Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis.

AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Evidence for polygenic and oligogenic basis of Australian sporadic amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with phenotypic and genetic heterogeneity. Approximately 10% of cases are familial, while remaining cases are classified as sporadic. To date, >30 genes and several hundred genetic variants have been implicated in ALS. METHODS: Seven hundred and fifty-seven sporadic ALS cases were recruited from Australian neurology clinics. Detailed clinical data and whole genome sequencing (WGS) data were available from 567 and 616 cases, respectively, of which 426 cases had both datasets available. As part of a comprehensive genetic analysis, 853 genetic variants previously reported as ALS-linked mutations or disease-associated alleles were interrogated in sporadic ALS WGS data. Statistical analyses were performed to identify correlation between clinical variables, and between phenotype and the number of ALS-implicated variants carried by an individual. Relatedness between individuals carrying identical variants was assessed using identity-by-descent analysis. RESULTS: Forty-three ALS-implicated variants from 18 genes, including C9orf72, ATXN2, TARDBP, SOD1, SQSTM1 and SETX, were identified in Australian sporadic ALS cases. One-third of cases carried at least one variant and 6.82% carried two or more variants, implicating a potential oligogenic or polygenic basis of ALS. Relatedness was detected between two sporadic ALS cases carrying a SOD1 p.I114T mutation, and among three cases carrying a SQSTM1 p.K238E mutation. Oligogenic/polygenic sporadic ALS cases showed earlier age of onset than those with no reported variant. CONCLUSION: We confirm phenotypic associations among ALS cases, and highlight the contribution of genetic variation to all forms of ALS.

Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis and frontotemporal dementia and transgenic TDP-43 mice.

OBJECTIVE: Since the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. METHODS: Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology. RESULTS: No causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease. CONCLUSIONS: Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS.

Reduction of Intersex in a Wild Fish Population in Response to Major Municipal Wastewater Treatment Plant Upgrades.

Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007-2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013-2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure.

Genetic and Pathological Assessment of hnRNPA1, hnRNPA2/B1, and hnRNPA3 in Familial and Sporadic Amyotrophic Lateral Sclerosis.

BACKGROUND: Mutations in the genes encoding the heterogeneous nuclear ribonucleoproteins hnRNPA1 and hnRNPA2/B1 have been reported in a multisystem proteinopathy that includes amyotrophic lateral sclerosis (ALS) and inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia. Mutations were also described in the prion-like domain of hnRNPA1 in patients with classic ALS. Another hnRNP protein, hnRNPA3, has been found to be associated with the ALS/frontotemporal dementia protein C9orf72. OBJECTIVE: To further assess their role in ALS, we examined these hnRNPs in spinal cord tissue from sporadic (SALS) and familial ALS (FALS) patients, including C9orf72 repeat expansion-positive patients, and controls. We also sought to determine the prevalence of HNRNPA1, HNRNPA2B1, and HNRNPA3 mutations in Australian ALS patients. METHODS: Immunostaining was used to assess hnRNPs in ALS patient spinal cords. Mutation analysis of the HNRNPA1, HNRNPA2B1, and HNRNPA3 genes was performed in FALS and of their prion-like domains in SALS patients. RESULTS: Immunostaining of spinal motor neurons of ALS patients with the C9orf72 repeat expansion showed significant mislocalisation of hnRNPA3, and no differences in hnRNPA1 or A2/B1 localisation, compared to controls. No novel or known mutations were identified in HNRNPA1, HNRNPA2B1, or HNRNPA3 in Australian ALS patients. CONCLUSIONS: hnRNPA3 pathology was identified in motor neurons of ALS patients with C9orf72 repeat expansions, implicating hnRNPA3 in the pathogenesis of C9orf72-linked ALS. hnRNPA3 warrants further investigation into the pathogenesis of ALS linked to C9orf72. This study also determined that HNRNP mutations are not a common cause of FALS and SALS in Australia.

