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Article

SOD-1 Variants in Amyotrophic Lateral Sclerosis: Systematic Re-Evaluation According to ACMG-AMP Guidelines

by
Paola Ruffo
,
Benedetta Perrone
and
Francesca Luisa Conforti
*
Medical Genetics Laboratory, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy
*
Author to whom correspondence should be addressed.
Genes 2022, 13(3), 537; https://doi.org/10.3390/genes13030537
Submission received: 14 January 2022 / Revised: 8 March 2022 / Accepted: 15 March 2022 / Published: 18 March 2022
(This article belongs to the Special Issue Functional Studies for Interpreting Genetic Variants Associated with Genetic Disorders)
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Abstract

:
Amyotrophic lateral sclerosis (ALS) is the most common type of motor neuron disease whose causes are unclear. The first ALS gene associated with the autosomal dominant form of the disease was SOD1. This gene has a high rate of rare variants, and an appropriate classification is essential for a correct ALS diagnosis. In this study, we re-evaluated the classification of all previously reported SOD1 variants (n = 202) from ALSoD, project MinE, and in-house databases by applying the ACMG-AMP criteria to ALS. New bioinformatics analysis, frequency rating, and a thorough search for functional studies were performed. We also proposed adjusting criteria strength describing how to apply them to SOD1 variants. Most of the previously reported variants have been reclassified as likely pathogenic and pathogenic based on the modified weight of the PS3 criterion, highlighting how in vivo or in vitro functional studies are determining their interpretation and classification. Furthermore, this study reveals the concordance and discordance of annotations between open databases, indicating the need for expert review to adapt the study of variants to a specific disease. Indeed, in complex diseases, such as ALS, the oligogenic inheritance, the presence of genes that act as risk factors and the reduced penetration must be considered. Overall, the diagnosis of ALS remains clinical, and improving variant classification could support genetic data as diagnostic criteria.

Graphical Abstract

1. Introduction

Neurodegenerative diseases are a heterogeneous set of diseases of the central nervous system, characterized by the progressive degeneration of neurons’ structure and function. ALS, also known as Charcot disease or Lou Gehrig’s disease, is the most common motor neuron disease with a global annual incidence ranging from 1 to 3 cases per 100,000 population [1]. It is characterized by the progressive neuromuscular junctions dismantling and the motor neurons degeneration in the motor cortex, brain stem, and spinal cord. Loss of motor neurons leads to progressive paralysis and death from respiratory failure within 3–5 years of the onset of disease [2]. To date, despite ALS being known to be a multifactorial disease associated with genetic predisposition, environmental factors, and lifestyle, its causes are unclear. In 10% of cases, ALS is caused by a genetic mutation that can characterize several generations of the same family (familial ALS (fALS)) or the disease arises suddenly and without any known familial link (sporadic ALS (sALS)) [3]. The main genes directly associated with the onset of the disease and which account for almost 50% of fALS and 6% of sALS cases in the world are SOD1, TARDBP, FUS, and C9orf72 [4].
The first ALS gene associated with the adult-onset autosomal dominant form of the disease was mapped to the chromosome 21q22.1–22.2 and encodes for the cytoplasmic Cu/Zn superoxide dismutase (SOD1). Mutations in this gene were first described and linked to the disease in 1993 [5], and their frequencies range from 1% to 2% and from 13% to 20% in sALS and fALS patients, respectively [6]. SOD1 gene encodes a homodimer enzyme composed of 154 highly conserved amino acids between species. SOD1 mutants show a gain of function in which the protein appears to take on a new toxic function, probably related to an increased tendency for mutated proteins to assemble and form aggregates in motor neurons [7].
The advancement of next-generation sequencing (NGS) technologies and the extension of screening tests have made it possible to associate over 100 genes to the disease and thousands of variants [8]. The enormous increase in data and the massive number of unknown variants obtained using these approaches require an interpretative step that allows for clinical classification of the variants as pathogenic or benign. The guidelines proposed in 2015 by the American College of Medical Genetics and Genomics and by the Association for Molecular Pathology (ACMG-AMP) [9] represent the criteria to be used for the classification of variants associated with Mendelian disorder in different categories—namely, benign, likely benign, variants of uncertain significance, likely pathogenic, and pathogenic. Among all, one of the criteria considered very important is based on the characterization of gene variants through functional studies (PS3), and the studies in which experimental tests are carried out are fundamental, as they help the interpretation and classification of pathogenic gene variants.
Since 2015, other publications have highlighted the need to re-evaluate some criteria in the classification of variants [10,11,12]. In particular, detailed guidance was provided to determine the appropriate strength of evidence of functional studies that can be used in the interpretation of clinical variants [13]. However, despite the introduction of these dedicated guidelines and algorithms, the variant interpretation process is complex and not always an efficient process. In addition, the existence of discordant variant classifications across public databases is one of the well-documented limitations in the interpretation of the pathogenicity of variants.
Based on this premise, a periodic re-evaluation of the genetic variants underlying complex diseases is necessary, especially in complex diseases such as ALS where mutation–phenotype correlations are growing rapidly.
To date, over 200 SOD1 variants have been identified. Most are present in heterozygosity [14], confirming the autosomal dominant inheritance of the gene in this motor neuron disease [15], while others represent an exception, such as the recessive variant D91A (p.Asp91Ala) that shows frequent homozygosity in Scandinavian populations and Nordic countries [16] and the compound heterozygous variant D97N (p.Asp97Asn) [17].
This study provides an updated evaluation of all previously reported SOD1 variants in the Amyotrophic Lateral Sclerosis Online Genetics Database (ALSoD, https://alsod.ac.uk/, 20 December 2021), project MinE (http://databrowser.projectmine.com/, 22 December 2021) and in-house databases [18] by applying the ACMG-AMP guidelines with criteria adapted for SOD1. We also assess the concordance of annotations between open databases (ClinVar and LOVD), highlighting ongoing sources of discordance.

2. Materials and Methods

2.1. Structured Search

SOD1 variants associated with ALS were extrapolated from the ALSoD, project MinE, and in-house databases [18]. All these substitutions were from patients diagnosed with definite, probable, and probable lab-supported ALS based on the revised El Escorial criteria [19]. We investigated all the genetic variants listed by ALSoD, while we selected only the variants matching the “LoF + Missense” criterion listed by Project MinE. In addition, a PubMed literature search was conducted using the keywords “SOD1 variant” and “SOD1 mutation”. Studies aimed at investigating the functional significance of genetic variants and abstracts related to genetic variants underlying ALS were also considered in the study.

