ALS is a heterogeneous disease with ethnic and sociodemographic differences. In this study, the ALS group has an average age of 57.3 ± 12.8 years, younger patients represent 4% of the group, and 55.4% are males. ALS disease affects mainly men, with a male-to-female ratio between 1 and 2, and the initial symptoms usually manifest between 55–75 years [2, 7, 38]. Despite the prevalence in the elderly, young people with an average age of 20 years are also affected, representing 1–10% of all cases, classified as juvenile ALS . In this study, the average age and gender proportions in the case group correspond to the literature . However, age and gender are not significantly different between groups (Table 1).
The reason for the difference in occurrence between the genders is not yet known. Some studies associate a lower prevalence of ALS in women with a hormonal protective effect or lower exposure to risk factors [41, 42]. Recent studies suggest that estrogen acts as a protective factor in neurodegenerative processes by countering mechanisms of oxidative stress and excitotoxicity [43–45]. Despite the lower incidence of women among ALS patients in general, they are more associated with a disease-specific classification, the FTD-ALS cases, which has been justified by genetic factors . However, further studies are needed to elucidate gender biases in ALS.
Looking at the sociodemographic make-up of the case group, white people comprised the most substantial proportion, representing 48.5% of the individuals, and 44.6% were brown. These results corroborate other studies that report a higher incidence of the disease in white populations, which has been associated with genetic factors [5, 47]. Besides, the white population has more easy access to health programs than the black population, traditionally considered a minority in a society that often neglects social rights .
Among the modifiable variables analyzed, alcohol consumption as a risk factor for ALS is still controversial. Some studies show the consumption of alcohol as a protective factor for the disease [49, 50]. However, in the present study, alcohol consumption was more reported in the case group; thus, it is a possible risk condition for the disease (p = 0.01). Such a hypothesis does have a potential mechanistic explanation. Excessive alcohol consumption increases the sensitivity of glutamate receptors and increases the concentration of glutamate in the central nervous system, leading to excitotoxicity and neuronal death . Additionally, it is known that alcohol consumption can activate astrocytes through alterations in proteins expressed in microglia, implying in neuroinflammatory mechanisms .
Smoking as a risk factor for ALS is also controversial. Some studies relate smoking to an increased risk for the disease, possibly linked to the toxic effects of cigarette substances [49, 53]. Smoking results in neuronal death since nicotine interacts with acetylcholine receptors in the central nervous system, causing the dysregulation of Ca2+, Na+, and K+ ion concentrations, increased cell permeability, oxidative stress, and neuroinflammation [54, 55]. Nevertheless, there was no statistically significant difference in identification as a smoker between the groups in this study (p = 0.28).
Other factors were investigated in the case group, such as physical activity and occupation before the diagnosis of ALS. Some studies indicate that physical activity could contribute to the risk of disease development, as it results in increased metabolic rates in the body, which is related to the production of reactive oxygen species and oxidative stress [56, 57]. In this sense, the hypermetabolic condition was reported as a characteristic of ALS pathogenesis, and it has been discussed as a determining factor in neurodegenerative processes [58, 59]. Besides, physical activity has been associated with neuronal death, due to its relation to the increase of pro-inflammatory cytokine expression resulting in neuroinflammation processes. Additionally, it is known that physical injuries during such practices may contribute to the genesis of the pathological process [57, 60]. In the present study, physical activity was reported by most ALS individuals, representing 53.5% in the case group (Table 3). Similar results were found in studies by  and .
In the professional occupation analysis of the ALS group, most individuals reported working in professions that require physical exertion or repetitive activities, like general services (36.6%), housework (13.9%), administrative services (12.9%), commerce (9.9%), teachers (7.9%), and health professionals (5.9%). In general, activities in which professionals are exposed to chemicals and require significant physical performance are considered risk factors for ALS [62, 63]. The present study corroborates the literature regarding occupational exposure, in which professions such as health professionals, teachers, scientists, and athletes are most affected by the disease [64, 65]. However, further research is needed on the association of occupational factors and clinical factors with disease susceptibility.