Increased Resolution of Structural MRI at 3T Improves Estimation of Regional Cortical Degeneration in Individual Dementia Patients Using Surface Thickness Maps.

BACKGROUND: Objective measurement of regional cortical atrophy in individual patients would be a highly desirable adjunct for diagnosis of degenerative dementias. OBJECTIVE: We hypothesized that increasing the resolution of magnetic resonance scans would improve the sensitivity of cortical atrophy detection for individual patients. METHODS: 46 participants including 8 semantic-variant primary progressive aphasia (svPPA), seven posterior cortical atrophy (PCA), and 31 cognitively unimpaired participants underwent clinical assessment and 3.0T brain scans. SvPPA and PCA were chosen because there is overwhelming prior knowledge of the expected atrophy pattern. Two sets of T1-weighted images with 0.8 mm3 (HighRes) and conventional 1.0 mm3 (ConvRes) resolution were acquired. The cortical ribbon was segmented using FreeSurfer software to obtain surface-based thickness maps. Inter-sequence performance was assessed in terms of cortical thickness and sub-cortical volume reproducibility, signal-to-noise and contrast-to-noise ratios. For clinical cases, diagnostic effect size (Cohen's d) and lesion distribution (z-score and t-value maps) were compared between HighRes and ConvRes scans. RESULTS: The HighRes scans produced higher image quality scores at 90 seconds extra scan time. The effect size of cortical thickness differences between patients and cognitively unimpaired participants was 15-20% larger for HighRes scans. HighRes scans showed more robust patterns of atrophy in expected regions in each and every individual patient. CONCLUSIONS: HighRes T1-weighted scans showed superior precision for identifying the severity of cortical atrophy in individual patients, offering a proof-of-concept for clinical translation. Studying svPPA and PCA, two syndromes with well-defined focal atrophy patterns, offers a method to clinically validate and contrast automated algorithms.

Pareidolias are a function of visuoperceptual impairment.

Pareidolias, or the misperception of ambiguous stimuli as meaningful objects, are complex visual illusions thought to be phenomenologically similar to Visual Hallucination (VH). VH are a major predictor of dementia in Parkinson's Disease (PD) and are included as a core clinical feature in Dementia with Lewy Bodies (DLB). A newly developed Noise Pareidolia Test (NPT) was proposed as a possible surrogate marker for VH in DLB patients as increased pareidolic responses correlated with informant-corroborated accounts of VH. This association could, however, be mediated by visuoperceptual impairment. To understand the drivers of performance on the NPT, we contrasted performances in patient groups that varied both in terms of visuoperceptual ability and rates of VH. N = 43 patients were studied of whom n = 13 had DLB or PD with Dementia (PDD); n = 13 had PD; n = 12 had typical, memory-onset Alzheimer's Disease (tAD); and n = 5 had Posterior Cortical Atrophy (PCA) due to Alzheimer's disease. All patient groups reported pareidolias. Within the Lewy body disorders (PD, DLB, PDD), there was no significant difference in pareidolic response rates between hallucinating and non-hallucinating patients. Visuoperceptual deficits and pareidolic responses were most frequent in the PCA group-none of whom reported VH. Regression analyses in the entire patient cohort indicated that pareidolias were strongly predicted by visuoperceptual impairment but not by the presence of VH. These findings suggest that pareidolias reflect the underlying visuoperceptual impairment of Lewy body disorders, rather than being a direct marker for VH.