2.2. ACMG-AMP Pathogenicity Analysis

The analysis and clinical classification of ALS variants were based on the ACMG-AMP guidelines (Supplementary Table S1). The assigned criteria were then combined, according to the scoring rules expressed in Supplementary Figure S1, to choose a classification from the 5-level system.

2.3. Frequency Analysis of Variants

For the evaluation of the minor allelic frequency (MAF), gnomAD v2.1 (gnomad.broadinstitute.org) was used. According to the ACMG-AMP guidelines, if a variant exists in a large general population or a control cohort, it can be considered a criterion 2 moderate pathogen (PM2). Recently, Oza et al. [20] recommended downgrading the weight of the PM2 criterion to a supporting weight in guidance from the Sequence Variant Interpretation (SVI) working group (https://clinicalgenome.org/site/assets/files/5182/pm2_-_svi_recommendation_-_approved_sept2020.pdf, 27 January 2022). In our context, SOD1 variants completely absent from all databases or with a MAF < 0.001 met the moderate pathogen criterion 2 (PM2) [21,22].

2.4. Computational Tools Analysis

The pathogenicity of the variants was also evaluated using multiple in silico genomic tools, including SIFT [23], Polyphen-2 [24], MutationTaster [25], MutationAssessor [26], FATHMM [27], PROVEAN [28], and CADD [29]. Splicing prediction analysis was performed using SpiP and SpliceAI tools [30]. The convergence should be considered as supporting evidence of pathogenicity or benignity. An adverse consequence predicted with more than six tools is considered to be evidence to support pathogenicity (PP3), while at least three total and concordant predictors are required to support a benign state (BP4). However, if the in silico predictions disagree, this test should not be used for the classification of a variant.

2.5. Analysis of Functional Studies

Functional studies conducted in vitro or in vivo may show a detrimental effect on protein function, supporting the pathogenicity of the tested variant (PS3), or no effect, thus supporting a benign impact of the variant (BS3). As recommended by Brnich et al., several factors must be considered for the assignment of this criterion [13]. It is necessary to delineate the genetic mechanism of the disease to choose the appropriate functional analysis, as well as the experimental model to be used on which to perform functional tests. Assays performed must be coded in biological replicates, and an adequate number of control cases should be considered. Finally, the test result must be in line with the pathogenetic mechanism of the disease [13].
ALS is a complex biological system, and the precise mechanisms underlying selective cell death in the disease are unknown. Current understanding indicates that there may be a complex interplay between genetic factors, oxidative stress, excitotoxicity, protein aggregation, impaired axonal transport, and mitochondrial damage. Over the years, several in vitro and in vivo experimental models have been exploited to study the molecular processes underlying motor neuron degradation and disease progression, providing essential small pieces to the ALS puzzle [31,32]. However, only an integrated and complimentary use of different experimental models could improve our understanding of the pathogenic mechanism of the disease.
According to Brnich’s recommendations and experimental evidence on SOD1 variants, we assigned three different weights to the PS3 criterion: PS3_Strong, PS3_Moderate, and PS3_Supporting. PS3_Strong was assigned to variants in which in vivo functional assay results showed toxic SOD1 function and disruption of mitochondrial dynamics, protein folding, axonal transport, and cellular metabolism. The PS3_Moderate and PS3_Supporting criteria were assigned to variants investigated by functional in vitro studies, following the flowchart shown in Supplementary Figure S2.

2.6. Critical Functional Domain Position

Pathogenic SOD1 variants associated with ALS are spread over the entire length of the gene. Hotspot mutations are characterized by the presence of many missense variants, in-frame, and nonsynonymous variants, most of which have a pathogenicity classification. The universal protein source, UniProt (https://www.uniprot.org/, 29 January 2022), and bioinformatics tools can help identify mutations in established hotspots or functional domains. The PM1 rule considers reported variants within the domain and is assigned with the ratio pathogenic to total nonpathogenic variants is greater than 1.5, with at least 5 pathogenic variants within the domain. We established that the PM1 criterion can be upgraded to strong (PM1_Strong) when at least 14 pathogenic variants are reported in the functional domain; the moderate weight (PM1_Moderate) of this criterion was assigned in the presence of at least 7 pathogenic missense variants. Finally, we considered PM1_Supporting when 5–6 pathogenic mutations are reported in the domain (Supplementary Table S2). The distribution of SOD1 variants is shown in Figure 1A.

2.7. Evaluation of the PVS1 Criterion

A strong weight in the classification of ALS genes is represented by the PVS1 criterion. This is attributed to a “Null variant in a gene where loss of function (LOF) is a known mechanism of disease”. Gain of function and several mechanisms are confirmed to be involved in ALS [33,34,35]. However, much evidence about SOD1 loss of function (LOF) suggests it may play a role in ALS pathogenesis, probably increasing the susceptibility to neurodegeneration. Overall, the major effect of SOD1 mutations in ALS is linked to the protein aggregation but these may also lead to loss of function of SOD1 activity or loss of the nuclear function, where SOD1 acts as a transcription factor [6].
According to the recommendations provided by the ClinGen SVI working group [10] and taking into account Guissart’s considerations [36], we adapted ACMG-AMP guidelines for SOD1 by evaluating the right strength related to the PVS1 criterion and distinguishing the variants with PVS1_Very strong and PVS1_Strong. In particular, we assigned PVS1_Very strong criterion when variants do not undergo the nonsense-mediated mRNA decay (NMD) and PVS1_Strong when variants undergo NMD. A representation of the SOD1 gene showing the localization of premature termination codons (PTCs) that cause ALS and NMD escaping is reported in Figure 1B.

2.8. Comparison between Databases

To assess the concordance between annotations generated by ClinVar and LOVD databases, we compared a list of 202 variants of the SOD1 gene across several online resources. The databases investigated generate an automated interpretation of the variants. Specifically, Leiden Open Variation Database (LOVD—https://www.lovd.nl/, 5 January 2022) is a software database for collecting and visualizing variants in the DNA sequence [37]. ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/, 7 January 2022) is an archive that aggregates information on genomic variation and the various correlations with human health [38].