ALS patients were also classified into major disease variations according to the recommendations of the El Escorial World Federation of Neurology and information contained in medical records. We observed a higher percentage of sALS (94.7%) and fALS (5.9%). Moreover, 72.3% fit into the cALS category, 23.8% were bALS, and 4% were juvenile cases. These data corroborate the prevalence of the classifications described in the literature, where sALS represents ~ 90 to 95% of all ALS cases, and ~ 5 to 10% are fALS. The other classifications also follow the same pattern, in which the classical form of the disease is most prevalent ~ 50 to 75%, the bulbar form affects ~ 20 to 25% of all cases, and juveniles vary from ~ 1 to 10% [3, 39].
Also, in the case group, individuals were asked about their past medical history and cases of neurodegenerative diseases in the family. Research on patients’ previous diseases and the occurrence of neurodegenerative diseases in the families of ALS patients has grown considerably in recent years due to the discovery of the common etiopathogenesis among some diseases, which aroused scientists’ attention to a better understanding of the mechanisms involved in ALS [66, 67]. The present study showed that 46.5% of the patients in the case group reported some type of disease before the ALS diagnosis, and 40.6% reported cases of neurodegenerative diseases in the family. Significant results of neurological disease history in family and ALS were found in the studies by ,  and .
Regarding drug treatment, 75.2% of ALS patients reported taking riluzole, and 24.8% reported not having followed the treatment. Riluzole is the only drug approved for ALS treatment in Brazil. Nevertheless, studies report no significant increase in patient survival attributed to this drug treatment – only about two to three months [14, 70]. Other medications are currently being studied for ALS treatment, such as Edaravone (Radicut), Masitinib , Tirasemtiv , and others. However, the progressive character of the disease and the lack of effective medication contribute to the short life expectancy after the diagnosis, which varies between three and five years.
Concerning the genotyping analyses, this study demonstrates a higher prevalence of genotype (T/T) in ALS patients, making it a possible risk factor for the disease (OR = 1,83; 95% CI = 1.27–2.65) (p = 0.02). Similarly, the allele frequency analysis showed that the T allele confers risk for disease susceptibility (OR = 1.54; 95% CI = 1.05–2.29) (p = 0.03) (Table 2). The same results were found in studies by  and .
The hypothesis that MTHFR C677T polymorphism may contribute to ALS pathogenesis is supported by evidence showing that this polymorphism causes an imbalance of the intracellular folate pathway , which is associated with several pathogenic mechanisms, such as hypomethylation, increased Hcy levels in the organism, and altered nucleotide synthesis. These mechanisms have been implicated in central nervous system malformation, cognitive impairment, depression, epilepsy, Down syndrome, and the development of neurodegenerative diseases [75, 76].
Intracellular folate dysregulation could be driven by several factors, especially alterations in genes acting on its biochemical pathway . Remarkably, the MTHFR gene stands out due to its coding of the enzyme MTHFR, which plays a crucial role in folate intracellular metabolism by converting 5,10-MTHF into 5-MTHF, the main circulating form of folic acid and most abundant form in the body . The enzyme MTHFR directly regulates the intracellular methylation cycle, since the 5-MTHF reduced by the enzyme is an intermediate necessary for the methylation of Hcy to produce methionine. Such a process is essential to cellular homeostasis, and vital to the synthesis of SAM, the main active molecule in folic acid intracellular methylation processes . Also, the enzyme MTHFR indirectly influences purine synthesis via the Methylenetetrahydrofolate dehydrogenase (MTHFD) pathway through the use of the 5,10-MTHF molecule, which can be used in both pathways .