Characterising the Genetic Landscape of Amyotrophic Lateral Sclerosis: A Catalogue and Assessment of Over 1,000 Published Genetic Variants.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with genetic and phenotypic heterogeneity. Pathogenic genetic variants remain the only validated cause of disease, the majority of which were discovered in familial ALS patients. While causal gene variants are a lesser contributor to sporadic ALS, an increasing number of risk alleles (low penetrance genetic variants associated with a small increase in disease risk) and variants of uncertain significance have been reported. OBJECTIVE: To examine the pathogenic potential of genetic variation in ALS, we sought to characterise variant- and gene-level attributes of previously reported ALS-implicated variants. METHODS: A list of 1,087 genetic variants reported in ALS to March 2021 was compiled through comprehensive literature review. Individual variants were annotated using in silico tools and databases across variant features including pathogenicity scores, localisation to protein domains, evolutionary conservation, and minor allele frequencies. Gene level attributes of genic tolerance, gene expression in ALS-relevant tissues and gene ontology terms were assessed for 33 ALS genes. Statistical analysis was performed for each characteristic, and we compared the most penetrant variants found in familial cases with risk alleles exclusive to sporadic cases, to explore genetic variant features that associate with disease penetrance. RESULTS: We provide spreadsheet (hg19 and GRCh38) and variant call format (GRCh38) resources for all 1,087 reported ALS-implicated variants, including detailed summaries for each attribute. We demonstrate that the characteristics of variants found exclusively in sporadic ALS cases are less severe than those observed in familial ALS. CONCLUSIONS: We provide a comprehensive, literature-derived catalogue of genetic variation in ALS thus far and reveal crucial attributes that contribute to ALS pathogenicity. Our variant- and gene-level observations highlight the complexity of genetic variation in ALS, and we discuss important implications and considerations for novel variant interpretation.

Wild fish responses to wastewater treatment plant upgrades in the Grand River, Ontario.

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

Short tandem repeat expansions in sporadic amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic short tandem repeat (STR) expansions cause over 20 neurodegenerative diseases. To determine the contribution of STRs in sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), we used ExpansionHunter, REviewer, and polymerase chain reaction validation to assess 21 neurodegenerative disease-associated STRs in whole-genome sequencing data from 608 patients with sporadic ALS, 68 patients with sporadic FTD, and 4703 matched controls. We also propose a data-derived outlier detection method for defining allele thresholds in rare STRs. Excluding C9orf72 repeat expansions, 17.6% of clinically diagnosed ALS and FTD cases had at least one expanded STR allele reported to be pathogenic or intermediate for another neurodegenerative disease. We identified and validated 162 disease-relevant STR expansions in C9orf72 (ALS/FTD), ATXN1 [spinal cerebellar ataxia type 1 (SCA1)], ATXN2 (SCA2), ATXN8 (SCA8), TBP (SCA17), HTT (Huntington's disease), DMPK [myotonic dystrophy type 1 (DM1)], CNBP (DM2), and FMR1 (fragile-X disorders). Our findings suggest clinical and pathological pleiotropy of neurodegenerative disease genes and highlight their importance in ALS and FTD.

Clinical testing panels for ALS: global distribution, consistency, and challenges.

Objective: In 2021, the Clinical Genome Resource (ClinGen) amyotrophic lateral sclerosis (ALS) spectrum disorders Gene Curation Expert Panel (GCEP) was established to evaluate the strength of evidence for genes previously reported to be associated with ALS. Through this endeavor, we will provide standardized guidance to laboratories on which genes should be included in clinical genetic testing panels for ALS. In this manuscript, we aimed to assess the heterogeneity in the current global landscape of clinical genetic testing for ALS. Methods: We reviewed the National Institutes of Health (NIH) Genetic Testing Registry (GTR) and members of the ALS GCEP to source frequently used testing panels and compare the genes included on the tests. Results: 14 clinical panels specific to ALS from 14 laboratories covered 4 to 54 genes. All panels report on ANG, SOD1, TARDBP, and VAPB; 50% included or offered the option of including C9orf72 hexanucleotide repeat expansion (HRE) analysis. Of the 91 genes included in at least one of the panels, 40 (44.0%) were included on only a single panel. We could not find a direct link to ALS in the literature for 14 (15.4%) included genes. Conclusions: The variability across the surveyed clinical genetic panels is concerning due to the possibility of reduced diagnostic yields in clinical practice and risk of a missed diagnoses for patients. Our results highlight the necessity for consensus regarding the appropriateness of gene inclusions in clinical genetic ALS tests to improve its application for patients living with ALS and their families.