2.9. Statistical Analysis

The chi-square concordance test was performed for discordance–concordance and variant classification across the various databases, using the SPSS 20.0 test (SPSS, Chicago, IL, USA). Pathogenic/likely pathogenic SOD1 variants were compared using Fisher’s test. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Summary of Variants

In this study, 202 sequence variants of SOD1 (NM_000454.5) were collected and analyzed. These variants have a MAF < 0.001 considering gnomAD 2.1 populations. Table 1 lists all variant sequences investigated and corresponding classification according to the ACMG-AMP guidelines.
The variants analyzed were five splice regions (2%), one nonsense (1%), eight frameshifts (4%), three stop-gained (1%), one synonymous (1%), and three intron variants (1%), while all remaining were missense variants (90%) (Figure 2).
In total, 13 variants (6.4%) were classified as variants of uncertain significance (VUS), with insufficient or conflicting evidence regarding the role of molecular alteration in disease. Of these, nine were missense variants (c.59A>G, c.89T>C, c.179G>A, c.298A>G, c.172T>C, c.179G>T, c.328G>T, c.35A>C, c.172T>G), three were intron variants (c.358-11A>G, c.358-304C>G, c.358-10T>G) and one was a splice region variant (c.240-7T>G). The variants classified as likely pathogenic were 136 (67.3%), of which 2 were frameshifts (1.5%), 1 was stop-gained (0.7%), 3 were splice region variants (2.2%), and 130 were missense variants (95.6%). The remaining 53 (26.3%) were considered pathogenic and included 6 frameshifts (11.3%), 1 splice region (1.9%), 2 nonsense (3.8%), 2 stop-gained (3.8%), 1 synonymous variant (1.9%), and 41 missense variants (77.3%).

3.2. Contribution of Each Criterion to the Final Classification

The contribution of each criterion to the final classification is shown in Figure 3. The PM2 criterion was assigned to all 202 variants analyzed in this study. Pathogenic variants of the SOD1 gene are often identified in a single patient or an isolated family. This evidence underlines the rarity of the variants and therefore allows the PM2 criterion to acquire a moderate level of evidence of pathogenicity.
The PM1 criterion was assigned to 186 variants (92%), with different weights depending on the number of pathogenic variants located in the functional domain of the gene. The PM1_Strong rule was assigned to 68 variants (36.6%), of which 32 (47.1%) were classified as likely pathogenic, 33 (48.5%) as pathogenic, and 3 (4.4%) as VUS. The PM1_ Moderate was assigned to 108 variants (58%); in this category, 97 (89.9%) were likely pathogenic, 10 (9.3%) as pathogenic, and only 1 (0.9%) was classified as VUS. The PM1_Supporting weight was assigned to 10 mutations (5.4%), of which 6 terms (60%) were classified as likely pathogenic and 4 (40%) as VUS.
The PP2 criterion characterized 185 variants (91.6%): 146 (79%) as likely pathogenic, 33 (17.9%) as pathogenic, and 6 (3.1%) as VUS.
Computational tools predicted a disease-causing role (PP3) in 179 (88.6%) cases examined. Of these variants, 138 variants (77%) were classified as likely pathogenic, 39 (21.8%) as pathogenic, and 2 (1.1) as VUS.
In the present study, the PM5 criterion assigned to a “novel missense change at an aminoacidic residue where a different missense change determined to be pathogenic has been seen before” [9], showed a moderate impact in 114 variants (56.4%). Of these, 91 (79.8%) were likely pathogenic, while the remaining 23 (20%) were pathogenic.
According to the ACMG-AMP guidelines, the PP5 criterion was assigned to 90 variants (90/202; 44.6%) for which different studies were available in the literature. The presence of the PP5 criterion allowed the classification of 65 (72.2%) likely pathogenic, 23 (25.5%) pathogenic, and of the remaining 2 (2.2%) VUS variants. The splice region c.240-7T>G and the intron c.358-11A>G variants were characterized as VUS, with the PM2 and PP5 criteria, since no data from the literature were available.
Considering previously published functional studies for the evaluation of the variant effect and the impact of the mutation on the clinical phenotype, the PS3 criterion was assigned to 32 (32/202; 15.8%) variants (Supplementary Table S3). PS3_Strong was assigned to 10 (31.3%) variants classified as pathogenic. These pathogenic variants were characterized by the attendance of multiple functional studies performed in various experimental models (Supplementary Table S3) [39,40,41,42,43,44,45,46,47,48,49]. The p.Gly94Ala mutation, known as G93A, was used to create the first ALS-associated SOD1 transgenic mouse. This model, which expresses large amounts of mutant SOD1, develops adult-onset neurodegeneration of spinal motor neurons and progressive motor deficits leading to paralysis, and it is now widely used both for pathogenic molecular mechanisms and for preclinical studies useful for the specific therapy of affected patients [31,39,40,41]. This mutation is currently the one studied in the largest number of animal models including C. Elegans, Saccharomyces Cerevisiae, Swine, and Danio rerio (Zebrafish) models. In the same way, experimental models of the transgenic mouse G93A and H46R (p.His47arg) show degeneration of the upper and lower motoneurons associated with a gravity directly proportional to the altered protein expression [31,39,42]. In addition, in vivo experiments show that SOD1-D91A (p.Asp91Ala), the most common mutation affecting SOD1 linked to ALS, is less toxic than other tested mutants, while homozygous mice develop fatal motor neuron disease similar to that observed in homozygous-D91A human ALS patients [41,43].
A Zebrafish model with G94R (p.Gly94Arg) variant has been developed, and this shows a mutated expression of SOD1 at a physiological level, which allows targeted pre-clinical studies [31,49]. For the variants G86R (p.Gly86Arg) [31,45,46,47] and G38R (p.Gly38Arg) [31,47,48], there are several functional studies carried out on transgenic mouse models in which there are high expression levels of the mutated gene and differences in the age of onset and progression of the disease as well as lifespan. For the G86R variant, studies have also been carried out using the C. Elegans nematode model. In particular, the presence of this missense variant produces an abnormal function of neurons ad a cellular dysfunction at the muscular level. The same variant was tested on Drosophila melanogaster models showing a progressive deterioration of motor functions with a specific clinical phenotype. The same experimental model was also used to study the effect of variants G38R and I114T (p.Ile114thr), determining a decrease in the lifespan of the midge accompanied by abnormalities in movement (Supplementary Table S3).
The effect of other mutations, classified with the PS3_Moderate or PS3_Supporting criterion, was evaluated by functional in vitro studies [50,51,52,53,54,55,56,57,58,59,60,61,62,63] assessing the correlation between the specific genetic alteration and the modification of oxidative stress, mitochondrial and cellular metabolism in different clinical phenotypes (Supplementary Table S3). PS3 with moderate weight was attributed to 20 (62.5%) variants: 10 (50%) were classified as likely pathogenic, while the remaining 10 (50%) as pathogenic. Finally, the PS3_Supporting weight was assigned to two (6.2%) variants classified as likely pathogenic (Supplementary Figure S2).
Another criterion with a strong impact on the classification, the PS1, was assigned to only eight SOD1 variants (4%). Of these, seven (87.5%) were classified as pathogenic and one (12.5%), the c.355G>T, was classified as likely pathogenic.
In our study, it was possible to attribute the PVS1 rule to 11 (5.4%) variants, 10 classified as pathogenic (92.8%) and 1 as likely pathogenic (7.1%). Only the variant c.275_276delAA expected to undergo NMD, while the other 10 mutations (c.301G>T, c.441T>A, c.409A>T, c.380T>A, c.424G>T, c.383_384insACCC, c.384_385insTGGG, c.320_321insT, c.335dup, c.383_392dup) did not undergo NMD, presenting the PVS1_Very strong criterion. The stop-gain variant c.301G>T, although known as a pathogenic mechanism (PVS1) and absent from population databases (PM2), was classified as likely pathogenic (Supplementary Table S4).
The frameshift variants c.272_274dupACA and c.56_58delTCA cause a change in the length of the protein for which the PM4 criterion can be applied. Combined with the PM and PP criteria, these two variants were classified as likely pathogenic.

