Genetic, Structural, and Functional Evidence Link TMEM175 to Synucleinopathies.

OBJECTIVE: The TMEM175/GAK/DGKQ locus is the 3rd strongest risk locus in genome-wide association studies of Parkinson disease (PD). We aimed to identify the specific disease-associated variants in this locus, and their potential implications. METHODS: Full sequencing of TMEM175/GAK/DGKQ followed by genotyping of specific associated variants was performed in PD (n = 1,575) and rapid eye movement sleep behavior disorder (RBD) patients (n = 533) and in controls (n = 1,583). Adjusted regression models and a meta-analysis were performed. Association between variants and glucocerebrosidase (GCase) activity was analyzed in 715 individuals with available data. Homology modeling, molecular dynamics simulations, and lysosomal localization experiments were performed on TMEM175 variants to determine their potential effects on structure and function. RESULTS: Two coding variants, TMEM175 p.M393T (odds ratio [OR] = 1.37, p = 0.0003) and p.Q65P (OR = 0.72, p = 0.005), were associated with PD, and p.M393T was also associated with RBD (OR = 1.59, p = 0.001). TMEM175 p.M393T was associated with reduced GCase activity. Homology modeling and normal mode analysis demonstrated that TMEM175 p.M393T creates a polar side-chain in the hydrophobic core of the transmembrane, which could destabilize the domain and thus impair either its assembly, maturation, or trafficking. Molecular dynamics simulations demonstrated that the p.Q65P variant may increase stability and ion conductance of the transmembrane protein, and lysosomal localization was not affected by these variants. INTERPRETATION: Coding variants in TMEM175 are likely to be responsible for the association in the TMEM175/GAK/DGKQ locus, which could be mediated by affecting GCase activity. ANN NEUROL 2020;87:139-153.

Fine-Mapping of SNCA in Rapid Eye Movement Sleep Behavior Disorder and Overt Synucleinopathies.

OBJECTIVE: Rapid eye movement sleep behavior disorder (RBD) is a prodromal synucleinopathy, as >80% will eventually convert to overt synucleinopathy. We performed an in-depth analysis of the SNCA locus to identify RBD-specific risk variants. METHODS: Full sequencing and genotyping of SNCA was performed in isolated/idiopathic RBD (iRBD, n = 1,076), Parkinson disease (PD, n = 1,013), dementia with Lewy bodies (DLB, n = 415), and control subjects (n = 6,155). The iRBD cases were diagnosed with RBD prior to neurodegeneration, although some have since converted. A replication cohort from 23andMe of PD patients with probable RBD (pRBD) was also analyzed (n = 1,782 cases; n = 131,250 controls). Adjusted logistic regression models and meta-analyses were performed. Effects on conversion rate were analyzed in 432 RBD patients with available data using Kaplan-Meier survival analysis. RESULTS: A 5'-region SNCA variant (rs10005233) was associated with iRBD (odds ratio [OR] = 1.43, p = 1.1E-08), which was replicated in pRBD. This variant is in linkage disequilibrium (LD) with other 5' risk variants across the different synucleinopathies. An independent iRBD-specific suggestive association (rs11732740) was detected at the 3' of SNCA (OR = 1.32, p = 4.7E-04, not statistically significant after Bonferroni correction). Homozygous carriers of both iRBD-specific SNPs were at highly increased risk for iRBD (OR = 5.74, p = 2E-06). The known top PD-associated variant (3' variant rs356182) had an opposite direction of effect in iRBD compared to PD. INTERPRETATION: There is a distinct pattern of association at the SNCA locus in RBD as compared to PD, with an opposite direction of effect at the 3' of SNCA. Several 5' SNCA variants are associated with iRBD and with pRBD in overt synucleinopathies. ANN NEUROL 2020;87:584-598.

Comprehensive Analysis of Familial Parkinsonism Genes in Rapid-Eye-Movement Sleep Behavior Disorder.