The MTHFR gene is more expressed in the brain, muscle, placenta, and stomach, possibly due to the greater need for folate homeostasis in these tissues . More than 20 genetic variations in the gene are known to result in the coding of non-functional enzymes. Among these, the C677T single nucleotide polymorphism – SNP is the main alteration studied. Cytosine is replaced with thymine at position 677 in exon 4, causing the change in coding for the amino acid alanine to valine. This change results in the production of a thermolabile enzyme with activities reduced by 35% in the case of heterozygous (C/T) and 65% in homozygous (T/T) at 35 ºC [22, 24]. Various diseases are associated with this production, such as cardiovascular disease [25, 26], diabetes , hypertension , congenital anomalies , cancers [27, 28], and neurodegenerative diseases such as Alzheimer [79, 80] and Parkinson [81, 82].
The C677T polymorphism directly interferes with the available concentration of 5-MTHF, a molecule that is used in the bioconversion of Hcy to methionine and is essential in the production of SAM . Low SAM synthesis is a limiting factor in the methylation cycle, which is crucial for DNA interactions, chromatin structure, and transcription rates, all of which are involved in epigenetic mechanisms that are essential for neuronal development and differentiation [22, 83].
Epigenetic changes during embryonic development are known to result in life-long functional changes, predisposing individuals to various neurological disorders . Although not well understood, some studies report that folate cycle dysregulation and epigenetic changes are associated with alterations in stem cell differentiation and proliferation rates of neurons, the formation of neuromuscular junctions, neural cell specialization, and decreased cells in cortical regions [74, 75, 84]. Thus, the importance of epigenetic changes for motor neurons should be considered, as they are highly differentiated with large prolongations in the central nervous system and responsible for specialized functions in the contraction of effector muscles in the periphery .
Furthermore, hypomethylation resulting from the MTHFR C677T polymorphism could be associated with ALS pathogenesis since it may affect proteins that compound the neuronal cytoskeleton such as tubulin, β-actin , myelin, and cell membrane phospholipids, thus supporting the neurodegenerative process through structural changes and demyelination of the nervous system [83, 87]. Moreover, disturbances in the methylation process are associated with lower synthesis of inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and serotonin, which causes neuroplasticity dysregulation and exposes motor neurons to glutamate excitotoxicity in ALS [88–90].
Additionally, HHcy due to the MTHFR C677T polymorphism is known to affect cellular redox states, associated with higher production of reactive oxygen species and oxidative stress  (Fig. 1). HHcy also affects ion channels, inducing intracellular calcium influx associated with mitochondrial dysregulation, DNA damage, and cell death [91–93]. Studies on ALS have reported HHcy in patients plasma and cerebrospinal fluids [94, 95], discussed as a factor in the predisposition of motor neurons to the process of excitotoxicity and oxidative stress, due to interactions in glutamatergic receptors, which causes higher sensitivity in the release process of glutamate neurotransmitters [94, 96].
Moreover, the increased Hcy level in the bloodstream could contribute to muscle denervation and neurodegeneration, given that the concentration of this amino acid is ten times higher in the bloodstream of ALS patients than in their central nervous system . This suggests that Hcy might act at neuromuscular junctions and skeletal muscles .
Of the main process arising from the MTHFR gene polymorphism liked to HHcy, oxidative stress presents itself as a mechanism implicated in many intracellular alterations in ALS neurodegeneration . In the transsulfuration pathway, Hcy is converted to cystathionine and later to cysteine, the precursor molecule in protein and glutathione (GSH) synthesis. Glutathione is an essential antioxidant compound that acts against oxidative imbalance and cell damage by capturing reactive oxygen species (ROS) [98, 99]. Intracellular folate deficiency due to MTHFR gene polymorphism compromises glutathione metabolism since SAM allosterically regulates GSH production [100, 101] (Fig. 1). In brain tissue, the conversion of cystathionine to cysteine does not occur, and the process of GSH synthesis is dependent on cysteine transporters . In this way, studies report low levels of glutathione in brain tissue, causing greater susceptibility of neurons to oxidative stress and development of ALS [103, 104] (Fig. 1). On the other hand, folate deficiency and HHcy contribute to oxidative stress, once the Hcy interacts with cysteine transporters preventing GSH synthesis .