3.3. Concordance–Discordance between Open Databases and Reclassification Based on ACMG-AMP Guidelines

LOVD contains a total of 47 variants, of which only 18 are classified as pathogenic, likely pathogenic, or VUS. The comparison of these variants (18/202, 9%) revealed a discordant classification (p-value < 0.001) in two substitutions. The variant c.59A>G, defined as likely benign in LOVD databases, was reclassified as VUS, while the c.272A>C was reclassified from VUS to pathogenic (Supplementary Table S5).
Evaluating SOD1 variant classifications in ClinVar database, 72 (35.6%) were concordant, and 19 (9.4%) variants were discordant (p-value < 0.001). In particular, 12 VUS were reclassified as likely pathogenic (n = 10) and pathogenic (n = 3), while 1 pathogenic variant was reclassified as VUS. Seven variants, defined as conflicting interpretations of pathogenicity by ClinVar, were reclassified: six as likely pathogenic/pathogenic and only one as VUS (Supplementary Table S5). Finally, 111 terms (55%), searched for rs ID, did not match in ClinVar.

4. Discussion

In this study, for the first time, ALS-associated SOD1 variants were systematically re-evaluated to perform clinical interpretation according to the ACMG-AMP guidelines. We also provided an updated assessment of variant concordance–discordance between bioinformatics tools used to evaluate variants’ roles and the criteria application provided by the above-mentioned guidelines adapted for SOD1.
Previous research has reported that only 34% of variants have a concordant classification according to the ACMG-AMP guidelines [64]. The high percentage of discordance between classifications can be explained by how individual laboratories interpret and analyze variants data, based on published levels of evidence and the misuse of ACMG-AMP guidelines.
Various scientific evidence supports the idea that ALS is caused by a combination of genetic and environmental factors [65,66]. To understand and quantify the value of the contribution of genetics in the pathogenesis of the disease several studies have been conducted [46]. Although a large number of genes have been related to ALS, other variants are to be discovered, which could play adjunct roles in increasing risk or susceptibility to the disease. Specific mutations not identifiable by GWAS or massive sequencing methods, such as repeated expansions [67] or variants in noncoding regions, could belong to this category [68,69].
Human SOD1 is widely known to be an important protein in the antioxidant system. The main hallmark of fALS is the presence of SOD1 aggregates in the spinal cord of patients [70], and the chemically modified form of the protein in the spinal cord of sporadic ALS patients has also been found [71]. SOD1 mutations were the first to be directly associated with the disease pathogenesis and the classification of variants in such a complex pathology remains a challenge. The heterogeneity of clinical data and the difference in approaches used for the interpretation of variants are the main challenges in variant classification [72].
SOD1 gene has a high rate of rare variants, and an appropriate classification is essential for a correct ALS diagnosis. Furthermore, one of the greatest challenges in interpreting pre-existing assessments is the heterogeneity in the approaches used for variant analysis. In this study, we applied a standardized in silico analysis and assessed the frequency of the variants, as well as considering the presence of functional and segregation studies. Results showed that 136 (67.3%) variants met the criteria of likely pathogenic, 53 (26.3%) were classified as pathogenic (Figure 4), and the remaining variants (n = 13; 6.4%) were categorized as VUS.
In our study, the PS3 criterion proved to be fundamental for the pathogenicity classification of 32 variants, representing 62.5% of total pathogenic terms. However, the near absence of functional and segregation data for a large number of terms remains an important limiting factor, considering that functional evidence is the main scoring driver for pathogenic/likely pathogenic for missense variants. Compared with all the variants analyzed, it is clear that there are few functional studies, conducted in vitro or in vivo, which show a detrimental effect on the protein. Furthermore, the PS3 criterion is not automatically assigned in the various databases, as functional evidence must be searched by the geneticist consulting PubMed and other available sites (Mastermind, etc.). After the manual revision, the strength of the criterion can also be changed according to the relevance of the evidence.
The attribution of the PM1 criterion was based on scientific evidence showing the absence of specific mutational hotspot mutations in SOD1 linked to ALS, indicating the whole protein involved [73]. In SOD1, several variants have been described to cluster in all five exons of the gene identified as hotspots (Figure 1A). Rather than defining clear function domains, these regions are defined by an increase in pathogenic variations identified in ALS patients. For this reason, we considered three different levels of strength of the criterion: PM1_Strong (≥14 variants), PM1_Moderate (at least 7 variants), and PM1_Supporting (5–6 variants).
All of the variants present in this study with a MAF value less than 0.001 and absents from all databases were considered to have a moderate pathogenicity level. The definition of a MAF threshold for ALS, in which significant confounding factors coexist (late-onset of the disease, age-dependent, variant-related reduced penetrance, and oligogenic inheritance), cannot ignore many factors difficult to evaluate, such as the inheritance model (monoallelic for most SOD1-ALS variants), the prevalence of the disease, and the penetrance. Failing consensus on allele frequency thresholds, the PM2 criterion was assigned to these cases.
For most of the missense variants involved in our study and classified as pathogenic or likely pathogenic, the PP5 criterion was considered supporting evidence with a very strong value, as the variants are present in several studies in the literature. However, this criterion must be used with caution as “the evidence is not available to the laboratory to perform an independent evaluation“ [11].
A strong impact on the evaluation of a mutation is expressed by the PS1 criterion, which is attributed when the same amino acid variation of a well-known causative variant occurs. The variant could act directly through the specific change of DNA or could modify the protein sequence through various mechanisms, such as an alteration in splicing. To support the pathogenicity of genetic variants, it is necessary to have well-established and reproducible functional studies.
The PVS1 criterion is considered the greatest impact, characterized by identifying a variant that causes a loss-of-function (LoF) allele. Although the gain of function is causative for amyotrophic lateral sclerosis, SOD1 loss of function may play a role in ALS, contributing to the onset and progression of the disease [73,74]. Many nonsense or frameshift mutations may lead to a LOF of SOD1 by affecting its structures and/or interactions pattern [6]. mRNA containing premature termination codons are known to be rapidly degraded by nonsense-mediated mRNA decay and premature termination codons that escape NMD in SOD1, causing the production of truncated SOD1 protein, which may have high toxicity [36] (Figure 1B).
An element of increasing importance is constituted by databases that report identified variants with their associated phenotypes and the interpretation of their functional role. In ALS, this concept is even more important since the vast majority of the variants identified are new and/or private, and for this reason, it is very important to share the knowledge obtained in dedicated databases. Another aspect to note is the presence/absence of SOD1 variants in these different databases. This study shows that many variants of this gene are absent in online archives used and, therefore, stresses that it is necessary to revise these data, so they are also open source, allowing the scientific community to search for updated information of genetic mutations, which are extremely important to the disease. A comparison of the annotations between open databases and our reclassification based on ACMG-AMP guidelines with the criteria adapted for SOD1 revealed a different rate of discordance. In particular, comparing ClinVar, we found discordance between pathogenic/likely pathogenic variants and VUS in 9.4% of terms. A case in point is the most common SOD1 mutation, p.Asp91Ala, which is still reported in ClinVar with conflicting interpretations of pathogenicity despite extensive evidence demonstrating pathogenicity according to the ACMG-AMP guidelines. In this public repository, many variants are associated with classification according to the ACMG-AMP guidelines. This archive accepts data derived from different working groups, the relevant literature, and documents from other web resources. To ensure the quality of the declarations, a review phase is carried out that involves evaluation by groups of experts. The variant data are then updated based on the new information presented, and therefore, an adequate time is required for the process. [39].
Based on the data obtained, it is evident that the mere application of the guidelines for interpreting genetic variants developed and refined so far cannot be considered sufficient in ALS to address various problems related to the implementation of new genetic analysis technologies in a diagnostic and clinical context. A manual reinterpretation of variants is essential to adapt the study to a particular disease, considering the specific characteristics of the gene and allowing for a consistent application. In this way, the different ACMG-AMP criteria acquire a different weight based on the type of pathology, scientific evidence, and knowledge of the variant itself.

