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Trinh, J., Hicks, A.A., König, I.R., Delcambre, S., Lüth, T., Schaake, S., Wasner, K., Ghelfi, J., Borsche, M., Vilariño-Güell, C., Hentati, F., Germer, E.L., Bauer, P., Takanashi, M., Kostić, V., Lang, A.E., Brüggemann, N., Pramstaller, P.P., Pichler, I., Rajput, A., Hattori, N., Farrer, M.J., Lohmann, K., Weissensteiner, H., May, P., Klein, C. & Grünewald, A.

Biallelic mutations in PINK1/PRKN cause recessive Parkinson's disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA (mtDNA) integrity and inflammation as disease modifiers in carriers of mutations in these genes. MtDNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson's disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mtDNA variant load (AUC = 0.83, CI:0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbor more heteroplasmic mtDNA variants in blood (p = 0.0006, Z = 3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in iPSC-derived (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and postmortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Lastly, the heteroplasmic mtDNA variant load correlated with IL6 levels in PINK1/PRKN mutation carriers (r = 0.57, p = 0.0074). PINK1/PRKN mutations predispose individuals to mtDNA variant accumulation in a dose- and disease-dependent manner.

Liao, C., Castonguay, C.E., Heilbron, K., Vuokila, V., Medeiros, M., Houle, G., Akcimen, F., Ross, J.P., Catoire, H., Diez-Fairen, M., Kang, J., Mueller, S.H., Girard, S.L., Hopfner, F., Lorenz, D., Clark, L.N., Soto-Beasley, A.I., Klebe, S., Hallett, M., Wszolek, Z.K., Pendziwiat, M., Lorenzo-Betancor, O., Seppi, K., Berg, D., Vilariño-Güell, C., Postuma, R.B., Bernard, G., Dupre, N., Jankovic, J., Testa, C.M., Ross, O.A., Arzberger, T., Chouinard, S., Louis, E.D., Mandich, P., Vitale, C., Barone, P., Garcia-Martin, E., Alonso-Navarro, H., Agundez, J.A.G., Jimenez-Jimenez, F.J., Pastor, P., Rajput, A., Deuschl, G., Kuhlenbaumer, G., Meijer, I.A., Dion, P.A. & Rouleau, G.A.

Importance: Essential tremor (ET) is one of the most common movement disorders, affecting 5% of the general population older than 65 years. Common variants are thought to contribute toward susceptibility to ET, but no variants have been robustly identified.
Objective: To identify common genetic factors associated with risk of ET.
Design, Setting, and Participants: Case-control genome-wide association study. Inverse-variance meta-analysis was used to combine cohorts. Multicenter samples collected from European populations were collected from January 2010 to September 2019 as part of an ongoing study. Included patients were clinically diagnosed with or reported having ET. Control individuals were not diagnosed with or reported to have ET. Of 485250 individuals, data for 483054 passed data quality control and were used.
Main Outcomes and Measures: Genotypes of common variants associated with risk of ET.
Results: Of the 483054 individuals included, there were 7177 with ET (3693 [51.46%] female; mean [SD] age, 62.66 [15.12] years), and 475877 control individuals (253785 [53.33%] female; mean [SD] age, 56.40 [17.6] years). Five independent genome-wide significant loci and were identified and were associated with approximately 18% of ET heritability. Functional analyses found significant enrichment in the cerebellar hemisphere, cerebellum, and axonogenesis pathways. Genetic correlation (r), which measures the degree of genetic overlap, revealed significant common variant overlap with Parkinson disease (r, 0.28; P = 2.38 x 10-8) and depression (r, 0.12; P = 9.78 x 10-4). A separate fine-mapping of transcriptome-wide association hits identified genes such as BACE2, LRRN2, DHRS13, and LINC00323 in disease-relevant brain regions, such as the cerebellum.
Conclusions and Relevance: The results of this genome-wide association study suggest that a portion of ET heritability can be explained by common genetic variation and can help identify new common genetic risk factors for ET.


Kamma, E., Becquart, P., Traboulsee, A., Schabas, A., Vavasour, I., Laule, C., Vilariño-Güell, C. & Quandt, J.A.

CD5 antigen-like (CD5L) protein is a macrophage-secreted protein with roles in immunomodulation and lipid homeostasis. We compared serum CD5L levels in healthy controls to individuals diagnosed with clinically isolated syndrome, relapsing remitting (RR), secondary progressive (SP), and primary progressive (PP) multiple sclerosis (MS). CD5L was increased in SPMS relative to controls, RRMS, and PPMS. SPMS CD5L was associated with longer disease duration independent of age, sex, or disease severity. The positive relationship between CD5L and disease duration in SPMS suggests a chronic peripheral inflammatory profile compared to other subtypes, particularly PPMS, warranting investigation of functional roles for CD5L in MS.

Vilariño-Güell, C., Encarnacion, M., Bernales, C.Q., Kamma, E., Becquart, P. & Quandt, J.

BACKGROUND: The development of effective treatments for multiple sclerosis (MS), and in particular its progressive forms, is hampered by the lack of etiologically relevant cellular and animal models of human disease. Models that recapitulate the biological and pathological processes leading to the onset and progression of MS in patients are likely to afford better translational efficacy. Following the discovery of the NR1H3 p.Arg415Gln pathogenic mutation for progressive MS in two Canadian families, we developed a knock-in mouse model harboring a homologous mutation in the endogenous gene to provide a more physiologically relevant model of human MS.

METHODS: Gene expression was evaluated in constitutive heterozygote (which recapitulates the human disease genotype) and homozygote Nr1h3 p.Arg413Gln knock-in mice on a C57BL/6 background, and compared to wild-type littermates. AmpliSeq Transcriptome Mouse Gene Expression kits analyzed on an Ion Proton sequencer were used to generate the gene expression profiles of spleen, liver, brain and spinal cord tissue from three-month-old male and female mice. Differential expression between genotypes was assessed with DESeq2, and Gene Ontologies pathways enrichment analysis performed with DAVID v6.8. Benjamini-Hochberg false discovery rate (FDR) correction for multiple testing was applied.

RESULTS: Transcriptome analysis of spleen tissue from Nr1h3 p.Arg413Gln mice revealed 23 significantly dysregulated genes (FDR<0.05) with greater than a two-fold change in expression. These include CD5 antigen-like (Cd5l), complement component 6 (C6), procollagen C-endopeptidase enhancer 2 (Pcolce2), interleukin 22 receptor, alpha 2 (Il22ra2), and T cell immunoglobulin and mucin domain containing 4 (Timd4). Gene Ontology enrichment analysis support upregulation of cell cycle pathways and downregulation of immune system response in splenic cells. The liver transcriptome identified 27 significantly dysregulated genes with greater than a two-fold change in expression compared to wild-type littermates. Cd5l, Timd4, C-C motif chemokine receptor 3 (Ccr3), ADAM metallopeptidase domain 11 (Adam11) and macrophage expressed 1 (Mpeg1) were amongst those most significantly dysregulated. Enrichment analysis supported altered immune function with upregulation of sterol and steroid metabolic processes and downregulation of fatty acid biosynthesis and inflammatory and immune system responses. Although brain and spinal cord transcriptome profiles identified several genes significantly dysregulated in Nr1h3 mice compared to wild-type littermates (FDR<0.05), none presented greater than two-fold changes in gene expression.

DISCUSSION: The analysis of the Nr1h3 p.Arg413Gln mouse model of MS suggests that the predominance of a pro-inflammatory over a healing or reparative phenotype, combined with deficiencies in myelination and remyelination, are the biological mechanisms implicated in the onset of MS and the development of a more severe progressive disease course observed in patients with NR1H3 mutations. Association of NR1H3 common variants with MS risk indicates that the disruption of these biological and immunological processes is not only informative for familial forms of disease but MS patients at large. Differences in transcriptome profiles underline the value of this model for the development and validation of novel therapeutic strategies and ultimately treatments with the potential to delay or even halt the onset of progressive MS and to ameliorate the severity of clinical symptoms.


Yun, Y., Zhang, Y., Zhang, C., Huang, L., Tan, S., Wang, P., Vilariño-Güell, C., Song, W. & Sun, X.

Posttranscriptional regulation of gene expression plays an important role in the maturation, transport, stability and translation of coding and noncoding RNAs. RNA-binding protein (RBP) is a key factor of the regulation. Regulator of calcineurin 1 (RCAN1) is a multifunctional protein involved in neurodegeneration, mitochondrial dysfunction, inflammation and protein glycosylation, and plays an important role in the pathogenesis of Down syndrome and Alzheimer's disease. In this report, we discovered that RCNA1 is a novel RNA-binding protein. A 23 nucleotide sequence of adenine nucleotide translocator (ANT1) mRNA was identified as the binding motif of RCAN1. Furthermore, we found that R1SR13, as the RNA aptamer of RCAN1 identified by SELEX, blocked RCAN1-induced inhibition of the nuclear factor of activated T cells (NFAT) and NF-kappaB signaling pathways, and reduced neuronal apoptosis. Taken together, our results demonstrate that RCAN1 is a novel RNA-binding protein and the RNA aptamer of RCAN1 plays a neuroprotective role.

Diez-Fairen, M., Houle, G., Ortega-Cubero, S., Bandres-Diga, S., Alvarez, I., Carcel, M., Ibañez, L., Fernandez, M.V., Budde, J.P., Trotta, J.R., Tonda, R., Chong, J.X., Bamshad, M.J., Nickerson, D.A., University of Washington Center for Mendelian Genomics (UWCMG) Aquilar, M., Tartari, J.P., Gironell, A., Garcia-Martin, E., Agundez, J.A.G., Alonso-Navarro, H., Jimenez-Jimenez, F.J., Fernandez, M., Valldeoriola, F., Marti, M.J., Tolosa, E., Coria, F., Pastor, M.A., Vilariño-Güell, C., Rajput, A., Dion, P.A., Cruchaga, C., Rouleau, G.A & Pastor, P.

INTRODUCTION: Essential tremor (ET) is one of the most common movement disorders. Despite its high prevalence and heritability, its genetic etiology remains elusive with only a few susceptibility genes identified and poorly replicated. Our aim was to find novel candidate genes involved in ET predisposition through whole exome sequencing.

METHODS: We studied eight multigenerational families (N = 40 individuals) with an autosomal-dominant inheritance using a comprehensive strategy combining whole exome sequencing followed by case-control association testing of prioritized variants in a separate cohort comprising 521 ET cases and 596 controls. We further performed gene-based burden analyses in an additional dataset comprising 789 ET patients and 770 healthy individuals to investigate whether there was an enrichment of rare deleterious variants within our candidate genes.

RESULTS: Fifteen variants co-segregated with disease status in at least one of the families, among which rs749875462 in CCDC183, rs535864157 in MMP10 and rs114285050 in GPR151 showed a nominal association with ET. However, we found no significant enrichment of rare variants within these genes in cases compared with controls. Interestingly, MMP10 protein is involved in the inflammatory response to neuronal damage and has been previously associated with other neurological disorders.

CONCLUSIONS: We prioritized a set of promising genes, especially MMP10, for further genetic and functional studies in ET. Our study suggests that rare deleterious coding variants that markedly increase susceptibility to ET are likely to be found in many genes. Future studies are needed to replicate and further infer biological mechanisms and potential disease causality for our identified genes.


Becquart, P., Vilariño-Güell, C. & Quandt, J.A.

Understanding the biological changes responsible for failures in repair and the development of progressive MS is paramount for therapeutic intervention. In a well characterized experimental autoimmune encephalomyelitis (EAE) model of MS the clinical phenotype features an acute attack with partial recovery followed by a chronic or progressive disease phase. Neuropathology-focused gene expression profiles were generated from spinal cord, hindbrain and forebrain of mice 25 days after the induction of EAE, the time when recovery plateaus and transitions to a chronic or worsening phase. Differences in gene expression were most pronounced in the spinal cord of EAE mice compared to sham-immunized animals, with a subset of genes also found to be differentially expressed in the hindbrain and the forebrain, albeit with smaller fold-changes in expression. Our data suggests that changes in complement components, chemoattractant cytokines and especially enrichment in microglial cells may be the primary drivers of processes that limit recovery in EAE.

Popplewell, L.F., Encarnacion, M., Bernales, C.Q., Sadovnick, A.D., Traboulsee, A.L., Quandt, J.A. & Vilariño-Güell, C.

Genetic and functional analysis of the inflammasome suggest a role for this multiprotein complex in the biological mechanisms leading to the onset and progression of multiple sclerosis (MS). Nucleotide-binding, leucine-rich repeat (NLR) receptors trigger the activation and assembly of specific inflammasomes in response to danger signals. Mining exome sequencing data from 326 MS patients identified 17 rare missense or nonsense variants in NLR family pyrin domain containing 1 (NLRP1), NLRP3, NLRP6, NLRP7 and NLR family CARD domain containing 4 (NLRC4). Genotyping these variants in 2,503 MS cases and 1,076 healthy controls did not result in statistically significant differences between groups and segregation analysis within MS families was largely unsupportive of co-segregation of these variants with disease. However, the identification of MS patients harboring rare homozygote variants in NLRP1 (p.Ile601Phe and p.Ser1387Ile), a variant in NLRP3 (p.Leu832Ile) resulting in the substitution of a critical amino acid for the formation of its leucine-rich repeat domain, and several MS patients with NLRC4 variants (p.Arg310Ter and p.Glu600Ter) causing protein truncations, suggest that rare protein-altering variants in inflammasome-activating NLR receptors may contribute to MS risk.