BACKGROUND: There is only partial overlap in the genetic background of isolated rapid-eye-movement sleep behavior disorder (iRBD) and Parkinson's disease (PD). OBJECTIVE: To examine the role of autosomal dominant and recessive PD or atypical parkinsonism genes in the risk of iRBD. METHODS: Ten genes, comprising the recessive genes PRKN, DJ-1 (PARK7), PINK1, VPS13C, ATP13A2, FBXO7, and PLA2G6 and the dominant genes LRRK2, GCH1, and VPS35, were fully sequenced in 1039 iRBD patients and 1852 controls of European ancestry, followed by association tests. RESULTS: We found no association between rare heterozygous variants in the tested genes and risk of iRBD. Several homozygous and compound heterozygous carriers were identified, yet there was no overrepresentation in iRBD patients versus controls. CONCLUSION: Our results do not support a major role for variants in these genes in the risk of iRBD. © 2020 International Parkinson and Movement Disorder Society.

Oligogenicity, C9orf72 expansion, and variant severity in ALS.

"Oligogenic inheritance" is used to describe cases where more than one rare pathogenic variant is observed in the same individual. While multiple variants can alter disease presentation, the necessity of multiple variants to instigate pathogenesis has not been addressed in amyotrophic lateral sclerosis (ALS). We sequenced ALS-associated genes in C9orf72-expansion-positive and negative ALS patients, alongside unaffected controls, to test the importance of oligogenicity and variant deleteriousness in ALS. We found that all groups had similar numbers of rare variants, but that variant severity was significantly higher in C9orf72-negative ALS cases, suggesting sufficiency of C9orf72 expansion to cause ALS alone.

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.

SMPD1 mutations, activity, and α-synuclein accumulation in Parkinson's disease.

BACKGROUND: SMPD1 (acid-sphingomyelinase) variants have been associated with Parkinson's disease in recent studies. The objective of this study was to further investigate the role of SMPD1 mutations in PD. METHODS: SMPD1 was sequenced in 3 cohorts (Israel Ashkenazi Jewish cohort, Montreal/Montpellier, and New York), including 1592 PD patients and 975 controls. Additional data were available for 10,709 Ashkenazi Jewish controls. Acid-sphingomyelinase activity was measured by a mass spectrometry-based assay in the New York cohort. α-Synuclein levels were measured in vitro following CRISPR/Cas9-mediated knockout and siRNA knockdown of SMPD1 in HeLa and BE(2)-M17 cells. Lysosomal localization of acid-sphingomyelinase with different mutations was studied, and in silico analysis of their effect on acid-sphingomyelinase structure was performed. RESULTS: SMPD1 mutations were associated with PD in the Ashkenazi Jewish cohort, as 1.4% of PD patients carried the p.L302P or p.fsP330 mutation, compared with 0.37% in 10,709 Ashkenazi Jewish controls (OR, 3.7; 95%CI, 1.6-8.2; P = 0.0025). In the Montreal/Montpellier cohort, the p.A487V variant was nominally associated with PD (1.5% versus 0.14%; P = 0.0065, not significant after correction for multiple comparisons). Among PD patients, reduced acid-sphingomyelinase activity was associated with a 3.5- to 5.8-year earlier onset of PD in the lowest quartile versus the highest quartile of acid-sphingomyelinase activity (P = 0.01-0.001). We further demonstrated that SMPD1 knockout and knockdown resulted in increased α-synuclein levels in HeLa and BE(2)-M17 dopaminergic cells and that the p.L302P and p.fsP330 mutations impair the traffic of acid-sphingomyelinase to the lysosome. CONCLUSIONS: Our results support an association between SMPD1 variants, acid-sphingomyelinase activity, and PD. Furthermore, they suggest that reduced acid-sphingomyelinase activity may lead to α-synuclein accumulation. © 2019 International Parkinson and Movement Disorder Society.

Full sequencing and haplotype analysis of MAPT in Parkinson's disease and rapid eye movement sleep behavior disorder.