5. Conclusions

In this study, we re-evaluated the classification of all previously reported SOD1 variants (n = 202) in ALSoD, Project MinE, and in-house databases by applying the ACMG-AMP criteria to ALS. Our results underline the importance of classifying variants involved in ALS and SOD1 variants with caution.
Specific guidelines for ALS would allow a more adequate classification of the large number of variants of uncertain significance, which have been constantly discovered. This can be accomplished by carrying out genetic research on large cohorts of patients and controls and by sharing the data obtained with the scientific community. Furthermore, it is crucial to develop and implement new analysis strategies that could facilitate the discovery and the validation of genetic factors involved in the pathogenesis of this disease that is still unknown. To date, the diagnosis of ALS remains, based on clinical, not genetic criteria. The improvement of the existing variant classification systems available could provide a better basis for using genetic findings as diagnostic criteria.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13030537/s1. Table S1: The ACMG-AMP criteria and their relevance in ALS, Table S2: Assessment of the PM1 criterion in SOD1 variants, Table S3: List of in vivo and in vitro functional studies considered for the PS3 criterion attribution, Table S4: Characteristics of the SOD1 variants relevant to the PVS1 criterion, Table S5: List of SOD1 variants and concordance–discordance between open databases and reclassification, Figure S1: ACMG-AMP guidelines for combining criteria to classify sequencing variants, Figure S2: Decision tree for functional evidence PS3.

Author Contributions

Conceptualization, P.R. and F.L.C.; methodology, P.R.; formal analysis, P.R.; resources, P.R. and B.P.; data curation, P.R.; writing—original draft preparation, P.R.; writing—review and editing, P.R. and F.L.C.; supervision, F.L.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a special award to the Department of Pharmacy, Health and Nutritional Sciences of University of Calabria (Italy) (Department of Excellence, Italian Law 232/2016) from the Italian Ministry of Education, University and Research (MIUR) and by MIUR ex 60% to F.L.C.

Institutional Review Board Statement

This study did not require ethical approval.