Becquart, P., Johnston, J.,Vilariño-Güell, C. & Quandt, J.A.

OBJECTIVE: We examined expression of aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a basicloop-helix transcription factor implicated in neuronal development and axonal health, in oligodendrocyte(OL) cultures and over the course of chronic experimental autoimmune encephalomyelitis(EAE), the murine model of multiple sclerosis (MS).
METHODS: We assessed OL ARNT2 expression in EAE compared with sham-immunized controls and also in OL primary cultures and over the course of dibutyryl cyclic adenosine monophosphate (dbcAMP)-mediated maturation of the immortalized Oli-neu cell line. We also tested the functional role of ARNT2 in influencing OL characteristics using small interfering RNA (siRNA).
RESULTS: ARNT2 is localized to Olig2+ cells in healthy spinal cord gray and white matter. Despite a significant expansion of Olig2+ cells in the white matter at peak disease, ARNT2 is reduced by almost half in OLs, along with a reduction in the percentage of ARNT2+/Olig2+ cells. Mature OLs in mixed cortical cultures or OLs matured from embryonic progenitors express negligible ARNT2. Similarly, Oli-neu cells express high levels of ARNT2, which are reduced following dbcAMP maturation. siRNA-mediated knockdown of ARNT2 affected OL viability, which led to an enrichment of myelin-producing OLs.
CONCLUSION: The analysis of ARNT2 expression in OLs demonstrates that OL ARNT2 expression is altered in EAE and during OL maturation. Findings point to ARNT2 as an important mediator of OL viability and differentiation and warrant further characterization as a target for intervention in demyelinating disorders such as MS.



Germer, E.L., Imhoff, S., Vilariño-Güell, C., Kasten, M., Seibler, P., Brüggemann, N., Klein, C. & Trinh, J.

INTRODUCTION: Genome-wide association studies (GWAS) have identified multiple loci associated with PD risk. The presence of rare variants within these loci that may account for the increased susceptibility requires further investigation.
METHODS: Using exome sequencing, we performed a comprehensive rare variant screen of genes located within 56 novel PD loci. We first analyzed exomes from 109 subjects in the discovery cohort (85 diagnosed with PD and 24 healthy controls), and filtered for rare coding variants with MAF<0.01 and CADD>15. Further investigation of exome data from a replication cohort of 2,859 European patients with PD (IPDGC) and 24,146 non-Finnish European controls from gnomAD were used for association testing of specific rare variants found in the discovery cohort.
RESULTS: Our genetic screening identified 54 potential disease-relevant variants in 71 genes in 109 subjects. Six out of 54 variants were found in two or more patients and were not observed in healthy controls: DNAH1 p.A3639T, STAB1 p.S1089G, ANK2 p.V3634D, ANK2 p.R3906W, SH3GL2 p.G276V, NOD2 p.G908R. Replication in the IPDGC confirmed the association with PD risk for three out of the six identified variants (STAB1 p.S1089G, SH3GL2 p.G276V, and NOD2 p.G908R) (p<10-3).
CONCLUSION: Our study suggests that some of the associations identified in PD risk loci can be ascribed to rare variants with likely functional effects that modify PD risk.

Encarnacion, M., Bernales, C.Q., Traboulsee, A.L., Sadovnick, A.D. & Vilariño-Güell, C.

Over the last few years, the implementation of whole exome sequencing (WES) technologies has nominated several pathogenic mutations for familial forms of multiple sclerosis (MS). Recently, Garcia-Rosa et al. described a homozygous loss-of-function mutation in galanin receptor 2 (GALR2 p.Trp249Leu, rs61745847) in a female patient who developed MS at 22 years of age.


Gharagozloo, M., Mahmoud, S., Simard, C., Yamamoto, K., Bobbala, D., Ilangumaran, S., Smith, M., Lamontagne, A., Jarjoura, S., Denault, J.B., Blais, V., Gendron, L., Vilariño-Güell, C., Sadovnick, A.D., Ting, J., Calabresi, P.A., Amrani, A. & Gris, D.

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1-/- mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity.

Vilariño-Güell, C., Zimprich, A., Martinelli-Boneschi, F., Herculano, B., Wang, Z., Matesanz, F., Urcelay, E., Vandenbroeck, K., Leyva, L., Gris, D., Massaad, C., Quandt, J.A., Traboulsee, A.L., Encarnacion, M., Bernales, C.Q., Follett, J., Yee, I.M., Criscuoli, M.G., Deutschländer, A., Reinthaler, E.M., Zrzavy, T., Mascia, E., Zauli, A., Esposito, F., Alcina, A., Izquierdo, G., Espino-Paisán, L., Mena, J., Antigüedad, A., Urbaneja-Romero, P., Ortega-Pinazo, J., Song, W. & Sadovnick, A.D.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.


Vilariño-Güell C., Encarnacion M., Bernales C.Q. & Sadovnick A.D.

A recent article described a missense variant in FKBP6 (p.R183C, rs147213094) co-segregating with multiple sclerosis (MS) in eight individuals from a large Dutch multi-incident family. Replication analysis in a Canadian cohort of European ancestry consisting of 2,383 MS patients and 1,055 healthy controls identified FKBP6 p.R183C in 15 patients (MAF=0.31%) and 6 controls (MAF=0.28%). Thirteen patients with the FKBP6 mutation had a positive family history of MS. Co-segregation analysis in these families identified the p.R183C mutation in 1 out of 17 additional family member diagnosed with MS, and 14 out of 60 unaffected relatives, thus not supporting co-segregation with MS.

Schmouth J.F., Houle G., Ambalavanan A., Leblond C.S., Spiegelman D., Laurent S.B., Bourassa C.V., Panisset M., Chouinard S., Dupré N., Vilariño-Güell C., Rajput A., Dion P.A. & Rouleau G.A.

Essential Tremor is a prevalent neurological disorder of unknown etiology. Studies suggest that genetic factors contribute to this pathology. To date, no causative mutations in a gene have been reproducibly reported. All three structures of the olivocerebellar motor circuitry have been linked to Essential Tremor. We postulated that genes enriched for their expression in the olivocerebellar circuitry would be more susceptible to harbor mutations in Essential Tremor patients. A list of 11 candidate genes, enriched for their expression in the olivocerebellar circuitry, was assessed for their variation spectrum and frequency in a cohort of Canadian Essential Tremor cases. Our results from this list of 11 candidate genes do not support an association for Essential Tremor in our cohort of Canadian cases. The heterogenic nature of ET and modest size of the cohort used in this study are two confounding factors that could explain these results.

Nygaard H.B., Omay Z.E., Wu X., Kent B.A., Bernales C.Q., Evans D.M., Farrer M.J., Vilariño-Güell C. & Strittmatter S.M.

Centenarians represent a unique cohort to study the genetic basis for longevity and factors determining the risk of neurodegenerative disorders, including Alzheimer’s disease (AD). The highest estimated genetic contribution to longevity is found in centenarians and supercententenarians, but few genetic variants have been shown to clearly impact this phenotype. Moreover, while the genetic risk for AD and other dementias is now well understood, the frequency of known dementia risk variants in centenarians is not known. To address these questions, we performed whole exome sequencing on 100 individuals of 98-108 years age in search of genes with large effect sizes towards the aging phenotype. Overall, we were unable to identify a rare protein-altering variant or individual genes with an increased burden of rare variants associated with exceptional longevity. Gene burden analysis revealed three genes of nominal statistical significance associated with extreme aging, including LYST, MDN1, and RBMXL1. Several genes with variants conferring an increased risk for AD and other dementias were identified, including TREM2, EPHA1, ABCA7, PDL3, MAPT, and NOTCH3. Larger centenarian studies will be required to further elucidate the genetic basis for longevity, and factors conferring protection against age-dependent neurodegenerative syndromes.


Reinthaler, E.M., Graf, E., Zrzavy, T., Wienland, T., Hotzy, C., Schmied, C., Keilani, M., Leumetzer, F., Lill, C.M., Kopecky, C., Deutschlander, A., Vilariño-Güell C., Sadovnick, A.D., Zimprich, F., Ahram, M., Tomkinson, B., Strom, T.M., Kristoferitsch, W., Lassmann, H. & Zimprich, A.

OBJECTIVE: To ascertain the genetic cause of a consanguineous family from Syria suffering from a sterile brain inflammation mimicking a mild non-progressive form of Multiple Sclerosis (MS).

METHODS: We used homozygosity mapping and next-generation sequencing to detect the disease-causing gene in the affected siblings. We additionally performed RNA and protein expression studies, enzymatic activity assays, immunohistochemistry, and targeted sequencing of further MS cases from Austria, Germany, Canada and Jordan.

RESULTS: In this study, we describe the identification of a homozygous missense mutation (c.82T>G, p.Cys28Gly) in the tripeptidyl peptidase II (TPP2) gene in all three affected siblings of the family. Sequencing of all TPP2-coding exons in 826 MS cases identified one further homozygous missense variant (c.2027C>T, p.Thr676Ile) in a Jordanian MS patient. TPP2 protein expression in whole blood was reduced in the affected siblings. In contrast, TPP2 protein expression in post-mortem brain tissue from MS patients without TPP2 mutations was highly upregulated.

CONCLUSION: The homozygous TPP2 mutation (p.Cys28Gly) is likely responsible for the inflammation phenotype in this family. TPP2 is an ubiquitously expressed serine peptidase that removes tripeptides from the N-terminal end of longer peptides. TPP2 is involved in various biological processes including the destruction of MHC class I epitopes. Recessive loss-of-function mutations in TPP2 were described in patients with Evans Syndrome, a rare autoimmune disease affecting the hematopoietic system. Based on the gene expression results in our MS autopsy brain samples, we further suggest that TPP2 may play a broader role in the inflammatory process in MS.

Gil-Varea E., Urcelay E., Vilariño-Güell C., Costa C., Matesanz F., Rodríguez-Antigüedad A., Oksenberg J., Saiz A., Villar L.M., García-Merino J.A., Ramió-Torrentà L., Triviño J.C., Malhotra S., Fissolo N., Montalban X. & Comabella M.

BACKGROUND: It remains unclear whether disease course in multiple sclerosis (MS) is influenced by genetic polymorphisms. Here, we aimed to identify genetic variants associated with benign and aggressive disease courses in MS patients.

METHODS: MS patients were classified into benign and aggressive phenotypes according to clinical criteria. We performed exome sequencing in a discovery cohort, which included 20 MS patients, 10 with benign and 10 with aggressive disease course, and genotyping in 2 independent validation cohorts. The first validation cohort encompassed 194 MS patients, 107 with benign and 87 with aggressive phenotypes. The second validation cohort comprised 257 patients, of whom 224 patients had benign phenotypes and 33 aggressive disease courses. Brain immunohistochemistries were performed using disease course associated genes antibodies.

RESULTS: By means of single-nucleotide polymorphism (SNP) detection and comparison of allele frequencies between patients with benign and aggressive phenotypes, a total of 16 SNPs were selected for validation from the exome sequencing data in the discovery cohort. Meta-analysis of genotyping results in two validation cohorts revealed two polymorphisms, rs28469012 and rs10894768, significantly associated with disease course. SNP rs28469012 is located in CPXM2 (carboxypeptidase X, M14 family, member 2) and was associated with aggressive disease course (uncorrected p value < 0.05). SNP rs10894768, which is positioned in IGSF9B (immunoglobulin superfamily member 9B) was associated with benign phenotype (uncorrected p value < 0.05). In addition, a trend for association with benign phenotype was observed for a third SNP, rs10423927, in NLRP9 (NLR family pyrin domain containing 9). Brain immunohistochemistries in chronic active lesions from MS patients revealed expression of IGSF9B in astrocytes and macrophages/microglial cells, and expression of CPXM2 and NLRP9 restricted to brain macrophages/microglia.

CONCLUSIONS: Genetic variants located in CPXM2, IGSF9B, and NLRP9 have the potential to modulate disease course in MS patients and may be used as disease activity biomarkers to identify patients with divergent disease courses. Altogether, the reported results from this study support the influence of genetic factors in MS disease course and may help to better understand the complex molecular mechanisms underlying disease pathogenesis.


Bernales C.Q., Encarnacion M., Criscuoli M.G., Yee I.M., Traboulsee A.L., Sadovnick A.D. & Vilariño-Güell C.

The implementation of exome sequencing technologies have started to unravel the genetic etiology of familial multiple sclerosis (MS). A homozygote p.G587S mutation in NLRP1 has been suggested as potentially causative for the onset of MS in an affected sibling pair, who later developed malignant melanoma. To validate the proposed role of recessive NLRP1 mutations in the pathological mechanisms of MS, we examined exome sequencing data from 326 MS patients from Canada for the identification of NLRP1 missense and nonsense variants. This analysis did not identify the previously described p.G587S mutation, however three patients with potential NLRP1 compound heterozygote mutations were observed. Haplotype and segregation analysis indicates that the variants observed in these patients were inherited in cis, and do not segregate with disease within families. Thus, the analysis of MS patients from Canada failed to identify potentially pathogenic mutations in NLRP1, including the previously described p.G587S mutation. Further studies are necessary to confirm a role of NLRP1 in the pathophysiology of MS.



Houle G., Ambalavanan A., Schmouth J.F., Leblond C.S., Spiegelman D., Laurent S.B., Bourassa C.V., Grayson C., Panisset M., Chouinard S., Dupré N., Vilariño-Güell C., Rajput A., Girard S.L., Dion P.A. & Rouleau G.A.

