOD1 gene in Italian SALS was 4.5%.

About FALS the percentage of SOD1-mutated FALS patients was 14.7% [18]. In the most recent screening of 480 SALS patients in 48 FALS has been found that the percentage of mutations was totally 2.1% [19].

Novel mutations are continuous discovered, the last one in Italian patient has been described in January 2012 [20].

**Figure 2.** Distribution of *SOD1* mutations detected in sporadic ALS patients.

#### **2.2. Phenotype**

In 1991, Siddique and collaborators [15] identified a linkage between familial ALS and the SOD1 locus on chromosome 21q22 and demonstrated genetic locus heterogeneity in FALS

**Figure 1.** Summarize the percentage of mutations in Italian ALS patients, (a) SALS, (b) FALS [2, 9, 10, 11, 12, 13, 14].

SOD1 FUS TARDBP ANG C9orf72 OPTN non-mutated Other genes (VAPB, FIG4, SPG11, PFN1)

**Italian SALS**

SOD1 FUS TARDBP ANG C9orf72 OPTN non-mutated

**Italian FALS**

0,3% 17,9%

23,9% 1,2%

4,5%

0,7%2,7% 1,0% 5,1%

3,5%

4,4%

4,0%

2,3%

In 1993, Rosen and collaborators [16] have reported tight genetic linkage between ALS and *SOD1* gene, establishing *SOD1* as the first causative gene for ALS. More than 150 *SOD1* mutations have been reported in 68 of the 153 codons, spread over all five exons (ALS Online

studying 23 ALS families.

(b)

(a)

46,0%

82,5%

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Genetic Database, ALSOD: http://alsod.iop.kcl.ac.uk/).

Patients with SOD1 mutations, FALS and SALS, show a phenotypic heterogeneity even within the same mutation although some sporadic missense mutations carry a consistently worse or better prognosis. A lot of mutations have been described during the time, we have focused in this paragraph on the most interesting in the Italian population for eventually presence of correlation between genotype and phenotype, both for FALS and SALS.

Patients with FALS and G41S mutations have similar clinical phenotype with early upper and lower motor neuron involvement in one or both lower limbs, rapidly spreading to upper limbs, appearance of bulbar signs within 1 years and death a few months later [21].

In 2010 Battistini et al. [22] described a family with 9 members ALS affected, in which there was evidence of a missense mutation in exon 4 (L106F) in SOD1 gene. In this family there was autosomal dominant inheritance. The clinical presentation was characterized by relatively early age of onset, spinal onset with proximal distribution weakness, bulbar involvement and a rapid disease course about two years.

**3. TAR DNA-Binding Protein 43 (TARDBP gene)**

with a very small amount being present in the cytoplasm [34-35].

**3.1. Genotype**

time in 2008 [34, 36].

codes for a protein of 414 amino acids.

by exon 6. The most common mutation is A382T.

individuals of mainly Northern European origin (2.7% vs. 1%).

**Figure 3.** Distribution of TDP-43 mutations detected in sporadic ALS patients [41].

population studies (about 3 to 4% of FALS cases) [37, 38].

TAR DNA-binding protein 43 is homologous to the heterogeneous nuclear ribonucleoproteins (hnRNPs) [33], which are involved in RNA processing, and its abnormal cellular distribution

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The protein is highly conserved, widely expressed and predominantly localized to the nucleus

The human *TARDBP* gene (Entrez Gene ID 23435) is located on chromosome 1p36.22, and it

Mutations in *TARDBP* gene associated with ALS disease have been discovered fro the first

The proposed mutational frequency is ~5% for FALS and 0.5-2% for SALS. To date, more than 30 different TARDBP mutations have been described, all of which are missense substitutions. With a single exception, all of them are clustered in the C-terminal glycine-rich region encoded

Mutations in *TARDBP* gene associated with ALS disease have been discovered fro the first time in 2009 and in the same year a Italian screening has been performed [9]. The Italian results showed a higher frequency of *TARDBP* mutations in SALS Italian patients compared to

The frequency of mutations in *TARDBP* gene in Italian patients (4.4%) are similar to other

Most *TARDBP* mutations are missense changes in exon 6, encoding for Gly-rich C-terminal region that allows to bind single-stranded DNA, RNA and proteins [39, 40] (Figure 3).

is one of the key feature of ALS and frontotemporal lobar degeneration (FTLD) [34].

Another mutation, L106P, has been found in a patient who presented similar clinical pattern with spinal onset with weakness mainly in proximal areas; however in this patient, 30 months after disease onset, weakness remained restricted to the upper limbs without pyramidal signs and it was consistent with brachial amyotrophic diplegia, a relatively slowly progressive variant of motor neuron disease [23].

Corrado et al. [2] suggested that the nonsense mutation in exon 5 was present in SALS patients with severe and rapid clinical course, analogous to what found for most SOD1 mutations leading to a truncated protein. Conversely, N65S and A95T are both associated to a slowly progressive course of the disease, similarly to other mutations (H46R, D76V, H13T, L144P, G93V, I151T, D90A, A89T) detected in patients with a disease duration >10 years. In addition N65S seems to be strictly correlated to a prevalent involvement of the lower motor neurons and only at the spinal cord.

In 2011 in their article, Del Grande et al. [3] showed a similar phenotype in three unrelated patients with sporadic SOD1 mutation D11Y: slow progression, initial distal limbs muscles involvement and predominant lower motor neurons signs. The topographic distribution in distal muscles was a constant feature over many years, with only late impairment of proximal or bulbar muscles (respiratory muscles involvement after 7-10 years). All three patients had slight pyramidal signs (hyperactive reflexes, Babinski sign without increase of muscular tone).

In 2011 Luigetti et al. [24] described a strange case report of a sporadic patient with SOD1 G93D mutation disclosing a rapid progression of the disease. The beginning of symptoms was weakness in upper limbs, without involvement of lower limbs or bulbar functions. Over a 2 year-follow up the patient showed a rapidly progressive course with involvement of lower limbs, bulbar and respiratory muscles and the patient died after 30 months since the onset.

This case is in contrast with literature data [25-27]: other patients with SOD1 mutation (FALS) presented a slowly progressive diseas with a long-lasting paucisymptomatic phase. The authors discovered a novel heterozygous ANG missense mutation (c. 433 C>T, p.R145C), so they hypothesised a role in pathogenesis and clinical phenotype [24].

Penco et al. [28] described a family with same mutation of SOD1, in which there was wide variability of disease expression among family members. The ANG IVS1+27 variant in the heterozygous state was found in the proband that disclosed an aggressive clinical course. Though this variant occurred in noncoding region and no prediction of splicing alteration was made, the authors speculated that this variant contributed to the clinical phenotype.

These findings support a possible pathogenetic role of ANG mutation with influence on clinical manifestations in patient with SOD1 mutation.

Often bulbar onset is associated with older age of disease presentation without significant difference of distribution between FALS and SALS [21].

These data are confirmed by international literature [29-32].