BACKGROUND: MAPT haplotypes are associated with PD, but their association with rapid eye movement sleep behavior disorder is unclear. OBJECTIVE: To study the role of MAPT variants in rapid eye movement sleep behavior disorder. METHODS: Two cohorts were included: (A) PD (n = 600), rapid eye movement sleep behavior disorder (n = 613) patients, and controls (n = 981); (B) dementia with Lewy bodies patients with rapid eye movement sleep behavior disorder (n = 271) and controls (n = 950). MAPT-associated variants and the entire coding sequence of MAPT were analyzed. Age-, sex-, and ethnicity-adjusted analyses were performed to examine the association between MAPT, PD, and rapid eye movement sleep behavior disorder. RESULTS: MAPT-H2 variants were associated with PD (odds ratios: 0.62-0.65; P = 0.010-0.019), but not with rapid eye movement sleep behavior disorder. In PD, the H1 haplotype odds ratio was 1.60 (95% confidence interval: 1.12-2.28; P = 0.009), and the H2 odds ratio was 0.68 (95% confidence interval: 0.48-0.96; P = 0.03). The H2/H1 haplotypes were not associated with rapid eye movement sleep behavior disorder. CONCLUSIONS: Our results confirm the protective effect of the MAPT-H2 haplotype in PD, and define its components. Furthermore, our results suggest that MAPT does not play a major role in rapid eye movement sleep behavior disorder, emphasizing different genetic background than in PD in this locus. © 2018 International Parkinson and Movement Disorder Society.

Rare deleterious variants in GRHL3 are associated with human spina bifida.

Neural tube defects, including spina bifida, are among the most common birth defects caused by failure of neural tube closure during development. They have a complex etiology involving largely undetermined environmental and genetic factors. Previous studies in mouse models have implicated the transcription factor Grhl3 as an important factor in the pathogenesis of spina bifida. In the present study, we conducted a resequencing analysis of GRHL3 in a cohort of 233 familial and sporadic cases of spina bifida. We identified two novel truncating variants: one homozygous frameshift variant, p.Asp16Aspfs*10, in two affected siblings and one heterozygous intronic splicing variant, p.Ala318Glyfs*26. We also identified five missense variants, one of which was demonstrated to reduce the activation of gene targets in a luciferase reporter assay. With the previously identified p.Arg391Cys variant, eight variants were found in GRHL3. Comparison of the variant rate between our cohort and the ExAC database identified a significant enrichment of deleterious variants in GRHL3 in the whole gene and the transactivation region in spina bifida patients. These data provide strong evidence for a role of GRHL3 as a predisposing factor to spina bifida and will help dissect the complex etiology and pathogenic mechanisms of these malformations.

Teneurin transmembrane protein 4 is not a cause for essential tremor in a Canadian population.

INTRODUCTION: Mutations in teneurin transmembrane protein 4 were reported to be a risk factor for essential tremor, but the relevance of this across different population remains to be examined. The aim of this study was to determine the frequency and spectrum of variations in teneurin transmembrane protein 4 in a cohort of Canadian essential tremor cases. METHODS: The coding portion of teneurin transmembrane protein 4 was sequenced in 269 unrelated essential tremor cases and 288 matched control individuals using a targeted and high-throughput sequencing approach. RESULTS: A total of 157 single nucleotide variations were identified, and from these 99 were a missense or nonsense mutation. A total of 68 cases were carriers of ≥1 rare missense or nonsense mutations, and 39 control individuals were carriers of the same types of variations. Gene-based association tests were used to jointly analyze the single nucleotide variations. CONCLUSIONS: Our results do not support a positive association between teneurin transmembrane protein 4 and the Canadian population. © 2017 International Parkinson and Movement Disorder Society.

Mutations in SYNE1 lead to a newly discovered form of autosomal recessive cerebellar ataxia.