A two-stage genome-wide association study (GWAS) that included 2,807 cases and 6,441 controls of European descent recently reported associations between essential tremor (ET) and specific intronic variants within three genes: STK32B, PPARGC1A, CTNNA3. To further assess their role as ET predisposing factors we screened the coding regions of these genes for rare variants. Looking at exome and whole genome sequencing data no rare deleterious variant were found to segregate with the disease in 14 autosomal dominant multiplex ET families. Next we used a targeted massive parallel sequencing approach to examine the protein-coding region of these genes in 269 ET cases and 287 control individuals. Thirty-four variants were identified. No difference emerged regarding the distributions of individual variants (or gene) between cases and controls. Thus no rare exonic variants further validated one of these genes as a risk factor for ET. The recent GWAS offers promising avenues but the genetic heterogeneity of ET is nonetheless challenging for the validation of risk factors, ultimately larger cohorts of cases should help to overcome this task.

Sadovnick A.D., Traboulsee A.L., Zhao Y., Bernales C.Q., Encarnacion M., Ross J.P., Yee I.M., Criscuoli M. & Vilariño-Güell C.

The genetic contribution to clinical outcomes for multiple sclerosis (MS) has yet to be defined. We performed exome sequencing analysis in 100 MS patients presenting opposite extremes of clinical phenotype (discovery cohort), and genotyped variants of interest in 2,016 MS patients (replication cohort). Linear and logistic regression analyses were used to identify significant associations with disease course, severity and onset. Our analysis of the discovery cohort nominated 38 variants in 21 genes. Replication analysis identified PSMG4 p.W99R and NLRP5 p.M459I to be associated with disease severity (p=0.002 and 0.008). CACNA1H p.R1871Q was found associated with patients presenting relapsing remitting MS at clinical onset (p=0.028) whereas NLRP5 p.M459I and EIF2AK1 p.K558R were associated with primary progressive disease (p=0.031 and 0.023). In addition, PSMG4 p.W99R and NLRP5 p.R761L were found to correlate with an earlier age at MS clinical onset, and MC1R p.R160W with delayed onset of clinical symptoms (p=0.010-0.041).

Puschmann A., Fiesel F.C., Caulfield T.R., Hudec R., Ando M., Truban D., Hou X., Ogaki K., Heckman M.G., James E.D., Swanberg M., Jimenez-Ferrer I., Hansson O., Opala G., Siuda J., Boczarska-Jedynak M., Friedman A., Koziorowski D., Rudzinska-Bar M., Aasly J.O., Lynch T., Mellick G.D., Mohan M., Silburn P.A., Sanotsky Y., Vilariño-Güell C., Farrer M.J., Chen L., Dawson V.L., Dawson T.M., Wszolek Z.K., Ross O.A. & Springer W.

We read with great interest that Gan-Or et al. have observed an association (albeit not reaching statistical significance) of the heterozygous PINK1 p.G411S mutation and rapid eye movement sleep behaviour disorder [RBD; effect size odds ratio (OR) of 2.49] in their multicentre cohort of 350 RBD patients and 869 controls subjects. Given the strong correlation of RBD with Parkinson’s disease, we interpret this as further support for our observation on 9142 individuals showing that heterozygous PINK1 p.G411S mutation confers a marked increase in Parkinson’s disease risk odds ratio = 2.89, P = 0.027.

Traboulsee A.L., Sadovnick A.D., Encarnacion M., Bernales C.Q., Yee I.M., Criscuoli M.G. & Vilariño-Güell C.

Several single gene disorders with clinical and radiological characteristics similar to those observed in multiple sclerosis (MS) patients have been described. To evaluate whether this phenotypic overlap can be ascribed to a common genetic etiology, 28 genes known to present pathogenic mutations for 24 of these disorders were sequenced in 270 MS patients. All identified variants were genotyped in 2,131 MS cases and 830 healthy controls, and those exclusively observed in patients were assessed for segregation within families. This analysis identified 9 rare variants in 6 genes segregating with disease in 13 families. Four different mutations were identified in CYP27A1, including a reported pathogenic mutation for cerebrotendinous xanthomatosis (p.R405W), which was observed in six patients from a multi-incident family, three diagnosed with MS, two with an undefined neurological disease and one seemingly healthy. A LYST p.V1678A and a PDHA1 p.K387Q mutation were both observed in five MS patients from three separate multi-incident families. In addition CLCN2 p.V174G, GALC p.D162E and POLG p.R361G were each identified in two MS patients from one family. This study suggests a shared genetic etiology between MS and the characterized single gene disorders, and highlights cholesterol metabolism and the synthesis of oxysterols as important biological mechanisms for familial MS.


Sadovnick A.D., Gu B.J., Traboulsee A.L., Bernales C.Q., Encarnacion M., Yee I.M., Criscuoli M., Huang X., Ou A., Milligan C.J., Petrou S., Wiley J.S. & Vilariño-Güell C.

Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study we set out to evaluate whether rare coding variants of major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T (p.T205M), P2RX7 rs201921967:A>G (p.N361S) and P2RX4 rs765866317:G>A (p.G135S)) segregating with disease in a multi-incident family with six family members diagnosed with MS (LOD=3.07). Functional analysis of this haplotype in HEK293 cells revealed impaired P2X7 surface expression (p<0.01), resulting in over 95% inhibition of ATP-induced pore function (p<0.001) and a marked reduction in phagocytic ability (p<0.05). In addition, transfected cells showed 40% increased peak ATP-induced inward current (p<0.01), and a greater Ca2+ response to the P2X4 135S variant compared to wild type (p<0.0001). Our study nominates rare genetic variants in P2RX4 and P2RX7 as major genetic contributors to disease, further supporting a role for these purinergic receptors in MS and suggesting the disruption of transmembrane cation channels leading to impairment of phagocytosis as the pathological mechanisms of disease.

Houle G., Schmouth J.F., Leblond C.S., Ambalavanan A., Spiegelman D., Laurent S.B., Bourassa C.V., Panisset M., Chouinard S., Dupre N., Vilariño-Güell C., Rajput A., Dion P.A. & Rouleau G.A.

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.

Puschmann A., Fiesel F.C., Caulfield T.R., Hudec R., Ando M., Truban D., Hou X., Ogaki K., Heckman M.G., James E.D., Swanberg M., I. J.-F., Hansson O., Opala G., Siuda J., Boczarska-Jedynak M., Friedman A., Koziorowski D., Aasly J.O., Lynch T., Mellick G.D., Mohan M., Silburn P.A., Sanotsky Y., Vilariño-Güell C., Farrer M.J., Chen L., Dawson V.L., Dawson T.M., Wszolek Z.K., Ross O.A. & Springer W.

It has been postulated that heterozygous mutations in recessive Parkinson’s genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson’s disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson’s disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson’s disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.


Muller S.H., Girard S.L., Hopfner F., Merner N.D., Bourassa C.V., Lorenz D., Clark L.N., Tittmann L., Soto-Ortolaza A.I., Klebe S., Hallett M., Schneider S.A., Hodkinson C.A., Lieb W., Wszolek Z.K., Pendziwiat M., Lorenzo-Bentacor O., Poewe W., Ortega-Cubero S., Seppi C., Rajput A., Hussl A., Rajput A.H., Berg D., Dion P.A., Wurster I., Shuman J.M., Srulijes K., Haubenberger D., Pastor P., Vilariño-Güell C., Postuma R.B., Panisset M., Winkelmann J., Testa C.M., Reischl E., Zeuner K.E., Ross O.A., Arzberger T., Chouinard S., Deuschl G., Louis E.D., Kuhlenbaumer G. & Rouleau G.A.

We conducted a genome-wide association study of essential tremor, a common movement disorder characterized mainly by a postural and kinetic tremor of the upper extremities. Twin and family history studies show a high heritability for essential tremor. The molecular genetic determinants of essential tremor are unknown. We included 2807 patients and 6441 controls of European descent in our two-stage genome-wide association study. The 59 most significantly disease-associated markers of the discovery stage were genotyped in the replication stage. After Bonferroni correction two markers, one (rs10937625) located in the serine/threonine kinase STK32B and one (rs17590046) in the transcriptional coactivator PPARGC1A were associated with essential tremor. Three markers (rs12764057, rs10822974, rs7903491) in the cell-adhesion molecule CTNNA3 were significant in the combined analysis of both stages. The expression of STK32B was increased in the cerebellar cortex of patients and expression quantitative trait loci database mining showed association between the protective minor allele of rs10937625 and reduced expression in cerebellar cortex. We found no expression differences related to disease status or marker genotype for the other two genes. Replication of two lead single nucleotide polymorphisms of previous small genome-wide association studies (rs3794087 in SLC1A2, rs9652490 in LINGO1) did not confirm the association with essential tremor.

Wang Z., Sadovnick A.D., Traboulsee A.L., Ross J.P., Bernales C.Q., Encarnacion M., Yee I.M., de Lemos M., Greenwood T., Lee J.D., Wright G., Ross C.J., Zhang S., Song W. & Vilariño-Güell C.

Identifying rare genetic variants that drive the onset of disease is challenging, even before considering the additional genetic and environmental influences that likely exist in complex diseases. We recently published a study proposing a rare variant in the NR1H3 gene (p.R415Q, rs61731956) as responsible for the onset of multiple sclerosis (MS) in two multi-incident families. This publication has generated much discussion, and fortunately the possibility to validate a finding or prove it spurious can occur rapidly in genetic studies. All novel discoveries must be replicated, and best efforts should be made to ensure that these replications use the appropriate samples and approach, and provide the correct interpretation of the results.

Trinh J., Gustavsson E.K., Vilariño-Güell C., Bortnick S., Latourelle J., McKenzie M.B., Szu Tu C., Nosova E., Khinda J., Milnerwood A., Lesage S., Brice A., Tazir M., Aasly J.O., Parkkinen L., Haytural H., Foroud T., Myers R.H., Sassi S.B., Hentati E., Nabli F., Farhat E., Amouri R., Hentati F. & Farrer M.J.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) mutation 6055G→A (Gly2019Ser) accounts for roughly 1% of patients with Parkinson’s disease in white populations, 13–30% in Ashkenazi Jewish populations, and 30–40% in North African Arab-Berber populations, although age of onset is variable. Some carriers have early-onset parkinsonism, whereas others remain asymptomatic despite advanced age. We aimed to use a genome-wide approach to identify genetic variability that directly affects LRRK2 Gly2019Ser penetrance.

METHODS: Between 2006 and 2012, we recruited Arab-Berber patients with Parkinson’s disease and their family members (aged 18 years or older) at the Mongi Ben Hamida National Institute of Neurology (Tunis, Tunisia). Patients with Parkinson’s disease were diagnosed by movement disorder specialists in accordance with the UK Parkinson’s Disease Society Brain Bank criteria, without exclusion of familial parkinsonism. LRRK2 carrier status was confirmed by Sanger sequencing or TaqMan SNP assays-on-demand. We did genome-wide linkage analysis using data from multi-incident Arab-Berber families with Parkinson’s disease and LRRK2 Gly2019Ser (with both affected and unaffected family members). We assessed Parkinson’s disease age of onset both as a categorical variable (dichotomised by median onset) and as a quantitative trait. We used data from another cohort of unrelated Tunisian LRRK2 Gly2019Ser carriers for subsequent locus-specific genotyping and association analyses. Whole-genome sequencing in a subset of 14 unrelated Arab-Berber individuals who were LRRK2 Gly2019Ser carriers (seven with early-onset disease and seven elderly unaffected individuals) subsequently informed imputation and haplotype analyses. We replicated the findings in separate series of LRRK2 Gly2019Ser carriers originating from Algeria, France, Norway, and North America. We also investigated associations between genotype, gene, and protein expression in human striatal tissues and murine LRRK2 Gly2019Ser cortical neurons.

FINDINGS: Using data from 41 multi-incident Arab-Berber families with Parkinson’s disease and LRRK2 Gly2019Ser (150 patients and 103 unaffected family members), we identified significant linkage on chromosome 1q23.3 to 1q24.3 (non-parametric logarithm of odds score 2·9, model-based logarithm of odds score 4·99, θ=0 at D1S2768). In a cohort of unrelated Arab-Berber LRRK2 Gly2019Ser carriers, subsequent association mapping within the linkage region suggested genetic variability within DNM3 as an age-of-onset modifier of disease (n=232; rs2421947; haplotype p=1·07 × 10–7). We found that DNM3 rs2421947 was a haplotype tag for which the median onset of LRRK2 parkinsonism in GG carriers was 12·5 years younger than that of CC carriers (Arab-Berber cohort, hazard ratio [HR] 1·89, 95% CI 1·20–2·98). Replication analyses in separate series from Algeria, France, Norway, and North America (n=263) supported this finding (meta-analysis HR 1·61, 95% CI 1·15–2·27, p=0·02). In human striatum, DNM3 expression varied as a function of rs2421947 genotype, and dynamin-3 localisation was perturbed in murine LRRK2 Gly2019Ser cortical neurons.

INTERPRETATION: Genetic variability in DNM3 modifies age of onset for LRRK2 Gly2019Ser parkinsonism and informs disease-relevant translational neuroscience. Our results could be useful in genetic counselling for carriers of this mutation and in clinical trial design.