Genetic determinants of antimicrobial resistance in three multi-drug resistant strains of Cutibacterium acnes isolated from patients with acne: a predictive in silico study.

Objectives: Using available whole genome data, the objective of this in silico study was to identify genetic mechanisms that could explain the antimicrobial resistance profile of three multi-drug resistant (MDR) strains (CA17, CA51, CA39) of the skin bacterium Cutibacterium acnes previously recovered from patients with acne. In particular, we were interested in detecting novel genetic determinants associated with resistance to fluoroquinolone and macrolide antibiotics that could then be confirmed experimentally. Methods: A range of open source bioinformatics tools were used to 'mine' genetic determinants of antimicrobial resistance and plasmid borne contigs, and to characterise the phylogenetic diversity of the MDR strains. Results: As probable mechanisms of resistance to fluoroquinolones, we identified a previously described resistance associated allelic variant of the gyrA gene with a 'deleterious' S101L mutation in type IA1 strains CA51 (ST1) and CA39 (ST1), as well as a novel E761R 'deleterious' mutation in the type II strain CA17 (ST153). A distinct genomic sequence of the efflux protein YfmO which is potentially associated with resistance to MLSB antibiotics was also present in CA17; homologues in CA51, CA39, and other strains of Cutibacterium acnes , were also found but differed in amino acid content. Strikingly, in CA17 we also identified a circular 2.7 kb non-conjugative plasmid (designated pCA17) that closely resembled a 4.8 kb plasmid (pYU39) from the MDR Salmonella enterica strain YU39. Conclusions: This study has provided a detailed explanation of potential genetic determinants for MDR in the Cutibacterium acnes strains CA17, CA39 and CA51. Further laboratory investigations will be required to validate these in silico results, especially in relation to pCA17.

Citizen Schools' partner-dependent expanded learning model.

Citizen Schools, a national nonprofit organization, shares lessons learned through years of experience developing and sustaining effective partnerships with schools around the country.

Genetic Analysis of Tryptophan Metabolism Genes in Sporadic Amyotrophic Lateral Sclerosis.

The essential amino acid tryptophan (TRP) is the initiating metabolite of the kynurenine pathway (KP), which can be upregulated by inflammatory conditions in cells. Neuroinflammation-triggered activation of the KP and excessive production of the KP metabolite quinolinic acid are common features of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In addition to its role in the KP, genes involved in TRP metabolism, including its incorporation into proteins, and synthesis of the neurotransmitter serotonin, have also been genetically and functionally linked to these diseases. ALS is a late onset neurodegenerative disease that is classified as familial or sporadic, depending on the presence or absence of a family history of the disease. Heritability estimates support a genetic basis for all ALS, including the sporadic form of the disease. However, the genetic basis of sporadic ALS (SALS) is complex, with the presence of multiple gene variants acting to increase disease susceptibility and is further complicated by interaction with potential environmental factors. We aimed to determine the genetic contribution of 18 genes involved in TRP metabolism, including protein synthesis, serotonin synthesis and the KP, by interrogating whole-genome sequencing data from 614 Australian sporadic ALS cases. Five genes in the KP (AFMID, CCBL1, GOT2, KYNU, HAAO) were found to have either novel protein-altering variants, and/or a burden of rare protein-altering variants in SALS cases compared to controls. Four genes involved in TRP metabolism for protein synthesis (WARS) and serotonin synthesis (TPH1, TPH2, MAOA) were also found to carry novel variants and/or gene burden. These variants may represent ALS risk factors that act to alter the KP and lead to neuroinflammation. These findings provide further evidence for the role of TRP metabolism, the KP and neuroinflammation in ALS disease pathobiology.