Informed Consent Statement

This study did not involve humans.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank the Project MinE GWAS Consortium.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. (A) Schematic representation of SOD1 showing the localization of variants that were assigned PM1. The amino acid changes are represented in red. The different weights of the criterion are represented by the different colors: dark blue, PM1_Strong; light blue, PM1_Moderate; green, PM1_Supporting. The numbers under the exons represent the amino acid distribution; (B) localization of variants that undergo/do not undergo NMD. The variants localized in SOD1 region where PTCs trigger NMD are represented in purple, the variants localized in the region that do not undergo NMD are colored in black.
Figure 1. (A) Schematic representation of SOD1 showing the localization of variants that were assigned PM1. The amino acid changes are represented in red. The different weights of the criterion are represented by the different colors: dark blue, PM1_Strong; light blue, PM1_Moderate; green, PM1_Supporting. The numbers under the exons represent the amino acid distribution; (B) localization of variants that undergo/do not undergo NMD. The variants localized in SOD1 region where PTCs trigger NMD are represented in purple, the variants localized in the region that do not undergo NMD are colored in black.
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Figure 2. Distribution of the different variant types analyzed.
Figure 2. Distribution of the different variant types analyzed.
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Figure 3. Contribution of each criterion to the final classification.
Figure 3. Contribution of each criterion to the final classification.
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Figure 4. Pie chart of different variant distribution types identified: (a) likely pathogenic variants; (b) pathogenic variants.
Figure 4. Pie chart of different variant distribution types identified: (a) likely pathogenic variants; (b) pathogenic variants.
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Table
Table 1. SOD1 variants in Amyotrophic Lateral Sclerosis: Systematic re-evaluation according to ACMG-AMP guidelines.
Table 1. SOD1 variants in Amyotrophic Lateral Sclerosis: Systematic re-evaluation according to ACMG-AMP guidelines.
Coding Change (NM_000454.5)Protein Change (NP_000445.1)Variant IDTypePS4PM3PP4PM1PM2PP2PM4PM5PM6PP1PP3PP5PS1PS2PS3PVS1Benign CriteriaRe-Classification
c.13G>Ap.Ala5Thrrs121912444Missense variant *MXX X XX *S P
c.13G>Tp.Ala5Serrs121912444Missense variant *MXX X XX P
c.13_14delGCinsTTp.Ala5Phe Missense variant *MXX X X LP
c.14C>Tp.Ala5Valrs121912442Missense variant *MXX X XX *S P
c.16G>Tp.Val6Leurs1568807314Missense variant *MXX X X LP
c.19T>Ap.Cys7Serrs1312702973Missense variant *MXX X X LP
c.19T>Gp.Cys7Glyrs1312702973Missense variant *MXX X X LP
c.20G>Ap.Cys7Tyrrs121912448Missense variant *MXX X X LP
c.21C>Gp.Cys7Trp Missense variant *MXX X X LP
c.23T>Ap.Val8Glurs1568807330Missense variant *MXX X X LP
c.25C>Gp.Leu9Valrs1568807333Missense variant *SXX X XX P
c.26T>Ap.Leu9Glnrs1568807342Missense variant *SXX X XX P
c.31G>Cp.Gly11Argrs1568807350Missense variant *SXX X LP
c.32G>Cp.Gly11Alars1555836167Missense variant *SXX X LP
c.34G>Tp.Asp12Tyrrs762628133Missense variant *SXX X LP
c.35A>Cp.Asp12Alars1568807374Missense variant *SXX VUS
c.37G>Cp.Gly13Argrs121912456Missense variant *MXX XX LP
c.43G>Ap.Val15*Metrs1568807400Missense variant *MXX X XX *M LP
c.44T>Cp.Val15Alars1202989817Missense variant *MXX X LP
c.44T>Gp.Val15Glyrs1202989817Missense variant *MXX X X *M LP
c.49G>Ap.Gly17Serrs121912453Missense variant *MXX X XX LP
c.50G>Cp.Gly17Alars1200906022Missense variant *MXX X XX LP
c.358-304C>G rs1555836889intron variant X VUS
c.56_58delTCAp.Ile19del frameshift variant *MX X X LP
c.59A>Gp.Asn20Serrs768029813Missense variant *MXX VUS
c.62T>Gp.Phe21Cysrs1555836169Missense variant *MXX XX LP
c.63C>Gp.Phe21Leurs1555836170Missense variant *MXX X X LP
c.64G>Ap.Glu22Lysrs121912450Missense variant *MXX X X *M LP
c.65A>Gp.Glu22Glyrs1568807435Missense variant *MXX X X *M LP
c.66G>Cp.Glu22Asp Missense variant *MXX X X LP
c.68A>Tp.Gln23Leurs1169198442Missense variant *MXX X X LP
c.68A>Gp.Gln23Argrs1169198442Missense variant *MXX X X LP
c.69G>Cp.Gln23Hisrs1424217272Missense variant *MXX X X LP
c.89T>Cp.Val30Alars1568809118Missense variant XX X VUS
c.95T>Cp.Val32Alars1428716759Missense variant *HXX XX LP
c.95T>Gp.Val32Glyrs1428716759Missense variant *HXX X X LP
c.112G>Ap.Gly38Argrs121912431Missense variant *MXX XXX *S P
c.112G>Cp.Gly38Argrs121912431Missense variant *MXX XXX *S P
c.113G>Tp.Gly38Valrs1555836517Missense variant *MXX X X LP
c.115C>Gp.Leu39Valrs121912432Missense variant *MXX X XX LP
c.116T>Ap.Leu39Glnrs1555836520Missense variant *MXX X X LP
c.116T>Gp.Leu39Argrs1555836520Missense variant *MXX X X *H LP
c.116T>Cp.Leu39Pro Missense variant *MXX X X LP
c.122A>Gp.Glu41Glyrs1568809149Missense variant *MXX X LP