The past decade has seen great advances in unraveling the biological basis of hereditary ataxias. Molecular studies of spinocerebellar ataxias (SCA) have extended our understanding of dominant ataxias. Causative genes have been identified for a few autosomal recessive ataxias: Friedreich's ataxia, ataxia with vitamin E deficiency, ataxia telangiectasia, recessive spastic ataxia of Charlevoix-Saguenay and ataxia with oculomotor apraxia type 1 (refs. 6,7) and type 2 (ref. 8). Nonetheless, genes remain unidentified for most recessive ataxias. Additionally, pure cerebellar ataxias, which represent up to 20% of all ataxias, remain poorly studied with only two causative dominant genes being described: CACNA1A (ref. 9) and SPTBN2 (ref. 10). Here, we report a newly discovered form of recessive ataxia in a French-Canadian cohort and show that SYNE1 mutations are causative in all of our kindreds, making SYNE1 the first identified gene responsible for a recessively inherited pure cerebellar ataxia.

Novel VPS13B Mutations in Three Large Pakistani Cohen Syndrome Families Suggests a Baloch Variant with Autistic-Like Features.

BACKGROUND: Cohen Syndrome (COH1) is a rare autosomal recessive disorder, principally identified by ocular, neural and muscular deficits. We identified three large consanguineous Pakistani families with intellectual disability and in some cases with autistic traits. METHODS: Clinical assessments were performed in order to allow comparison of clinical features with other VPS13B mutations. Homozygosity mapping followed by whole exome sequencing and Sanger sequencing strategies were used to identify disease-related mutations. RESULTS: We identified two novel homozygous deletion mutations in VPS13B, firstly a 1 bp deletion, NM_017890.4:c.6879delT; p.Phe2293Leufs*24, and secondly a deletion of exons 37-40, which co-segregate with affected status. In addition to COH1-related traits, autistic features were reported in a number of family members, contrasting with the "friendly" demeanour often associated with COH1. The c.6879delT mutation is present in two families from different regions of the country, but both from the Baloch sub-ethnic group, and with a shared haplotype, indicating a founder effect among the Baloch population. CONCLUSION: We suspect that the c.6879delT mutation may be a common cause of COH1 and similar phenotypes among the Baloch population. Additionally, most of the individuals with the c.6879delT mutation in these two families also present with autistic like traits, and suggests that this variant may lead to a distinct autistic-like COH1 subgroup.

Expanded ATXN3 frameshifting events are toxic in Drosophila and mammalian neuron models.

Spinocerebellar ataxia type 3 is caused by the expansion of the coding CAG repeat in the ATXN3 gene. Interestingly, a -1 bp frameshift occurring within an (exp)CAG repeat would henceforth lead to translation from a GCA frame, generating polyalanine stretches instead of polyglutamine. Our results show that transgenic expression of (exp)CAG ATXN3 led to -1 frameshifting events, which have deleterious effects in Drosophila and mammalian neurons. Conversely, transgenic expression of polyglutamine-encoding (exp)CAA ATXN3 was not toxic. Furthermore, (exp)CAG ATXN3 mRNA does not contribute per se to the toxicity observed in our models. Our observations indicate that expanded polyglutamine tracts in Drosophila and mouse neurons are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with (exp)CAG repeats.

Rare PSAP Variants and Possible Interaction with GBA in REM Sleep Behavior Disorder.