Wang Z., Sadovnick A.D., Traboulsee A.L., Ross J.P., Bernales C.Q., Encarnacion M., Yee I.M., de Lemos M., Greenwood T., Lee J.D., Wright G., Ross C.J., Zhang S., Song W. & Vilariño-Güell C.

Multiple sclerosis (MS) is an inflammatory disease characterized by myelin loss and neuronal dysfunction. Despite the aggregation observed in some families, pathogenic mutations have remained elusive. In this study we describe the identification of NR1H3 p.Arg415Gln in seven MS patients from two multi-incident families presenting severe and progressive disease, with an average age at onset of 34 years. Additionally, association analysis of common variants in NR1H3 identified rs2279238 conferring a 1.35-fold increased risk of developing progressive MS. The p.Arg415Gln position is highly conserved in orthologs and paralogs, and disrupts NR1H3 heterodimerization and transcriptional activation of target genes. Protein expression analysis revealed that mutant NR1H3 (LXRA) alters gene expression profiles, suggesting a disruption in transcriptional regulation as one of the mechanisms underlying MS pathogenesis. Our study indicates that pharmacological activation of LXRA or its targets may lead to effective treatments for the highly debilitating and currently untreatable progressive phase of MS.

Sadovnick A.D., Traboulsee A.L., Bernales C.Q., Ross J.P., Forwell A.L., Yee I.M., Guillot-Noel L., Fontaine B., Cournu-Rebeix I., Alcina A., Fedetz M., Izquierdo G., Matesanz F., Hilven K., Dubois B., Goris A., Astobiza I., Alloza I., Antiguedad A., Vandenbroeck K., Akkad D.A., Aktas O., Blaschke P., Buttmann M., Chan A., Epplen J.T., Gerdes L.A., Kroner A., Kubisch C., Kumpfel T., Lohse P., Rieckmann P., Zettl U.K., Zipp F., Bertram L., Lill C.M., Fernandez O., Urbaneja P., Leyva L., Alvarez-Cermeno J.C., Arroyo R., Garagorri A.M., Garcia-Martinez A., Villar L.M., Urcelay E., Malhotra S., Montalban X., Comabella M., Berger T., Fazekas F., Reindl M., Schmied M.C., Zimprich A. & Vilariño-Güell C.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here we describe the characterization of a multi-incident MS family which nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients and 886 controls from Canada identified ten additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports co-segregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients and 8797 controls from Canada, France, Spain, Germany, Belgium and Austria failed to identify significant association with disease (p=0.117), despite an overall higher prevalence in patients (OR=1.32; 95% CI=0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

Forwell A.L., Bernales C.Q., Ross J.P., Yee I.M., Encarnacion M., Lee J.D., Sadovnick A.D., Traboulsee A.L. & Vilariño-Güell C.

Oxysterols produced by CH25H during cholesterol metabolism have been shown to play an important role in the immune response. In this study we report the genetic characterization of CH25H in patients diagnosed with multiples sclerosis (MS) or neuromyelitis optica (NMO), for the identification of genetic variants affecting disease susceptibility and course. Exome analysis in 212 MS and 14 NMO patients identified a rare CH25H p.Q17H mutation in two NMO patients of Asian ancestry. In addition, association analysis of common CH25H variants identified a nominally significant association with MS patients presenting a clinical course consistent with primary progressive disease.


Lorenzo-Betancor O., Ogaki K., Soto-Ortolaza A.I., Labbe C., Walton R.L., Strongosky A.J., van Gerpen J.A., Uitti R.J., McLean P.J., Springer W., Siuda J., Opala G., Krygowska-Wajs A., Barcikowska M., Czyzewski K., McCarthy A., Lynch T., Puschmann A., Rektorova I., Sanotsky Y., Vilariño-Güell C., Farrer M.J., Ferman T.J., Boeve B.F., Petersen R.C., Parisi J.E., Graff-Radford N.R., Dickson D.W., Wszolek Z.K. & Ross O.A.

BACKGROUND: Recently, a novel mutation in exon 24 of DNAJC13 gene (p.Asn855Ser, rs387907571) has been reported to cause autosomal dominant Parkinson's disease (PD) in a multi-incident Mennonite family.
METHODS: In the present study the mutation containing exon of the DNAJC13 gene has been sequenced in a Caucasian series consisting of 1938 patients with clinical PD and 838 with pathologically diagnosed Lewy body disease (LBD).
RESULTS: Our sequence analysis did not identify any coding variants in exon 24 of DNAJC13. Two previously described variants in intron 23 (rs200204728 and rs2369796) were observed.
CONCLUSION: Our results indicate that the region surrounding the DNAJC13 p.Asn855Ser substitution is highly conserved and mutations in this exon are not a common cause of PD or LBD among Caucasian populations.

Trinh J., Vilariño-Güell C. & Ross O.A.

It has been almost two decades since the first genetic form of Parkinson’s disease (PD), a point mutation in the α-synuclein gene (SNCA), was identified. Remarkable technological advancements in recent times have accelerated genetic discovery for PD and nominated potential molecular pathways for therapeutic intervention strategies. To date, pathogenic mutations in six genes (SNCA, LRRK2, VPS35, PARKIN, PINK1 and DJ-1) have been confirmed to result in carriers presenting the clinical phenotype associated with PD, with the vast majority of these reporting a family history of the disease. Furthermore, a recent large-scale meta-analysis of population-based genome-wide association studies (GWAS) has identified 24 independent genomic loci (including the familial genes SNCA and LRRK2) that influence susceptibility to the common sporadic form of PD.

Lee J.D., Guimond C., Yee I.M., Vilariño-Güell C., Wu Z.-Y., Traboulsee A.L. & Sadovnick A.D.

BACKGROUND: Global variation in the incidence of multiple sclerosis (MS) is generally ascribed to differences in genetic and environmental risk factors. Here we investigate temporal trends in the incidence of MS and related disorders in British Columbia, Canada, from 1986 to 2010, focusing particularly on the Asian ethnic subpopulation.
METHODS: A longitudinal database was screened to identify newly diagnosed cases of MS and related disorders, including neuromyelitis optica and clinically isolated syndromes. Age-standardized, sex-specific mean annual incidence was calculated for the Asian and non-Asian population of British Columbia for 5-year intervals from 1986 to 2010. Temporal changes and cohort differences in incidence rates and demographic characteristics were evaluated.
RESULTS: During this period, the incidence of MS and related disorders in the non-Asian population remained relatively unchanged, from 10.41 (95% confidence interval [CI]: 9.87-10.97) to 9.91 (95% CI: 9.46-10.39) per 100,000 (p=0.167). In contrast, incidence in the Asian population doubled during the same period. This increase was driven by a precipitous rise in the incidence of MS in females from 0.71 (95% CI: 0.01-1.50) to 2.08 (95% CI: 1.43-2.91) per 100,000 (p=0.004), including both Canadian-born and immigrant Asians. The incidence of neuromyelitis optica did not change significantly during this period.
CONCLUSIONS: The incidence of MS may be increasing among females in the Asian ethnic population of British Columbia.

Steele J.C., Guella I., Szu-Tu C., Lin M.K., Thompson C., Evans D.M., Sherman H.E., Vilariño-Güell C., Gwinn K., Morris H., Dickson D.W. & Farrer M.J.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS)/Parkinsonism-dementia complex (PDC) has been described in Guam, Western Papua and the Kii peninsula of Japan. The etiology and pathogenesis of this complex neurodegenerative disease remains enigmatic.
METHODS: In this study we have used targeted genomic sequencing to evaluate the contribution of genetic variability in the pathogenesis of amyotrophic lateral sclerosis, parkinsonism and dementia in Guamanian Chamorro’s.
RESULTS: Genes previously linked/associated to amyotrophic lateral sclerosis, parkinsonism, dementia and related neurodegenerative syndromes were sequenced in Chamorro subjects living in the Mariana islands. Homozygous PINK1 p.L347P, heterozygous DCTN1 p.T54I, FUS p.P431L and HTT (42 CAG repeats) were identified as pathogenic mutations.
INTERPRETATION: The findings explain the clinical, pathologic and genetic heterogeneity observed in some multi-incident families and contribute to the excess incidence of neurodegeneration previously reported on Guam.

Gustavsson E.K., Trinh J., Guella I., Vilariño-Güell C., Appel-Cresswell S., Stoessl A.J., Tsui J.K., McKeown M., Rajput A., Rajput A.H., Aasly J.O. & Farrer M.J.

BACKGROUND: A novel mutation (p.N855S) in DNAJC13 has been linked to familial, late-onset Lewy body parkinsonism in a Dutch–German–Russian Mennonite multi-incident kindred.
METHODS: DNAJC13 was sequenced in 201 patients with parkinsonism and 194 controls from Canada. Rare (minor allele frequency < 0.01) missense variants identified in patients were genotyped in two Parkinson's disease case–controls cohorts.
RESULTS: Eighteen rare missense mutations were identified; four were observed in controls, three were observed in both patients and controls, and eleven were identified only in patients. Subsequent genotyping showed p.E1740Q and p.L2170W to be more frequent in patients, and p.R1516H being more frequent in controls. Additionally, p.P336A, p.V722L, p.N855S, p.R1266Q were seen in one patient each, and p.T1895M was found in two patients.
CONCLUSION: Although the contribution of rare genetic variation in DNAJC13 to parkinsonisms remains to be further elucidated, this study suggests that, in addition to p.N855S, other rare variants might affect disease susceptibility.

 Rajput A.H., Ross J.P., Bernales C.Q., Rayaprolu S., Soto-Ortolaza A.I., Ross O.A., van Gerpen J., Uitti R.J., Wszolek Z.K., Rajput A. & Vilariño-Güell C.

Exome-sequencing analyses have identified vacuolar protein sorting 35 homolog (VPS35) and DnaJ (Hsp40) homolog, subfamily C, member 13 (DNAJC13) harboring disease-causing variants for Parkinson disease (PD). Owing to the suggested clinical, pathological and genetic overlap between PD and essential tremor (ET) we assessed the presence of two VPS35 and DNAJC13 disease-causing variants in ET patients. TaqMan probes were used to genotype VPS35 p.D620N (rs188286943) and DNAJC13 p.N855S (rs387907571) in 571 ET patients of European descent, and microsatellite markers were used to define the disease haplotype in variant carriers. Genotyping of DNAJC13 identified two ET patients harboring the p.N855S variant previously identified in PD patients. Both patients appear to share the disease haplotype previously reported. ET patients with the VPS35 p.D620N variants were not observed. Although a genetic link between PD and ET has been suggested, DNAJC13 p.N855S represents the first disease-causing variant identified in both, and suggests the regulation of clathrin dynamics and endosomal trafficking in the pathophysiology of a subset of ET patients.


Lorenzo-Betancor, O., Ogaki, K., Soto-Ortolaza, A., Labbe, C., Vilariño-Güell, C., Rajput, A., Rajput, A.H., Pastor, P., Ortega, S., Lorenzo, E., Strongosky, A.J., van Gerpen, J.A., Uitti, R.J., Wszolek, Z.K. & Ross, O.A.

BACKGROUND AND OBJECTIVE: Genes encoding RNA-binding proteins, including FUS and TDP43, play a central role in different neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Recently, a mutation located in the nuclear export signal (NES) of the FUS gene has been reported to cause an autosomal dominant form of familial essential tremor (ET).
MATERIAL AND METHODS: We sequenced the exons coding the NES domains of five RNA-binding proteins (TARDBP, hnRNPA2B1, hnRNPA1, TAF15 and EWSR1) that have been previously implicated in neurodegeneration in a series of 257 ET cases and 376 healthy controls. We genotyped 404 additional ET subjects and 510 healthy controls to assess the frequency of the EWSR1 p.R471C substitution.
RESULTS: We identified a rare EWSR1 p.R471C substitution, which is highly conserved, in a single subject with familial ET. The pathogenicity of this substitution remains equivocal, as DNA samples from relatives were not available and the genotyping of 404 additional ET subjects did not reveal any further carriers. No other variants were observed with significant allele frequency differences compared to controls in the NES coding regions.
CONCLUSIONS: The present study demonstrates that the NES domains of RNA-binding proteins are highly conserved. The role of the EWSR1 p.R471C substitution needs to be further evaluated in future studies.

Trinh, J., Gustavsson, E.K., Guella, I., Vilariño-Güell, C., Evans, D., Encarnacion, M., Sherman, H., Hentati, F. & Farrer, M.J.

Parkinson's disease (PD) is a progressively debilitating neurodegenerative disease for which current treatments help with symptoms but fail to slow or halt progression. SNCA variability contributes to PD susceptibility, and possibly also to age at onset (AAO); but there is limited evidence that it contributes to disease progression or outcomes. The influence of MAPT appears limited to Caucasians in which an ancestral paracentric inversion resulted in non-recombining H1/H2 alleles. Recently, SNCA rs356219 and MAPT rs11079727 were proposed as modulators of disease onset in patients with LRRK2 p.G2019S parkinsonism. Hence, we tested whether SNCA/MAPT genetic variability influences susceptibility and AAO in idiopathic PD and LRRK2 p.G2019S parkinsonism.

Appel-Cresswell, S., Rajput, A.H., Sossi, V., Thompson, C., Silva, V., McKenzie, J., Dinelle, K., McCormick, S.E., Vilariño-Güell, C., Stoessl, A.J., Dickson, D.W., Robinson, C.A., Farrer, M.J. & Rajput, A.