As previously mentioned, the MTHFR C677T polymorphism can also affect DNA synthesis via the thymidylate pathway by MTHFD and TMS enzymes . In this pathway, the 5,10-MTHF is reduced and oxidized to 10-Formiltetrahydrofolate, which donates a methyl group for the conversion of deoxyuridine-5-monophosphate (dUMP) to dTMP [106, 107] (Fig. 2). Although not well understood, deficiency in the MTHFR enzyme is known to result in the accumulation of the 10-Formiltetrahydrofolate molecule due to the non-conversion of 5,10-MTHFR to 5-MTHF, stimulating dTMP synthesis . Thus, the synthesis of new thymidylate is an alternative for intracellular folate reactions in cases of MTHFR enzyme deficiency, possibly increasing the dTMP concentration in plasma.
The increased production of thymidylate contributes to oxidative stress, as its entire synthesis process is performed by oxidation-reduction reactions that depend on NADPH [20, 21]. NADPH is a major ROS generator that contributes to the development of neurodegenerative diseases, including ALS, due to the release of free radicals in the brain and the vulnerability of this tissue to oxidizing agents . Increased plasma thymidine levels have been reported in neurogastrointestinal mitochondrial encephalomyopathy disease (MNGIE), discussed as a cause in mitochondrial DNA imbalance associated with symptoms such as mental regression, ophthalmoplegia, and fatal gastrointestinal complications . Moreover, increased thymidylate was reported in carcinoma cells as an angiogenic factor associated with oxidative stress, stimulation of pro-inflammatory interleukin secretion, and vascular endothelial growth factor secretion (VEGF) .
In addition to analyzing the MTHFR C677T polymorphism as a possible risk factor for ALS, this study also identified associations between the genotypic profile and sociodemographic and clinical factors reported by the individuals in the case group. Interactions between genotype (T/T) and factors such as ethnicity in white (p = 0.005) and brown (p = 0.001) individuals, as well as physical activity (p = 0.006) were observed (Table 3). Other important variables, such as gender, alcohol consumption, smoking, ALS classification, neurological disease in the family, and previous pathologies showed no interactions with the genotypic profile presented in the case group.
The association of ethnicity with the MTHFR C677T polymorphism is consistent with the higher prevalence of this genetic variable in white people such as Hispanic, Italian, Californian, and others. Conversely, the lowest incidence was reported in Black people, including North America, Brazil, and sub-Saharan Africa . Since studies suggest that genetic influences can explain the higher frequency in white populations , it is possible that the variation in the MTHFR gene is a factor, in agreement with the results presented in our study.
Physical activity is considered a risk factor for ALS due to the increase in energy expenditure associated with oxidative stress (discussed earlier in this study). Moreover, the relationship between physical activity and genetic contributions in ALS susceptibility has been discussed . In this context, the results obtained in this study show an interaction between physical activity and MTHFR C677T polymorphism in disease susceptibility. This interaction perhaps results from the possibility that both of these risk factors contribute to oxidative stress.
ALS is a rapidly progressing disease and has a median survival time of three to five years. However, there are discrepant cases in which survival exceeds ten years, as was the case of the famous physicist Stephen Hawking [112, 113]. The precise causes of these longer survival cases are still unknown; however, genetic and environmental factors are possible causes .
In this study, we performed a comparative analysis between the C677T MTHFR polymorphism and the death, which showed a statistical trend towards more significant ALS progression and early death in patients with the heterozygous (C/T) genotype compared to the wild genotype (C/C) (p = 0.06) (Fig. 2). Until the data collection period, there were no cases of death with the genotype (T/T). A study by Tsang et al.  reports that the heterozygous genotype (C/T) exhibited the most significant effect of MTHFR C677T polymorphism on decreasing blood folate, which surprisingly corroborates our data. This result suggests a higher pathogenic risk and earlier death in heterozygous (C/T) than mutant homozygous (T/T) genotypes.