BACKGROUND: PSAP encodes saposin C, the co-activator of glucocerebrosidase, encoded by GBA. GBA mutations are associated with idiopathic/isolated REM sleep behavior disorder (iRBD), a prodromal stage of synucleinopathy. OBJECTIVE: To examine the role of PSAP mutations in iRBD. METHODS: We fully sequenced PSAP and performed Optimized Sequence Kernel Association Test in 1,113 iRBD patients and 2,324 controls. We identified loss-of-function (LoF) mutations, which are very rare in PSAP, in three iRBD patients and none in controls (uncorrected p = 0.018). RESULTS: Two variants were stop mutations, p.Gln260Ter and p.Glu166Ter, and one was an in-frame deletion, p.332_333del. All three mutations have a deleterious effect on saposin C, based on in silico analysis. In addition, the two carriers of p.Glu166Ter and p.332_333del mutations also carried a GBA variant, p.Arg349Ter and p.Glu326Lys, respectively. The co-occurrence of these extremely rare PSAP LoF mutations in two (0.2%) GBA variant carriers in the iRBD cohort, is unlikely to occur by chance (estimated co-occurrence in the general population based on gnomAD data is 0.00035%). Although none of the three iRBD patients with PSAP LoF mutations have phenoconverted to an overt synucleinopathy at their last follow-up, all manifested initial signs suggestive of motor dysfunction, two were diagnosed with mild cognitive impairment and all showed prodromal clinical markers other than RBD. Their probability of prodromal PD, according to the Movement Disorder Society research criteria, was 98% or more. CONCLUSION: These results suggest a possible role of PSAP variants in iRBD and potential genetic interaction with GBA, which requires additional studies.

LRRK2 p.M1646T is associated with glucocerebrosidase activity and with Parkinson's disease.

The LRRK2 p.G2019S Parkinson's disease (PD) variant is associated with elevated glucocerebrosidase (GCase) activity in peripheral blood. We aimed to evaluate the association of other LRRK2 variants with PD and its association with GCase activity. LRRK2 and GBA were fully sequenced in 1123 PD patients and 576 controls from the Columbia and PPMI cohorts, in which GCase activity was measured in dried blood spots by liquid chromatography-tandem mass spectrometry. LRRK2 p.M1646T was associated with increased GCase activity in the Columbia University cohort (ß = 1.58, p = 0.0003), and increased but not significantly in the PPMI cohort (ß = 0.29, p = 0.58). p.M1646T was associated with PD (odds ratio = 1.18, 95% confidence interval = 1.09-1.28, p = 7.33E-05) in 56,306 PD patients and proxy-cases, and 1.4 million controls. Our results suggest that the p.M1646T variant is associated with risk of PD with a small effect and with increased GCase activity in peripheral blood.

Association study of DNAJC13, UCHL1, HTRA2, GIGYF2, and EIF4G1 with Parkinson's disease.

Rare mutations in genes originally discovered in multigenerational families have been associated with increased risk of Parkinson's disease (PD). The involvement of rare variants in DNAJC13, UCHL1, HTRA2, GIGYF2, and EIF4G1 loci has been poorly studied or has produced conflicting results across cohorts. However, they are still being often referred to as "PD genes" and used in different models. To further elucidate the role of these 5 genes in PD, we fully sequenced them using molecular inversion probes in 2408 patients with PD and 3444 controls from 3 different cohorts. A total of 788 rare variants were identified across the 5 genes and 3 cohorts. Burden analyses and optimized sequence Kernel association tests revealed no significant association between any of the genes and PD after correction for multiple comparisons. Our results do not support an association of the 5 tested genes with PD. Combined with previous studies, it is unlikely that any of these genes plays an important role in PD. Their designation as "PARK" genes should be reconsidered.

Novel Associations of BST1 and LAMP3 With REM Sleep Behavior Disorder.

OBJECTIVE: To examine the role of genes identified through genome-wide association studies (GWASs) of Parkinson disease (PD) in the risk of isolated REM sleep behavior disorder (iRBD). METHODS: We fully sequenced 25 genes previously identified in GWASs of PD in a total of 1,039 patients with iRBD and 1,852 controls. The role of rare heterozygous variants in these genes was examined with burden tests. The contribution of biallelic variants was further tested. To examine the potential effect of rare nonsynonymous BST1 variants on the protein structure, we performed in silico structural analysis. Finally, we examined the association of common variants using logistic regression adjusted for age and sex. RESULTS: We found an association between rare heterozygous nonsynonymous variants in BST1 and iRBD (p = 0.0003 at coverage >50× and 0.0004 at >30×), driven mainly by 3 nonsynonymous variants (p.V85M, p.I101V, and p.V272M) found in 22 (1.2%) controls vs 2 (0.2%) patients. All 3 variants seem to be loss-of-function variants with a potential effect on the protein structure and stability. Rare noncoding heterozygous variants in LAMP3 were also associated with iRBD (p = 0.0006 at >30×). We found no association between rare heterozygous variants in the rest of genes and iRBD. Several carriers of biallelic variants were identified, yet there was no overrepresentation in iRBD. CONCLUSION: Our results suggest that rare coding variants in BST1 and rare noncoding variants in LAMP3 are associated with iRBD. Additional studies are required to replicate these results and to examine whether loss of function of BST1 could be a therapeutic target.