c.123A>Tp.Glu41Asp Missense variant *MXX X LP
c.124G>Ap.Gly42Serrs121912433Missense variant *MXX X XX *M LP
c.125G>Ap.Gly42Asprs121912434Missense variant *MXX X XX *M LP
c.131A>Gp.His44Argrs121912435Missense variant *MXX XX LP
c.137T>Cp.Phe46Serrs121912457Missense variant *MXX X X LP
c.137T>Gp.Phe46Cysrs121912457Missense variant *MXX XX LP
c.139C>Gp.His47Asprs748897491Missense variant *MXX X X LP
c.140A>Gp.His47Argrs121912443Missense variant *MXX XX *S P
c.142G>Tp.Val48Phers1555836523Missense variant *MXX X LP
c.143T>Cp.Val48Alars1568809169Missense variant *MXX X LP
c.146A>Gp.His49Argrs1568809172Missense variant *MXX X XX LP
c.147T>Gp.His49Glnrs1568809175Missense variant *MXX X XX LP
c.148G>Ap.Glu50Lysrs1568809178Missense variant *MXX X LP
c.164C>Gp.Thr55Argrs986277034Missense variant *HXX XX LP
c.172T>Cp.Cys58Argrs1568810255Missense variant *HXX X VUS
c.172T>Gp.Cys58Gly Missense variant *HXX X VUS
c.179G>Ap.Ser60Asnrs1413388444Missense variant *HXX X VUS
c.179G>Tp.Ser60Ilers1413388444Missense variant *HXX X VUS
c.184G>Cp.Gly62Argrs1568810268Missense variant *MXX X LP
c.193T>Cp.Phe65Leurs1030039318Missense variant *MXX XX LP
c.197A>Gp.Asn66Serrs1568810275Missense variant *MXX X X LP
c.199C>Gp.Pro67Alars1356474292Missense variant *MXX X X LP
c.199C>Tp.Pro67Serrs1356474292Missense variant *MXX X X LP
c.199G>Cp.Pro67Alars1356474292Missense variant *MXX X X LP
c.200C>Gp.Pro67Argrs1568810284Missense variant *MXX X X LP
c.203T>Cp.Leu68Prors1568810289Missense variant *MXX X X BP4LP
c.203T>Gp.Leu68Argrs1568810289Missense variant *MXX X LP
c.215A>Cp.His72Pro Missense variant *MXX X X LP
c.217G>Ap.Gly73Serrs121912455Missense variant *MXX XX LP
c.217G>Tp.Gly73Cysrs121912455Missense variant *MXX X X LP
c.218G>Ap.Gly73Asp Missense variant *MXX X X LP
c.223C>Tp.Pro75Ser Missense variant *MXX X *H LP
c.224C>Gp.Pro75Arg Missense variant *MXX X LP
c.229G>Tp.Asp77Tyrrs1601157750Missense variant *HXX X XX LP
c.230A>Tp.Asp77Valrs1568810316Missense variant *HXX X XX LP
c.230A>Gp.Asp77Gly Missense variant *HXX X X LP
c.240-7T>G rs1568810602splice region X X VUS
c.241C>Tp.His81Tyr Missense variant *MXX X X LP
c.242A>Gp.His81Argrs121912458Missense variant *MXX X XX LP
c.250G>Ap.Asp84Asnrs1555836789Missense variant *MXX X LP
c.251A>Tp.Asp84Val Missense variant *MXX X LP
c.251A>Gp.Asp84Glyrs1568810615Missense variant *MXX X LP
c.253T>Gp.Leu85Valrs121912452Missense variant *MXX X XX *S BP7P
c.255G>Cp.Leu85Phers1315541036Missense variant *SXX X XXX P
c.255G>Tp.Leu85Phe Missense variant *SXX X X X P
c.256G>Ap.Gly86Serrs121912436synonymous variant *SXX X XX P
c.256G>Cp.Gly86Argrs121912436Missense variant *SXX X XX *S P
c.259A>Gp.Asn87Asprs1555836792Missense variant *SXX XX LP
c.260A>Gp.Asn87Serrs11556620Missense variant *SXX XX *M P
c.260A>Tp.Asn87Ilers11556620Missense variant *MXX XX LP
c.261T>Ap.Asn87Lysrs1555836793Missense variant *SXX X X P
c.262G>Ap.Val88Metrs1568810641Missense variant *SXX X XX P
c.263T>Cp.Val88Alars1339283341Missense variant *SXX X XX P
c.268G>Ap.Ala90Thrrs1568810660Missense variant *SXX X XX P
c.269C>Tp.Ala90Valrs1280042397Missense variant *SXX X XX P
c.271G>Ap.Asp91Asnrs1343616996Missense variant *SXX X BP4LP
c.272A>Cp.Asp91Alars80265967Missense variant *SXX X X *S P
c.272A>Tp.Asp91Valrs80265967Missense variant *SXX X P
c.272_274dupACAp.Asp91_Lys92
insAsn		 frameshift variant *SX X LP
c.275_276delAAp.Lys92ArgfsTer9 frameshift variant X *S P
c.280G>Ap.Gly94Serrs121912437Missense variant *SXX X X *M P
c.280G>Tp.Gly94Cysrs121912437Missense variant *SXX X XX P
c.280G>Cp.Gly94Argrs121912437Missense variant *SXX X XX *S P
c.281G>Ap.Gly94Asprs121912438Missense variant *SXX X XX *M P
c.281G>Cp.Gly94Alars121912438Missense variant *SXX X XX *S P
c.284T>Cp.Val95Alars202198235Missense variant *SXX X LP
c.286G>Ap.Ala96Thrrs1568810686Missense variant *SXX X X LP
c.287C>Gp.Ala96Glyrs1568810690Missense variant *SXX XX LP
c.287C>Tp.Ala96Valrs1568810690Missense variant *SXX XX LP
c.289G>Ap.Asp97Asnrs121912459Missense variant *SXX X BP4LP
c.290A>Tp.Asp97Valrs1555836803Missense variant *SXX X BP4LP
c.292G>Ap.Val98*Metrs1555836806Missense variant *SXX XX LP
c.292G>Cp.Val98Leurs1555836806Missense variant *SXX X X LP
c.298A>Gp.Ile100Valrs760740095Missense variant *SXX VUS
c.301G>Ap.Glu101Lysrs76731700Missense variant *SXX X X *M P
c.301G>Tp.Glu101Terrs76731700stop gained X *VS P
c.301G>Cp.Glu101Gln Missense variant *SXX X LP
c.302A>Gp.Glu101Glyrs121912439Missense variant *SXX X XX *M P
c.304G>Ap.Asp102Asnrs1568810715Missense variant *SXX X XX P
c.304G>Cp.Asp102Hisrs1568810715Missense variant *SXX X XX P
c.304G>Tp.Asp102Tyrs1568810715Missense variant *SXX X X LP
c.305A>Gp.Asp102Glyrs1568810721Missense variant *SXX X XX *M P
c.313A>Tp.Ile105Phers121912445Missense variant *MXX XX LP
c.317C>Tp.Ser106Leurs1378590183Missense variant *MXX XX LP
c.319C>Gp.Leu107Valrs121912440Missense variant *SXX XX LP
c.319C>Tp.Leu107Phers121912440Missense variant *SXX X X LP
c.320_321insTp.Ser108LeufsTer15 frameshift variant X X *VS P