BACKGROUND: Families of Dutch-German-Russian Mennonite descent with multi-incident parkinsonism have been identified as harboring a pathogenic DNAJC13 p.N855S mutation and are awaiting clinical and pathophysiological characterization.
METHODS: Family members were examined clinically longitudinally, and 5 underwent dopaminergic PET imaging. Four family members came to autopsy.
RESULTS: Of the 16 symptomatic DNAJC13 mutation carriers, 12 had clinically definite, 3 probable, and 1 possible Parkinson's disease (PD). Symptoms included bradykinesia, tremor, rigidity, and postural instability, with a mean onset of 63 years (range, 40-85) and slow progression. Eight of ten subjects who required treatment had a good levodopa response; motor complications and nonmotor symptoms were observed. Dopaminergic PET imaging revealed rostrocaudal striatal deficits typical for idiopathic PD in established disease and subtle abnormalities in incipient disease. Pathological examinations revealed Lewy body pathology.
CONCLUSION: PD associated with a DNAJC13 p.N855S mutation presents as late-onset, often slowly progressive, usually dopamine-responsive typical PD.

Traboulsee, A.L., Bernales, C.Q., Ross, J.P., Lee, J.D., Sadovnick, A.D. & Vilariño-Güell, C.

Multiple sclerosis (MS) is a common demyelinating neurodegenerative disease with a strong genetic component. Previous studies have associated genetic variants in IL2RA and IL7R in the pathophysiology of the disease. In this study we describe the association between IL2RA (rs2104286) and IL7R (rs6897932) in the Canadian population. Genotyping 1,978 MS patients and 830 controls failed to identify any significant association between these variants and disease risk. However, stratified analysis for family history of disease, and disease course identified a trend towards association for IL2RA in patients without a family history (p = 0.05; odds ratio = 0.77), and a significant association between IL7R and patients who developed progressive MS (PrMS) (p = 0.002; odds ratio = 0.73). Although not statistically significant, the effect of IL2RA (rs2104286) in patients without a family history of MS indicates that the genetic components for familial and sporadic disease are perhaps distinct. This data suggests the onset of sporadic disease is likely determined by a large number of variants of small effect, whereas MS in patients with a family history of disease is caused by a few deleterious variants. In addition, the significant association between PrMS and rs6897932 indicates that IL7R may not be disease-causing but a determinant of disease course. Further characterization of the effect of IL2RA and IL7R genetic variants in defined MS subtypes is warranted to evaluate the effect of these genes on specific clinical outcomes and to further elucidate the mechanisms of disease onset and progression.

Araki, E., Tsuboi, Y., Daechsel, J., Milnerwood, A., Vilariño-Güell, C., Fujii, N., Mishima, T., Oka, T., Hara, H., Fukae, J. & Farrer, M.J.

BACKGROUND: Depression, parkinsonism, and hypoventilation (Perry syndrome) or familial motor neuron disease have been linked to mutations in dynactin P150Glued (DCTN1).
METHODS: We employed genealogic, clinical, neurologic, and MRI investigations, as well as analysis of genes implicated in parkinsonism. Cellular transfection, immunocytochemistry, and immunoprecipitation analysis of wild-type (WT) and mutant DCTN1 were also performed.
RESULTS: A novel heterozygous mutation, DCTN1 c.156T>G, encoding p.Phe52Leu, segregates with parkinsonism in a Japanese family. The substitution was not observed in affected probands with familial parkinsonism or control subjects and is evolutionarily conserved. In contrast to Perry syndrome, affected carriers have late-onset disease and slower progression, with frontotemporal atrophy revealed by MRI. In vitro studies suggest the mutant protein has impaired microtubule binding, compared to WT dynactin p150Glued .
CONCLUSIONS: DCTN1 mutations may contribute to disparate neurodegenerative diagnoses, including familial motor neuron disease, parkinsonism, and frontotemporal atrophy, and further studies of dynactin-mediated cargo transport may prove insightful.

Bernales, C.Q., Ross, J.P., Lee, J.D., Zhao, Y., Sadovnick, A.D., Traboulsee, A.L. & Vilariño-Güell, C.

A genome-wide association study in a small case-control series of patients with progressive multiple sclerosis (PrMS; primary progressive (PPMS) and secondary progressive (SPMS)) identified an intergenic variant (rs996343) with a putative role in disease course. Nominally significant associations were also identified in dipeptidyl-peptidase 6 (DPP6), which after further genetic characterization nominated rs2046748 as a determinant of MS course. To further define the effect of rs996343 and rs2046748 on disease progression, we genotyped a series of multiple sclerosis (MS) patients and unrelated healthy controls from Canada.

Nishioka, K., Funayama, M., Vilariño-Güell, C., Ogaki, K., Li, Y., Sasaki, R., Kokubo, Y., Kuzuhara, S., Kachergus, J.M., Cobb, S.A., Takahashi, H., Mizuno, Y., Farrer, M.J., Ross, O.A. & Hattori, N.

Pathogenic mutations in the EIF4G1 gene were recently reported as a cause of autosomal dominant parkinsonism. To assess the frequency of EIF4G1 mutations in the Japanese population we sequenced the entire gene coding region (31 exons) in 95 patients with an apparent autosomal dominant inherited form of Parkinson's disease. We detected three novel point mutations located in a poly-glutamic acid repeat within exon 10. These variants were screened through 224 Parkinson's disease cases and 374 normal controls from the Japanese population. We detected the poly-glutamic acid deletion in exon 10 in two additional patients with sporadic Parkinson's disease. Although the EIF4G1 variants identified in the present study were not observed in control subjects, co-segregation analyses and population-based screening data suggest they are not pathogenic. In conclusion, we did not identify novel or previously reported pathogenic mutations (including the p.A502V and p.R1205H mutants) within EIF4G1 in the Japanese population, thus future studies are warranted to elucidate the role of this gene in Parkinson's disease.

Ross, J.P., Rayaprolu, S., Bernales, C.Q., Soto-Ortolaza, A.I., van Gerpen, J., Uitti, R.J., Wszolek, Z.K., Rajput, A., Rajput, A.H., Rajput, M.L., Ross, O.A. & Vilariño-Güell, C.

A recent genome-wide association study of patients with essential tremor (ET) from Germany has nominated SLC1A2 rs3794087 as a novel risk factor for disease. This association was independently replicated in the Chinese population, albeit with an opposite direction of effect. To further define the role of SLC1A2 in ET, we genotyped rs3794087 in a North American series consisting of 1347 patients with ET and controls. Statistical analysis did not identify significant differences in genotype or allele frequencies between healthy controls and patients with ET (p > 0.36). These findings therefore do not support a role for SLC1A2 rs3794087 in susceptibility to ET in the North American population. Further studies in ethnically distinct populations of patients with ET are necessary to understand whether genetic variability in SLC1A2 affects disease risk for ET.

Ross, J.P., Bernales, C.Q., Lee, J.D., Sadovnick, A.D., Traboulsee, A.L. & Vilariño-Güell, C.

The analysis of genetic variability in CYP27B1 and its effect on risk of multiple sclerosis (MS) has yielded conflicting results. Here we describe a study to genetically characterize CYP27B1 and elucidate its role on MS risk in the Canadian population. Sequencing CYP27B1 failed to identify mutations known to cause loss of enzymatic activity, however genotyping of p.R389H in cases and controls identified the mutation in one multi-incident family (allele frequency=0.03%) in which the p.R389H mutation segregates with disease in five family members diagnosed with MS, thus providing additional support for CYP27B1 p.R389H in the pathogenicity of MS.

Rajput, A., Rajput, A.H., Rajput, M.L., Encarnacion, M., Bernales, C.Q., Ross, J.P., Farrer, M.J. & Vilariño-Güell, C.

BACKGROUND AND PURPOSE: Exome sequencing analysis has recently identified a nonsense mutation in fused in sarcoma (FUS) segregating with essential tremor (ET) within a large French-Canadian family. Further characterization of FUS resulted in the identification of additional mutations in ET patients; however, their pathogenicity still remains to be confirmed. The role of FUS in an independent cohort of ET patients from Canada was evaluated.
METHODS: The entire coding sequence of FUS in 217 patients diagnosed with ET was analyzed and two missense variants in 219 healthy controls were genotyped by Sanger sequencing.
RESULTS: Sequencing of FUS identified a previously reported non-pathogenic mutation p.G174_G175del in one ET patient and two healthy controls, and a novel p.R377W in one patient with family history of disease. This mutation is highly conserved and strongly predicted to be damaging by in silico analysis.
CONCLUSION: This study has identified a novel FUS p.R377W substitution in ET patients. Additional genotyping studies in a large number of ET patients and controls are necessary to conclusively define its pathogenicity.

Vilariño-Güell, C., Rajput, A., Milnerwood, A.J., Shah, B., Szu-Tu, C., Trinh, J., Yu, I., Encarnacion, M., Munsie, L.N., Tapia, L., Gustavsson, E.K., Chou, P., Tatarnikov, I., Evans, D.M., Pishotta, F.T., Volta, M., Beccano-Kelly, D., Thompson, C., Lin, M.K., Sherman, H.E., Han, H.J., Guenther, B.L., Wasserman, W.W., Bernard, V., Ross, C.J., Appel-Cresswell, S., Stoessl, A.J., Robinson, C.A., Dickson, D.W., Ross, O.A., Wszolek, Z.K., Aasly, J.O., Wu, R.M., Hentati, F., Gibson, R.A., McPherson, P.S., Girard, M., Rajput, M., Rajput, A.H. & Farrer, M.J.

A Saskatchewan multi-incident family was clinically characterized with Parkinson disease (PD) and Lewy body pathology. PD segregates as an autosomal-dominant trait, which could not be ascribed to any known mutation. DNA from three affected members was subjected to exome sequencing. Genome alignment, variant annotation and comparative analyses were used to identify shared coding mutations. Sanger sequencing was performed within the extended family and ethnically matched controls. Subsequent genotyping was performed in a multi-ethnic case-control series consisting of 2928 patients and 2676 control subjects from Canada, Norway, Taiwan, Tunisia, and the USA. A novel mutation in receptor-mediated endocytosis 8/RME-8 (DNAJC13 p.Asn855Ser) was found to segregate with disease. Screening of cases and controls identified four additional patients with the mutation, of which two had familial parkinsonism. All carriers shared an ancestral DNAJC13 p.Asn855Ser haplotype and claimed Dutch-German-Russian Mennonite heritage. DNAJC13 regulates the dynamics of clathrin coats on early endosomes. Cellular analysis shows that the mutation confers a toxic gain-of-function and impairs endosomal transport. DNAJC13 immunoreactivity was also noted within Lewy body inclusions. In late-onset disease which is most reminiscent of idiopathic PD subtle deficits in endosomal receptor-sorting/recycling are highlighted by the discovery of pathogenic mutations VPS35, LRRK2 and now DNAJC13. With this latest discovery, and from a neuronal perspective, a temporal and functional ecology is emerging that connects synaptic exo- and endocytosis, vesicular trafficking, endosomal recycling and the endo-lysosomal degradative pathway. Molecular deficits in these processes are genetically linked to the phenotypic spectrum of parkinsonism associated with Lewy body pathology.

Trinh, J., Amouri, R., Duda, J.E., Morley, J.F., Read, M., Donald, A., Vilariño-Güell, C., Thompson, C., Szu Tu, C., Gustavsson, E.K., Ben Sassi, S., Hentati, E., Zouari, M., Farhat, E., Nabli, F., Hentati, F. & Farrer, M.J.

Parkinson disease is a progressive neurodegenerative disease for which leucine-rich repeat kinase 2 (LRRK2 carriers) p.G2019S confers substantial genotypic and population attributable risk. With informed consent, we have recruited clinical data from 778 patients from Tunisia (of which 266 have LRRK2 parkinsonism) and 580 unaffected subjects. Motor, autonomic, and cognitive assessments in idiopathic Parkinson disease and LRRK2 patients were compared with regression models. The age-associated cumulative incidence of LRRK2 parkinsonism was also estimated using case-control and family-based designs. LRRK2 parkinsonism patients had slightly less gastrointestinal dysfunction and rapid eye movement sleep disorder. Overall, disease penetrance in LRRK2 carriers was 80% by 70 years but women become affected a median 5 years younger than men. Idiopathic Parkinson disease patients with younger age at diagnosis have slower disease progression. However, age at diagnoses does not predict progression in LRRK2 parkinsonism. LRRK2 p.G2019S mutation is a useful aid to diagnosis and modifiers of disease in LRRK2 parkinsonism may aid in developing therapeutic targets.

Heckman, M.G., Elbaz, A., Soto-Ortolaza, A.I., Serie, D.J., Aasly, J.O., Annesi, G., Auburger, G., Bacon, J.A., Boczarska-Jedynak, M., Bozi, M., Brighina, L., Chartier-Harlin, M.C., Dardiotis, E., Destee, A., Ferrarese, C., Ferraris, A., Fiske, B., Gispert, S., Hadjigeorgiou, G.M., Hattori, N., Ioannidis, J.P., Jasinska-Myga, B., Jeon, B.S., Kim, Y.J., Klein, C., Kruger, R., Kyratzi, E., Lin, C.H., Lohmann, K., Loriot, M.A., Lynch, T., Mellick, G.D., Mutez, E., Opala, G., Park, S.S., Petrucci, S., Quattrone, A., Sharma, M., Silburn, P.A., Sohn, Y.H., Stefanis, L., Tadic, V., Tomiyama, H., Uitti, R.J., Valente, E.M., Vassilatis, D.K., Vilariño-Güell, C., White, L.R., Wirdefeldt, K., Wszolek, Z.K., Wu, R.M., Xiromerisiou, G., Maraganore, D.M., Farrer, M.J. & Ross, O.A.