Analysis of common and rare VPS13C variants in late-onset Parkinson disease.

OBJECTIVE: We aimed to study the role of coding VPS13C variants in a large cohort of patients with late-onset Parkinson disease (PD) (LOPD). METHODS: VPS13C and its untranslated regions were sequenced using targeted next-generation sequencing in 1,567 patients with PD and 1,667 controls from 3 cohorts. Association tests of rare potential homozygous and compound heterozygous variants and burden tests for rare heterozygous variants were performed. Common variants were analyzed using logistic regression adjusted for age and sex in each of the cohorts, followed by a meta-analysis. RESULTS: No biallelic carriers of rare VPS13C variants were found among patients, and 2 carriers of compound heterozygous variants were found in 2 controls. There was no statistically significant burden of rare (minor allele frequency [MAF] <1%) or very rare (MAF <0.1%) coding VPS13C variants in PD. A VPS13C haplotype including the p.R153H-p.I398I-p.I1132V-p.Q2376Q variants was nominally associated with a reduced risk for PD (meta-analysis of the tagging SNP p.I1132V [odds ratio = 0.48, 95% confidence interval = 0.28-0.82, p = 0.0052]). This haplotype was not in linkage disequilibrium with the known genome-wide association study top hit. CONCLUSIONS: Our results do not support a role for rare heterozygous or biallelic VPS13C variants in LOPD. Additional genetic replication and functional studies are needed to examine the role of the haplotype identified here associated with reduced risk for PD.

Variants in the Niemann-Pick type C gene NPC1 are not associated with Parkinson's disease.

Biallelic variants in NPC1, a gene coding for a lysosomal transmembrane protein involved in cholesterol trafficking, may cause Niemann-Pick disease type C (NPC). A few cases of NPC1 variant carriers with Parkinson's disease (PD) have been reported. In addition, pathologic studies have demonstrated phosphorylated alpha-synuclein and Lewy pathology in brains of NPC patients. Therefore, we aimed to examine whether NPC1 genetic variants may be associated with PD. Full sequencing of NPC1 was performed in 2657 PD patients and 3647 controls from 3 cohorts, using targeted sequencing with molecular inversion probes. A total of 9 common variants and 126 rare variants were identified across the 3 cohorts. To examine their association with PD, regression models adjusted for age, sex, and origin were performed for common variants, and optimal sequence Kernel association test (SKAT-O) was performed for rare variants. After correction for multiple comparisons, common and rare NPC1 variants were not associated with PD. Our results do not support a link between heterozygous variants in NPC1 and PD.

Novel de novo SHANK3 mutation in autistic patients.

A number of studies have confirmed that genetic factors play an important role in autism spectrum disorder (ASD). More recently de novo mutations in the SHANK3 gene, a synaptic scaffolding protein, have been associated with the ASD phenotype. As part of our gene discovery strategy, we sequenced the SHANK3 gene in a cohort of 427 ASD subjects and 190 controls. Here, we report the identification of two putative causative mutations: one being a de novo deletion at an intronic donor splice site and one missense transmitted from an epileptic father. We were able to confirm the deleterious effect of the splice site deletion by RT-PCR using mRNA extracted from cultured lymphoblastoid cells. The missense mutation, a leucine to proline at amino acid position 68, is perfectly conserved across all species examined, and would be predicted to disrupt an alpha-helical domain. These results further support the role of SHANK3 gene disruption in the etiology of ASD.