Genetic analysis of GLT8D1 and ARPP21 in Australian familial and sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive degeneration of motor neurons. Recently, genetic variants in GLT8D1 and ARPP21 were associated with ALS in a cohort of European descent. A synergistic relationship was proposed between ALS associated variants in GLT8D1 and ARPP21. We aimed to determine the prevalence of genetic variation in GLT8D1 and ARPP21 in an Australian cohort of familial (n = 81) and sporadic ALS (n = 618) cases using whole-exome and whole-genome sequencing data. No novel mutations were identified in either gene, nor was there significant enrichment of protein-altering sequence variation among ALS cases. GLT8D1 and ARPP21 mutations are not a common cause of ALS in Australian familial and sporadic cohorts.

Identity by descent analysis identifies founder events and links SOD1 familial and sporadic ALS cases.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by the loss of upper and lower motor neurons resulting in paralysis and eventual death. Approximately 10% of ALS cases have a family history of disease, while the remainder present as apparently sporadic cases. Heritability studies suggest a significant genetic component to sporadic ALS, and although most sporadic cases have an unknown genetic aetiology, some familial ALS mutations have also been found in sporadic cases. This suggests that some sporadic cases may be unrecognised familial cases with reduced disease penetrance in their ancestors. A powerful strategy to uncover a familial link is identity-by-descent (IBD) analysis, which detects genomic regions that have been inherited from a common ancestor. IBD analysis was performed on 83 Australian familial ALS cases from 25 families and three sporadic ALS cases, each of whom carried one of three SOD1 mutations (p.I114T, p.V149G and p.E101G). We defined five unique 350-SNP haplotypes that carry these mutations in our cohort, indicative of five founder events. This included two founder haplotypes that carry SOD1 p.I114T; linking familial and sporadic cases. We found that SOD1 p.E101G arose independently in each family that carries this mutation and linked two families that carry SOD1 p.V149G. The age of disease onset varied between cases that carried each SOD1 p.I114T haplotype. Linking families with identical ALS mutations allows for larger sample sizes and increased statistical power to identify putative phenotypic modifiers.

Theme 3 In vitro experimental models.

Background: Ongoing disease gene discoveries continue to drive our understanding of the molecular and cellular mechanisms underlying ALS. Causative genes from 60% of ALS families have been identified using modern genetic techniques, but the causal gene defect is yet to be identified in the remaining 40% of families. These remaining families often do not follow true Mendelian inheritance patterns and are challenging to solve using traditional genetic analysis alone. In vitro and in vivo studies have become critical in assessing and validating these ALS candidate genes.Objectives: In this study, we aim to develop and validate the utility of an in vitro functional pipeline for the discovery and validation of novel ALS candidate genes.Methods: A panel of cell based-assays were applied to candidate genes to examine the presence/absence of known ALS pathologies in cell lines as well as human autopsy tissues. These include immunofluorescence, flow cytometry and western blotting to study toxicity, neuronal inclusion formation, interaction with TDP-43, aberrant protein degradation and accumulation in detergent-insoluble cellular fractions. Immunohistochemistry and immunofluorescence were also used to examine if candidates were present in neuronal inclusions from ALS patient spinal cord tissues.Results: The in vitro pipeline was applied to five candidate genes from an ALS family that is negative for known ALS gene mutations. Two candidates were prioritized as top candidates based on their capacity to induce known ALS cellular pathologies. In transfected cells, the variants in these two genes caused a significantly higher toxicity than wild type, formed detergent insoluble inclusions and was able to co-aggregate with TDP-43 in neuronal cells. The variants have also led to protein degradation defects. One of the candidates also co-localised with TDP-43-positive neuronal inclusions in sporadic ALS patient post-mortem tissues, a signature pathology of ALS.Discussion and conclusions: We have demonstrated the utility of a functional prioritization pipeline and successfully prioritized two novel candidate ALS genes. These genes, and its associated pathways, will be further investigated through the development of animal models to establish if there is support for its role in ALS. New ALS genes offer fresh diagnostic and therapeutic targets and tools for the generation of novel animal models to better understand disease biology and offer preclinical testing of candidate treatments for ALS in the future.