The best validated susceptibility variants for Parkinson's disease are located in the alpha-synuclein (SNCA) and microtubule-associated protein tau (MAPT) genes. Recently, a protective p.N551K-R1398H-K1423K haplotype in the leucine-rich repeat kinase 2 (LRRK2) gene was identified, with p.R1398H appearing to be the most likely functional variant. To date, the consistency of the protective effect of LRRK2 p.R1398H across MAPT and SNCA variant genotypes has not been assessed. To address this, we examined 4 SNCA variants (rs181489, rs356219, rs11931074, and rs2583988), the MAPT H1-haplotype-defining variant rs1052553, and LRRK2 p.R1398H (rs7133914) in Caucasian (n = 10,322) and Asian (n = 2289) series. There was no evidence of an interaction of LRRK2 p.R1398H with MAPT or SNCA variants (all p >/= 0.10); the protective effect of p.R1398H was observed at similar magnitude across MAPT and SNCA genotypes, and the risk effects of MAPT and SNCA variants were observed consistently for LRRK2 p.R1398H genotypes. Our results indicate that the association of LRRK2 p.R1398H with Parkinson's disease is independent of SNCA and MAPT variants, and vice versa, in Caucasian and Asian populations.


Sadovnick, A.D., Traboulsee, A.L., Lee, J.D., Ross, J.P., Bernales, C.Q. & Vilariño-Güell, C.

Multiple sclerosis (MS) is an inflammatory disease of the CNS characterized by myelin loss, varying degrees of axonal injury, and progressive neurological dysfunction. Due to the clinical and pathological overlap between MS and hereditary diffuse leukoencephalopathy with spheroids (HDLS), the clinical diagnosis of MS in patients with CSF1R mutations, and the biological role of CSF1R in the inflammatory response, the question of whether mutations in CSF1R are implicated in the pathological mechanisms necessary to trigger the onset of MS was evaluated.

Trinh, J., Vilariño-Güell, C., Donald, A., Shah, B., Yu, I., Szu-Tu, C., Aasly, J.O., Wu, R.M., Hentati, F., Rajput, A.H., Rajput, A. & Farrer, M.J.

A variant in Syntaxin 6 (a soluble N-ethylmaleimide-sensitive factor attachment protein receptor STX6) (rs1411478) has been shown to be associated with progressive supranuclear palsy (PSP). Although Parkinson's disease (PD) and PSP are distinct neurodegenerative diseases, they share some clinical and genetic features. In this study, we evaluated STX6 genetic variability in PD susceptibility in ethnically matched case-control series from Canada, Norway, Taiwan and Tunisia and we evaluated the presence of pathogenic mutations within families. No pathogenic mutations were found in STX6. Similarly, statistical analysis of rs1411478 failed to identify differences in genotype or allelic frequencies between cases and controls. Our results do not support a role for STX6 in PD.

Heckman, M.G., Soto-Ortolaza, A.I., Aasly, J.O., Abahuni, N., Annesi, G., Bacon, J.A., Bardien, S., Bozi, M., Brice, A., Brighina, L., Carr, J., Chartier-Harlin, M.C., Dardiotis, E., Dickson, D.W., Diehl, N.N., Elbaz, A., Ferrarese, C., Fiske, B., Gibson, J.M., Gibson, R., Hadjigeorgiou, G.M., Hattori, N., Ioannidis, J.P., Boczarska-Jedynak, M., Jasinska-Myga, B., Jeon, B.S., Kim, Y.J., Klein, C., Kruger, R., Kyratzi, E., Lesage, S., Lin, C.H., Lynch, T., Maraganore, D.M., Mellick, G.D., Mutez, E., Nilsson, C., Opala, G., Park, S.S., Petrucci, S., Puschmann, A., Quattrone, A., Sharma, M., Silburn, P.A., Sohn, Y.H., Stefanis, L., Tadic, V., Theuns, J., Tomiyama, H., Uitti, R.J., Valente, E.M., Van Broeckhoven, C., van de Loo, S., Vassilatis, D.K., Vilariño-Güell, C., White, L.R., Wirdefeldt, K., Wszolek, Z.K., Wu, R.M., Hentati, F., Farrer, M.J. & Ross, O.A.

BACKGROUND: Variants within the leucine-rich repeat kinase 2 gene are recognized as the most frequent genetic cause of Parkinson's disease. Leucine-rich repeat kinase 2 variation related to disease susceptibility displays many features that reflect the nature of complex, late-onset sporadic disorders like Parkinson's disease. METHODS: The Genetic Epidemiology of Parkinson's Disease Consortium recently performed the largest genetic association study for variants in the leucine-rich repeat kinase 2 gene across 23 different sites in 15 countries. RESULTS: Herein, we detail the allele frequencies for the novel risk factors (p.A419V and p.M1646T) and the protective haplotype (p.N551K-R1398H-K1423K) nominated in the original publication. Simple population allele frequencies not only can provide insight into the clinical relevance of specific variants but also can help genetically define patient groups. CONCLUSIONS: Establishing individual patient-based genomic susceptibility profiles that incorporate both risk factors and protective factors will determine future diagnostic and treatment strategies.

Fujioka, S., Sundal, C., Strongosky, A.J., Castanedes, M.C., Rademakers, R., Ross, O.A., Vilariño-Güell, C., Farrer, M.J., Wszolek, Z.K. & Dickson, D.W.

We recently reported a missense mutation and four variants in eukaryotic translation initiation factor 4-gamma (EIF4G1) associated with parkinsonism, dementia or both. In those with a positive family history, the mode of inheritance was autosomal dominant. Detailed neuropathologic descriptions of individuals with EIF4G1 genetic variants have not been reported. Herein, we report neuropathologic findings of three individuals from two American families with EIF4G1 variants. The patients had initial clinical presentations of dementia or parkinsonism and all had dementia at the time of autopsy. One family carried an EIF4G1 double variant, c.2056G>T (p.G686C) and c.3589C>T (p.R1197 W), and one family carried variant c.1505C>T (p.A502V). All three patients also carried at least one epsilon4 allele of apolipoprotein E. One individual presented with cognitive impairment without significant parkinsonism; one presented with memory problems followed by bradykinesia; and the third presented with cardinal signs of Parkinson's disease, followed more than a year later by cognitive dysfunction. Pathological examination showed diffuse cortical Lewy bodies and Lewy neurites in all patients. A small subset of Lewy bodies and Lewy neurites were immunopositive for eIF4G1. All patients had moderate to frequent non-neuritic, cortical amyloid plaques, mostly medial temporal neurofibrillary pathology (Braak neurofibrillary tangle stages of II to IV), and minimal or no TDP-43 pathology. The results suggest that in some patients variants in EIF4G1 can be associated with pathology that has a high likelihood of association with clinical features of dementia with Lewy bodies.

Appel-Cresswell, S., Vilariño-Güell, C., Encarnacion, M., Sherman, H., Yu, I., Shah, B., Weir, D., Thompson, C., Szu-Tu, C., Trinh, J., Aasly, J.O., Rajput, A., Rajput, A.H., Jon Stoessl, A. & Farrer, M.J.

BACKGROUND: Alpha-synuclein plays a central role in the pathophysiology of Parkinson's disease. Three missense mutations in SNCA, the gene encoding alpha-synuclein, as well as genomic multiplications have been identified as causes for autosomal-dominantly inherited Parkinsonism. METHODS: Here, we describe a novel missense mutation in exon 4 of SNCA encoding a H50Q substitution in a patient with dopa-responsive Parkinson's disease with a family history of parkinsonism and dementia. RESULTS: The variant was not observed in public databases or identified in unrelated subjects. CONCLUSIONS: The substitution's evolutionary conservation and protein modeling provide additional support for pathogenicity as the amino acid perturbs the same amphipathic alpha helical structure as the previously described pathogenic mutations.


Wider, C., Ross, O.A., Nishioka, K., Heckman, M.G., Vilariño-Güell, C., Jasinska-Myga, B., Erketin-Taner, N., Rademakers, R., Graff-Radford, N.R., Mash, D.C., Papapetropoulos, S., Duara, R., Uchikado, H., Wszolek, Z.K., Farrer, M.J. & Dickson, D.W.

PURPOSE: The study investigates the effects of genetic factors on the pathology of Alzheimer's disease (AD) and Lewy body (LB) diseases, including Parkinson's disease and dementia with Lewy bodies. METHODS: A multicentre autopsy series (762 brain samples) with AD, LB or vascular pathology was examined. The effects of the tau gene (MAPT) H1 haplotype, the H1 specific SNP rs242557, APOE and the alpha-synuclein gene (SNCA) 3'UTR SNP rs356165 on the burden of AD and LB pathology were assessed. Neurofibrillary tangles (NFTs) were counted in four brain regions, senile plaques in five and LBs in four. Braak NFT stage, brain weight and presence of vascular pathology were also documented. RESULTS: MAPT H1 associated with lower counts of NFTs in the middle frontal (p<0.001) and inferior parietal (p=0.005) cortices, and also with lower counts of senile plaques in the motor cortex (p=0.001). Associations of MAPT H1 with increased LB counts in the middle frontal cortex (p=0.011) and inferior parietal cortex (p=0.033) were observed but were not significant after multiple testing adjustment. The APOE epsilon4 allele was strongly associated with overall Alzheimer type pathology (all p</=0.001). SNCA rs356165 and the MAPT H1 specific SNP rs242557 did not associate with AD or LB pathology. CONCLUSION: This study shows for the first time that MAPT H1 is associated with reduced Alzheimer type pathology which could have important implications for the understanding of disease mechanisms and their genetic determinants.

Sharma, M., Ioannidis, J.P., Aasly, J.O., Annesi, G., Brice, A., Bertram, L., Bozi, M., Barcikowska, M., Crosiers, D., Clarke, C.E., Facheris, M.F., Farrer, M., Garraux, G., Gispert, S., Auburger, G., Vilariño-Güell, C., Hadjigeorgiou, G.M., Hicks, A.A., Hattori, N., Jeon, B.S., Jamrozik, Z., Krygowska-Wajs, A., Lesage, S., Lill, C.M., Lin, J.J., Lynch, T., Lichtner, P., Lang, A.E., Libioulle, C., Murata, M., Mok, V., Jasinska-Myga, B., Mellick, G.D., Morrison, K.E., Meitnger, T., Zimprich, A., Opala, G., Pramstaller, P.P., Pichler, I., Park, S.S., Quattrone, A., Rogaeva, E., Ross, O.A., Stefanis, L., Stockton, J.D., Satake, W., Silburn, P.A., Strom, T.M., Theuns, J., Tan, E.K., Toda, T., Tomiyama, H., Uitti, R.J., Van Broeckhoven, C., Wirdefeldt, K., Wszolek, Z., Xiromerisiou, G., Yomono, H.S., Yueh, K.C., Zhao, Y., Gasser, T., Maraganore, D. & Kruger, R.

BACKGROUND: Two recent studies identified a mutation (p.Asp620Asn) in the vacuolar protein sorting 35 gene as a cause for an autosomal dominant form of Parkinson disease . Although additional missense variants were described, their pathogenic role yet remains inconclusive. METHODS AND RESULTS: We performed the largest multi-center study to ascertain the frequency and pathogenicity of the reported vacuolar protein sorting 35 gene variants in more than 15,000 individuals worldwide. p.Asp620Asn was detected in 5 familial and 2 sporadic PD cases and not in healthy controls, p.Leu774Met in 6 cases and 1 control, p.Gly51Ser in 3 cases and 2 controls. Overall analyses did not reveal any significant increased risk for p.Leu774Met and p.Gly51Ser in our cohort. CONCLUSIONS: Our study apart from identifying the p.Asp620Asn variant in familial cases also identified it in idiopathic Parkinson disease cases, and thus provides genetic evidence for a role of p.Asp620Asn in Parkinson disease in different populations worldwide.

Puschmann, A., Englund, E., Ross, O.A., Vilariño-Güell, C., Lincoln, S.J., Kachergus, J.M., Cobb, S.A., Tornqvist, A.L., Rehncrona, S., Widner, H., Wszolek, Z.K., Farrer, M.J. & Nilsson, C.

The c.4309A>C mutation in the LRRK2 gene (LRRK2 p.N1437H) has recently been reported as the seventh pathogenic LRRK2 mutation causing monogenic Parkinson's disease (PD). So far, only two families worldwide have been identified with this mutation. By screening DNA from seven brains of PD patients, we found one individual with seemingly sporadic PD and LRRK2 p.N1437H mutation. Clinically, the patient had levodopa-responsive PD with tremor, and developed severe motor fluctuations during a disease duration of 19 years. There was severe and painful ON-dystonia, and severe depression with suicidal thoughts during OFF. In the advanced stage, cognition was slow during motor OFF, but there was no noticeable cognitive decline. There were no signs of autonomic nervous system dysfunction. Bilateral deep brain stimulation of the subthalamic nucleus had unsatisfactory results on motor symptoms. The patient committed suicide. Neuropathological examination revealed marked cell loss and moderate alpha-synuclein positive Lewy body pathology in the brainstem. There was sparse Lewy pathology in the cortex. A striking finding was very pronounced ubiquitin-positive pathology in the brainstem, temporolimbic regions and neocortex. Ubiquitin positivity was most pronounced in the white matter, and was out of proportion to the comparatively weaker alpha-synuclein immunoreactivity. Immunostaining for tau was mildly positive, revealing non-specific changes, but staining for TDP-43 and FUS was entirely negative. The distribution and shape of ubiquitin-positive lesions in this patient differed from the few previously described patients with LRRK2 mutations and ubiquitin pathology, and the ubiquitinated protein substrate remains undefined.