c.326G>Ap.Gly109Glurs1359299834Missense variant *SXX X X LP
c.326G>Tp.Gly109Valrs1359299834Missense variant *SXX XX P
c.328G>Tp.Asp110Tyrrs567432143Missense variant *SXX VUS
c.335G>Ap.Cys112Tyrrs1601158483Missense variant *SXX XX LP
c.335dupp.Cys112TrpfsTer11rs1568810771frameshift variant X XX *VS P
c.338T>Cp.Ile113Thrrs74315452Missense variant *SXX X XX P
c.339C>Gp.Ile113*Metrs1299542356Missense variant *MXX X X LP
c.340A>Tp.Ile114Phers1568810780Missense variant *MXX X X LP
c.341T>Cp.Ile114Thrrs121912441Missense variant *MXX XX *M LP
c.344G>Cp.Gly115Alars1568810789Missense variant *MXX X X LP
c.346C>Gp.Arg116Glyrs1301635320Missense variant *MXX X XX LP
c.346C>Tp.Arg116Cysrs1301635320Missense variant *MXX X X LP
c.358-11A>G rs369600566intron variant X X VUS
c.358-10T>G rs1197141604intron variant X X VUS
c.350C>Gp.Thr117Argrs1568810800Missense variant *MXX X LP
c.352C>Gp.Leu118Valrs199474723Missense variant *MXX *M LP
c.355G>Cp.Val119Leurs1235629842Missense variant *MXX XXX P
c.355G>Tp.Val119Leurs1235629842Missense variant *MXX XXX LP
c.355G>Ap.Val119Met Missense variant *MXX XX LP
c.358G>Tp.Val120Phe Missense variant *MXX X LP
c.358G>Cp.Val120Leurs1457889952Missense variant *MXX X X LP
c.361C>Ap.His121Asn Missense variant *MXX X LP
c.362A>Tp.His121Leurs1410925719Missense variant *MXX X LP
c.374A>Cp.Asp125Alars1568811366Missense variant *MXX X X LP
c.374A>Tp.Asp125Valrs1568811366Missense variant *MXX X XX *M LP
c.376G>Cp.Asp126Hisrs1568811372Missense variant *MXX X XX LP
c.376G>Ap.Asp126Asn Missense variant *MXX X XX LP
c.377A>C rs1164911383Missense variant *MXX X XX LP
c.380T>Ap.Leu127Terrs121912454stop gained X XX *VS P
c.380T>Cp.Leu127Serrs121912454Missense variant *MXX XX LP
c.382G>Cp.Gly128Argrs1568811389Missense variant *MXX X LP
c.383_384insACCCp.Lys129ProfsTer6 frameshift variant X X *VS P
c.383_392dupp.Asn132GlnfsTer5 frameshift variant X XX *VS P
c.384_385insTGGGp.Lys129TrpfsTer6 frameshift variant X X *VS P
c.385A>Gp.Lys129Glu Missense variant *MXX X LP
c.400G>Ap.Glu134Lys Missense variant *MXX XX LP
c.401A>Tp.Glu134Valrs1568811426Missense variant *MXX X X LP
c.403A>Gp.Ser135Glyrs1555836932Missense variant *MXX X X LP
c.404G>Ap.Ser135Asnrs121912451Missense variant *MXX XX LP
c.404G>Cp.Ser135Thrrs121912451Missense variant *MXX X X LP
c.409A>Tp.Lys137Terrs1555836934nonsense variant X X *VS P
c.412A>Gp.Thr138Alars1568811445Missense variant *MXX X X LP
c.413C>Gp.Thr138Argrs1568811454Missense variant *MXX X X LP
c.416G>Ap.Gly139Glurs1568811464Missense variant *MXX X LP
c.418A>Cp.Asn140Hisrs1568811471Missense variant *SXX X X LP
c.418A>Gp.Asn140Asprs1568811471Missense variant *SXX X X LP
c.420C>Ap.Asn140Lysrs1804449Missense variant *SXX X XXX *M P
c.422C>Gp.Ala141Glyrs1555836937Missense variant *SXX X LP
c.424G>Tp.Gly142Terrs1319062081stop gained X X *VS P
c.424G>Ap.Gly142Arg Missense variant *SXX X LP
c.425G>Ap.Gly142Glurs1568811489Missense variant *SXX X LP
c.425G>Cp.Gly142Alars1568811489Missense variant *SXX X LP
c.434T>Cp.Leu145Serrs121912446Missense variant *SXX X XX *M P
c.435G>Cp.Leu145Phers1482760341Missense variant *SXX X XXX *M P
c.436G>Ap.Ala146Thrrs121912447Missense variant *SXX X XX P
c.437C>Ap.Ala146Asprs1131690781Missense variant *SXX X XX P
c.437C>Gp.Ala146Glyrs1131690781Missense variant *SXX X X LP
c.439T>Cp.Cys147Argrs1568811515Missense variant *SXX X *M P
c.441T>Ap.Cys147Ter nonsense variant X X *VS P
c.442G>Cp.Gly148Argrs1568811520Missense variant *SXX X XX P
c.442G>Ap.Gly148Ser Missense variant *SXX X X LP
c.442G>Tp.Gly148Cys Missense variant *SXX X X LP
c.443G>Ap.Gly148Asprs1555836950Missense variant *SXX X XX P
c.445G>Ap.Val149Ilers567511139Missense variant *MXX X XX LP
c.446T>Gp.Val149Glyrs1476760624Missense variant *MXX X XX *M LP
c.446T>Cp.Val149Ala Missense variant *MXX X X LP
c.448A>Gp.Ile150Valrs1169917994Missense variant *MXX X X LP
c.449T>Cp.Ile150Thrrs1424014997Missense variant *MXX XX LP
c.455T>Cp.Ile152Thrrs121912449Missense variant *MXX XX LP
c.455T>Gp.Ile152Ser Missense variant *MXX XX LP
c.457G>Ap.Ala153Thrrs747094021Missense variant *MXX X LP
*M = *Moderate, *H = Supporting, *S = Strong, *VS = Very strong. LP = Likely pathogenic, P = Pathogenic, VUS = Variant of uncertain significance.
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MDPI and ACS Style

Ruffo, P.; Perrone, B.; Conforti, F.L. SOD-1 Variants in Amyotrophic Lateral Sclerosis: Systematic Re-Evaluation According to ACMG-AMP Guidelines. Genes 2022, 13, 537. https://doi.org/10.3390/genes13030537

AMA Style

Ruffo P, Perrone B, Conforti FL. SOD-1 Variants in Amyotrophic Lateral Sclerosis: Systematic Re-Evaluation According to ACMG-AMP Guidelines. Genes. 2022; 13(3):537. https://doi.org/10.3390/genes13030537

Chicago/Turabian Style

Ruffo, Paola, Benedetta Perrone, and Francesca Luisa Conforti. 2022. "SOD-1 Variants in Amyotrophic Lateral Sclerosis: Systematic Re-Evaluation According to ACMG-AMP Guidelines" Genes 13, no. 3: 537. https://doi.org/10.3390/genes13030537

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