Ross, O.A., Conneely, K.N., Wang, T., Vilariño-Güell, C., Soto-Ortolaza, A.I., Rajput, A., Wszolek, Z.K., Uitti, R.J., Louis, E.D., Clark, L.N., Farrer, M.J. & Testa, C.M.

BACKGROUND: Given the overlap between Parkinson's disease and essential tremor, we examined genetic variants in alpha-synuclein (SNCA) as risk determinants for essential tremor.METHODS: Samples from 661 essential tremor subjects and 1316 control subjects from 4 participating North American sites were included in this study. Parkinson's disease samples (n = 427) were compared against controls. Twenty variants were selected for association analysis within the SNCA locus. Individual logistic regression analyses against essential tremor diagnosis were run for each variant and then combined using meta-analysis. RESULTS: Our results do not show a significant association between variants in the SNCA locus and risk of essential tremor, whereas the established association of SNCA variants with Parkinson's disease risk was observed. CONCLUSIONS: Whereas genetic factors are likely to play a large role in essential tremor pathogenesis, our results do not support a role for common SNCA genetic variants in risk for essential tremor.

Sharma, M., Ioannidis, J.P., Aasly, J.O., Annesi, G., Brice, A., Van Broeckhoven, C., Bertram, L., Bozi, M., Crosiers, D., Clarke, C., Facheris, M., Farrer, M., Garraux, G., Gispert, S., Auburger, G., Vilariño-Güell, C., Hadjigeorgiou, G.M., Hicks, A.A., Hattori, N., Jeon, B., Lesage, S., Lill, C.M., Lin, J.J., Lynch, T., Lichtner, P., Lang, A.E., Mok, V., Jasinska-Myga, B., Mellick, G.D., Morrison, K.E., Opala, G., Pramstaller, P.P., Pichler, I., Park, S.S., Quattrone, A., Rogaeva, E., Ross, O.A., Stefanis, L., Stockton, J.D., Satake, W., Silburn, P.A., Theuns, J., Tan, E.K., Toda, T., Tomiyama, H., Uitti, R.J., Wirdefeldt, K., Wszolek, Z., Xiromerisiou, G., Yueh, K.C., Zhao, Y., Gasser, T., Maraganore, D. & Kruger, R.

OBJECTIVE:Eleven genetic loci have reached genome-wide significance in a recent meta-analysis of genome-wide association studies in Parkinson disease (PD) based on populations of Caucasian descent. The extent to which these genetic effects are consistent across different populations is unknown. METHODS:Investigators from the Genetic Epidemiology of Parkinson's Disease Consortium were invited to participate in the study. A total of 11 SNPs were genotyped in 8,750 cases and 8,955 controls. Fixed as well as random effects models were used to provide the summary risk estimates for these variants. We evaluated between-study heterogeneity and heterogeneity between populations of different ancestry. RESULTS:In the overall analysis, single nucleotide polymorphisms (SNPs) in 9 loci showed significant associations with protective per-allele odds ratios of 0.78-0.87 (LAMP3, BST1, and MAPT) and susceptibility per-allele odds ratios of 1.14-1.43 (STK39, GAK, SNCA, LRRK2, SYT11, and HIP1R). For 5 of the 9 replicated SNPs there was nominally significant between-site heterogeneity in the effect sizes (I(2) estimates ranged from 39% to 48%). Subgroup analysis by ethnicity showed significantly stronger effects for the BST1 (rs11724635) in Asian vs Caucasian populations and similar effects for SNCA, LRRK2, LAMP3, HIP1R, and STK39 in Asian and Caucasian populations, while MAPT rs2942168 and SYT11 rs34372695 were monomorphic in the Asian population, highlighting the role of population-specific heterogeneity in PD. CONCLUSION:Our study allows insight to understand the distribution of newly identified genetic factors contributing to PD and shows that large-scale evaluation in diverse populations is important to understand the role of population-specific heterogeneity.


Vilariño-Güell, C., Wider, C., Ross, O.A., Dachsel, J.C., Kachergus, J.M., Lincoln, S.J., Soto-Ortolaza, A.I., Cobb, S.A., Wilhoite, G.J., Bacon, J.A., Behrouz, B., Melrose, H.L., Hentati, E., Puschmann, A., Evans, D.M., Conibear, E., Wasserman, W.W., Aasly, J.O., Burkhard, P.R., Djaldetti, R., Ghika, J., Hentati, F., Krygowska-Wajs, A., Lynch, T., Melamed, E., Rajput, A., Rajput, A.H., Solida, A., Wu, R.M., Uitti, R.J., Wszolek, Z.K., Vingerhoets, F. & Farrer, M.J.

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 +/- 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.

Vilariño-Güell, C., Soto-Ortolaza, A.I., Rajput, A., Mash, D.C., Papapetropoulos, S., Pahwa, R., Lyons, K.E., Uitti, R.J., Wszolek, Z.K., Dickson, D.W., Farrer, M.J. & Ross, O.A.

Mutations in the microtubule-associated protein tau gene (MAPT) cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In addition, the major common MAPT-containing H1 haplotype is associated with increased risk for 2 parkinsonian disorders: progressive supranuclear palsy (PSP) characterized by 4-repeat tau pathology and Parkinson disease (PD) with α-synuclein pathology. However, the role of MAPT variation in other disorders with similar pathology or disease phenotype is unclear. We investigated the frequency of the MAPT H1 haplotype in both essential tremor (ET) and multiple system atrophy (MSA).

Winkelmann, J., Czamara, D., Schormair, B., Knauf, F., Schulte, E.C., Trenkwalder, C., Dauvilliers, Y., Polo, O., Hogl, B., Berger, K., Fuhs, A., Gross, N., Stiasny-Kolster, K., Oertel, W., Bachmann, C.G., Paulus, W., Xiong, L., Montplaisir, J., Rouleau, G.A., Fietze, I., Vavrova, J., Kemlink, D., Sonka, K., Nevsimalova, S., Lin, S.C., Wszolek, Z., Vilariño-Güell, C., Farrer, M.J., Gschliesser, V., Frauscher, B., Falkenstetter, T., Poewe, W., Allen, R.P., Earley, C.J., Ondo, W.G., Le, W.D., Spieler, D., Kaffe, M., Zimprich, A., Kettunen, J., Perola, M., Silander, K., Cournu-Rebeix, I., Francavilla, M., Fontenille, C., Fontaine, B., Vodicka, P., Prokisch, H., Lichtner, P., Peppard, P., Faraco, J., Mignot, E., Gieger, C., Illig, T., Wichmann, H.E., Muller-Myhsok, B. & Meitinger, T.

Restless legs syndrome (RLS) is a sensorimotor disorder with an age-dependent prevalence of up to 10% in the general population above 65 years of age. Affected individuals suffer from uncomfortable sensations and an urge to move in the lower limbs that occurs mainly in resting situations during the evening or at night. Moving the legs or walking leads to an improvement of symptoms. Concomitantly, patients report sleep disturbances with consequences such as reduced daytime functioning. We conducted a genome-wide association study (GWA) for RLS in 922 cases and 1,526 controls (using 301,406 SNPs) followed by a replication of 76 candidate SNPs in 3,935 cases and 5,754 controls, all of European ancestry. Herein, we identified six RLS susceptibility loci of genome-wide significance, two of them novel: an intergenic region on chromosome 2p14 (rs6747972, P = 9.03 x 10(-11), OR = 1.23) and a locus on 16q12.1 (rs3104767, P = 9.4 x 10(-19), OR = 1.35) in a linkage disequilibrium block of 140 kb containing the 5'-end of TOX3 and the adjacent non-coding RNA BC034767.

Wider, C., Vilariño-Güell, C., Heckman, M.G., Jasinska-Myga, B., Ortolaza-Soto, A.I., Diehl, N.N., Crook, J.E., Cobb, S.A., Bacon, J.A., Aasly, J.O., Gibson, J.M., Lynch, T., Uitti, R.J., Wszolek, Z.K., Farrer, M.J. & Ross, O.A.

Background and purpose: Recent evidence suggests that variation in the SNCA, MAPT, and GSK3B genes interacts in affecting risk for Parkinson disease (PD). In the current study, we attempt to validate previously published findings, evaluating gene-gene interactions between SNCA, MAPT, and GSK3B in association with PD. Methods: Three Caucasian PD patient-control series from the United States, Ireland, and Norway (combined n = 1020 patients and 1095 controls) were genotyped for SNCA rs356219, MAPT H1/H2-discriminating SNP rs1052553, and GSK3B rs334558 and rs6438552. Results: Our findings indicate that as previously reported, the SNCA rs356219-G allele and MAPT rs1052553 (H1 haplotype) were both associated with an increased risk of PD, whilst contrary to previous reports, GSK3B variants were not. No pair-wise interaction was observed between SNCA, MAPT, and GSK3B; the risk effects of SNCA rs356219-G and MAPT rs1052553-H1 were seen in a similar manner across genotypes of other variants, with no evidence suggesting synergistic, antagonistic, or deferential effects. Conclusions: In the Caucasian patient-control series examined, risk for PD was influenced by variation in SNCA and MAPT but not GSK3B. Additionally, those three genes did not interact in determining disease risk.

Sharma, S., Bandopadhyay, R., Lashley, T., Renton, A.E., Kingsbury, A.E., Kumaran, R., Kallis, C., Vilariño-Güell, C., O'Sullivan, S.S., Lees, A.J., Revesz, T., Wood, N.W. & Holton, J.L.

AIMS: Mutations in the gene encoding leucine-rich repeat kinase-2 (LRRK2) have been established as a common genetic cause of Parkinson's disease (PD). The distribution of LRRK2 mRNA and protein in the human brain has previously been described, although it has not been reported in PD cases with the common LRRK2 G2019S mutation. METHODS: To further elucidate the role of LRRK2 in PD, we determined the localization of LRRK2 mRNA and protein in post-mortem brain tissue from control, idiopathic PD (IPD) and G2019S positive PD cases. RESULTS: Widespread neuronal expression of LRRK2 mRNA and protein was recorded and no difference was observed in the morphological localization of LRRK2 mRNA or protein between control, IPD and G2019S positive PD cases. Using quantitative real-time polymerase chain reaction, we demonstrated that there is no regional variation in LRRK2 mRNA in normal human brain, but we have identified differential expression of LRRK2 mRNA with significant reductions recorded in limbic and neocortical regions of IPD cases compared with controls. Semi-quantitative analysis of LRRK2 immunohistochemical staining demonstrated regional variation in staining intensity, with weak LRRK2 immunoreactivity consistently recorded in the striatum and substantia nigra. No clear differences were identified in LRRK2 immunoreactivity between control, IPD and G2019S positive PD cases. LRRK2 protein was identified in a small proportion of Lewy bodies. CONCLUSIONS: Our data suggest that widespread dysregulation of LRRK2 mRNA expression may contribute to the pathogenesis of IPD.

Ross, O.A., Soto-Ortolaza, A.I., Heckman, M.G., Aasly, J.O., Abahuni, N., Annesi, G., Bacon, J.A., Bardien, S., Bozi, M., Brice, A., Brighina, L., Van Broeckhoven, C., Carr, J., Chartier-Harlin, M.C., Dardiotis, E., Dickson, D.W., Diehl, N.N., Elbaz, A., Ferrarese, C., Ferraris, A., Fiske, B., Gibson, J.M., Gibson, R., Hadjigeorgiou, G.M., Hattori, N., Ioannidis, J.P., Jasinska-Myga, B., Jeon, B.S., Kim, Y.J., Klein, C., Kruger, R., Kyratzi, E., Lesage, S., Lin, C.H., Lynch, T., Maraganore, D.M., Mellick, G.D., Mutez, E., Nilsson, C., Opala, G., Park, S.S., Puschmann, A., Quattrone, A., Sharma, M., Silburn, P.A., Sohn, Y.H., Stefanis, L., Tadic, V., Theuns, J., Tomiyama, H., Uitti, R.J., Valente, E.M., van de Loo, S., Vassilatis, D.K., Vilariño-Güell, C., White, L.R., Wirdefeldt, K., Wszolek, Z.K., Wu, R.M. & Farrer, M.J.

BACKGROUND: Background The leucine-rich repeat kinase 2 gene (LRRK2) harbours highly penetrant mutations that are linked to familial parkinsonism. However, the extent of its polymorphic variability in relation to risk of Parkinson's disease (PD) has not been assessed systematically. We therefore assessed the frequency of LRRK2 exonic variants in individuals with and without PD, to investigate the role of the variants in PD susceptibility. METHODS: LRRK2 was genotyped in patients with PD and controls from three series (white, Asian, and Arab-Berber) from sites participating in the Genetic Epidemiology of Parkinson's Disease Consortium. Genotyping was done for exonic variants of LRRK2 that were identified through searches of literature and the personal communications of consortium members. Associations with PD were assessed by use of logistic regression models. For variants that had a minor allele frequency of 0.5% or greater, single variant associations were assessed, whereas for rarer variants information was collapsed across variants. FINDINGS: 121 exonic LRRK2 variants were assessed in 15 540 individuals: 6995 white patients with PD and 5595 controls, 1376 Asian patients and 962 controls, and 240 Arab-Berber patients and 372 controls. After exclusion of carriers of known pathogenic mutations, new independent risk associations were identified for polymorphic variants in white individuals (M1646T, odds ratio 1.43, 95% CI 1.15-1.78; p=0.0012) and Asian individuals (A419V, 2.27, 1.35-3.83; p=0.0011). A protective haplotype (N551K-R1398H-K1423K) was noted at a frequency greater than 5% in the white and Asian series, with a similar finding in the Arab-Berber series (combined odds ratio 0.82, 0.72-0.94; p=0.0043). Of the two previously reported Asian risk variants, G2385R was associated with disease (1.73, 1.20-2.49; p=0.0026), but no association was noted for R1628P (0.62, 0.36-1.07; p=0.087). In the Arab-Berber series, Y2189C showed potential evidence of risk association with PD (4.48, 1.33-15.09; p=0.012). INTERPRETATION: The results for LRRK2 show that several rare and common genetic variants in the same gene can have independent effects on disease risk. LRRK2, and the pathway in which it functions, is important in the cause and pathogenesis of PD in a greater proportion of patients with this disease than previously believed. These results will help discriminate those patients who will benefit most from therapies targeted at LRRK2 pathogenic activity.

Puschmann, A., Pfeiffer, R.F., Stoessl, A.J., Kuriakose, R., Lash, J.L., Searcy, J.A., Strongosky, A.J., Vilariño-Güell, C., Farrer, M.J., Ross, O.A., Dickson, D.W. & Wszolek, Z.K.

BACKGROUND: Previous epidemiologic and genetic studies have suggested a link between Parkinson disease (PD), essential tremor (ET), and restless legs syndrome (RLS). METHODS: We describe the clinical, PET, and pathologic characteristics of an extensive kindred from Arkansas with hereditary PD, ET, and RLS. The pedigree contains 138 individuals. Sixty-five family members were examined neurologically up to 3 times from 2004 to 2010. Clinical data were collected from medical records and questionnaires. Genetic studies were performed. Five family members underwent multitracer PET. Two individuals with PD were examined postmortem. RESULTS: Eleven family members had PD with generally mild and slowly progressive symptoms. Age at onset was between 39 and 74 years (mean 59.1, SD 13.4). All individuals treated with l-dopa responded positively. Postural or action tremor was present in 6 individuals with PD, and in 19 additional family members. Fifteen persons reported symptoms of RLS. PET showed reduced presynaptic dopamine function typical of sporadic PD in a patient with PD and ET, but not in persons with ET or RLS. The inheritance pattern was autosomal dominant for PD and RLS. No known pathogenic mutation in PD-related genes was found. Fourteen of the family members with PD, ET, or RLS had depression. Neuropathologic examination revealed pallidonigral pigment spheroid degeneration with ubiquitin-positive axonal spheroids, TDP43-positive pathology in the basal ganglia, hippocampus, and brainstem, and only sparse Lewy bodies. CONCLUSION: Familial forms of PD, ET, RLS, and depression occur in this family. The genetic cause remains to be elucidated.

Nishioka, K., Ross, O.A., Vilariño-Güell, C., Cobb, S.A., Kachergus, J.M., Mann, D.M., Snowden, J., Richardson, A.M., Neary, D., Robinson, C.A., Rajput, A., Papapetropoulos, S., Mash, D.C., Pahwa, R., Lyons, K.E., Wszolek, Z.K., Dickson, D.W. & Farrer, M.J.

Clinicogenetic and pathological studies have shown that mutations of the glucocerebrosidase gene (GBA) are a risk factor for Parkinson's disease and Lewy body disorders. In the present study, we have identified GBA mutations in 6.8% (4/59) of cases with a pathological diagnosis of diffuse Lewy body disease. Taken with previous studies, it appears that GBA mutations are associated with a more diffuse pattern of Lewy body distribution involving the cerebral cortex than the brainstem/limbic distribution observed in typical Parkinson's disease.

Mata, I.F., Wilhoite, G.J., Yearout, D., Bacon, J.A., Cornejo-Olivas, M., Mazzetti, P., Marca, V., Ortega, O., Acosta, O., Cosentino, C., Torres, L., Medina, A.C., Perez-Pastene, C., Diaz-Grez, F., Vilariño-Güell, C., Venegas, P., Miranda, M., Trujillo-Godoy, O., Layson, L., Avello, R., Dieguez, E., Raggio, V., Micheli, F., Perandones, C., Alvarez, V., Segura-Aguilar, J., Farrer, M.J., Zabetian, C.P. & Ross, O.A.

Mutations in the LRRK2 gene are the most common genetic cause of Parkinson's disease, with frequencies displaying a high degree of population-specificity. Although more than 100 coding substitutions have been identified, only seven have been proven to be highly penetrant pathogenic mutations. Studies however are lacking in non-white populations. Recently, Lrrk2 p.Q1111H (rs78365431) was identified in two affected Hispanic brothers and absent in 386 non-Hispanic white healthy controls. We therefore screened this variant in 1460 individuals (1150 PD patients and 310 healthy controls) from 4 Latin American countries (Peru, Chile, Uruguay and Argentina). In our case-control series from Peru and Chile we observed an increased frequency of Lrrk2 p.Q1111H in patients (7.9%) compared to controls (5.4%) although the difference did not reach significance (OR 1.38; p = 0.10). In addition, the frequency of Lrrk2 p.Q1111H varied greatly between populations and further screening in a set of pure Amerindian and pure Spanish controls suggested that this variant likely originated in an Amerindian population. Further studies in other Latin American populations are warranted to assess its role as a risk factor for Parkinson's disease. Screening in Parkinson's disease patients from under-represented populations will increase our understanding of the role of LRRK2 variants in disease risk worldwide.

Elbaz, A., Ross, O.A., Ioannidis, J.P., Soto-Ortolaza, A.I., Moisan, F., Aasly, J., Annesi, G., Bozi, M., Brighina, L., Chartier-Harlin, M.C., Destee, A., Ferrarese, C., Ferraris, A., Gibson, J.M., Gispert, S., Hadjigeorgiou, G.M., Jasinska-Myga, B., Klein, C., Kruger, R., Lambert, J.C., Lohmann, K., van de Loo, S., Loriot, M.A., Lynch, T., Mellick, G.D., Mutez, E., Nilsson, C., Opala, G., Puschmann, A., Quattrone, A., Sharma, M., Silburn, P.A., Stefanis, L., Uitti, R.J., Valente, E.M., Vilariño-Güell, C., Wirdefeldt, K., Wszolek, Z.K., Xiromerisiou, G., Maraganore, D.M. & Farrer, M.J.

OBJECTIVE: We studied the independent and joint effects of the genes encoding alpha-synuclein (SNCA) and microtubule-associated protein tau (MAPT) in Parkinson disease (PD) as part of a large meta-analysis of individual data from case-control studies participating in the Genetic Epidemiology of Parkinson's Disease (GEO-PD) consortium. METHODS: Participants of Caucasian ancestry were genotyped for a total of 4 SNCA (rs2583988, rs181489, rs356219, rs11931074) and 2 MAPT (rs1052553, rs242557) single nucleotide polymorphism (SNPs). Individual and joint effects of SNCA and MAPT SNPs were investigated using fixed- and random-effects logistic regression models. Interactions were studied on both a multiplicative and an additive scale, and using a case-control and case-only approach. RESULTS: Fifteen GEO-PD sites contributed a total of 5,302 cases and 4,161 controls. All 4 SNCA SNPs and the MAPT H1-haplotype-defining SNP (rs1052553) displayed a highly significant marginal association with PD at the significance level adjusted for multiple comparisons. For SNCA, the strongest associations were observed for SNPs located at the 3' end of the gene. There was no evidence of statistical interaction between any of the 4 SNCA SNPs and rs1052553 or rs242557, neither on the multiplicative nor on the additive scale. INTERPRETATION: This study confirms the association between PD and both SNCA SNPs and the H1 MAPT haplotype. It shows, based on a variety of approaches, that the joint action of variants in these 2 loci is consistent with independent effects of the genes without additional interacting effects.

Dachsel, J.C., Wider, C., Vilariño-Güell, C., Aasly, J.O., Rajput, A., Rajput, A.H., Lynch, T., Craig, D., Krygowska-Wajs, A., Jasinska-Myga, B., Opala, G., Barcikowska, M., Czyzewski, K., Wu, R.M., Heckman, M.G., Uitti, R.J., Wszolek, Z.K., Farrer, M.J. & Ross, O.A.

BACKGROUND AND PURPOSE: Mutations of the LRRK2 gene are now recognized as major risk factors for Parkinson's disease. The Lrrk2 protein is a member of the ROCO family, which also includes Lrrk1 and Dapk1. Functional genetic variants of the DAPK1 gene (rs4877365 and rs4878104) have been previously associated with Alzheimer's disease. METHODS: Herein, we assessed the role of DAPK1 variants (rs4877365 and rs4878104) in risk of Parkinson's disease with Sequenom iPLEX genotyping, employing one Taiwanese series (391 patients with Parkinson's disease, 344 controls) and five separate Caucasian series' (combined sample size 1962 Parkinson's disease patients, 1900 controls). RESULTS: We observed no evidence of association for rs4877365 and rs4878104 and risk of Parkinson's disease in any of the individual series or in the combined Caucasian series under either an additive or recessive model. CONCLUSION: These specific DAPK1 intronic variants do not increase the risk of Parkinson's disease. However, further functional studies are required to elucidate the potential therapeutic implications with the dimerization of the Dapk1 and Lrrk2 proteins.

Chartier-Harlin, M.C., Dachsel, J.C., Vilariño-Güell, C., Lincoln, S.J., Lepretre, F., Hulihan, M.M., Kachergus, J., Milnerwood, A.J., Tapia, L., Song, M.S., Le Rhun, E., Mutez, E., Larvor, L., Duflot, A., Vanbesien-Mailliot, C., Kreisler, A., Ross, O.A., Nishioka, K., Soto-Ortolaza, A.I., Cobb, S.A., Melrose, H.L., Behrouz, B., Keeling, B.H., Bacon, J.A., Hentati, E., Williams, L., Yanagiya, A., Sonenberg, N., Lockhart, P.J., Zubair, A.C., Uitti, R.J., Aasly, J.O., Krygowska-Wajs, A., Opala, G., Wszolek, Z.K., Frigerio, R., Maraganore, D.M., Gosal, D., Lynch, T., Hutchinson, M., Bentivoglio, A.R., Valente, E.M., Nichols, W.C., Pankratz, N., Foroud, T., Gibson, R.A., Hentati, F., Dickson, D.W., Destee, A. & Farrer, M.J.

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.

Selected 2008-2010

Vilariño-Güell, C., Wider, C., Ross, O.A., Jasinska-Myga, B., Kachergus, J., Cobb, S.A., Soto-Ortolaza, A.I., Behrouz, B., Heckman, M.G., Diehl, N.N., Testa, C.M., Wszolek, Z.K., Uitti, R.J., Jankovic, J., Louis, E.D., Clark, L.N., Rajput, A. & Farrer, M.J.

Genetic variation in the leucine-rich repeat and Ig domain containing 1 gene (LINGO1) was recently associated with an increased risk of developing essential tremor (ET) and Parkinson disease (PD). Herein, we performed a comprehensive study of LINGO1 and its paralog LINGO2 in ET and PD by sequencing both genes in patients (ET, n=95; PD, n=96) and by examining haplotype-tagging single-nucleotide polymorphisms (tSNPs) in a multicenter North American series of patients (ET, n=1,247; PD, n= 633) and controls (n=642). The sequencing study identified six novel coding variants in LINGO1 (p.S4C, p.V107M, p.A277T, p.R423R, p.G537A, p.D610D) and three in LINGO2 (p.D135D, p.P217P, p.V565V), however segregation analysis did not support pathogenicity. The association study employed 16 tSNPs at the LINGO1 locus and 21 at the LINGO2 locus. One variant in LINGO1 (rs9652490) displayed evidence of an association with ET (odds ratio (OR) =0.63; P=0.026) and PD (OR=0.54; P=0.016). Additionally, four other tSNPs in LINGO1 and one in LINGO2 were associated with ET and one tSNP in LINGO2 associated with PD (P<0.05). Further analysis identified one tSNP in LINGO1 and two in LINGO2 which influenced age at onset of ET and two tSNPs in LINGO1 which altered age at onset of PD (P<0.05). Our results support a role for LINGO1 and LINGO2 in determining risk for and perhaps age at onset of ET and PD. Further studies are warranted to confirm these findings and to determine the pathogenic mechanisms involved.

Vilariño-Güell, C., Wider, C., Soto-Ortolaza, A.I., Cobb, S.A., Kachergus, J.M., Keeling, B.H., Dachsel, J.C., Hulihan, M.M., Dickson, D.W., Wszolek, Z.K., Uitti, R.J., Graff-Radford, N.R., Boeve, B.F., Josephs, K.A., Miller, B., Boylan, K.B., Gwinn, K., Adler, C.H., Aasly, J.O., Hentati, F., Destee, A., Krygowska-Wajs, A., Chartier-Harlin, M.C., Ross, O.A., Rademakers, R. & Farrer, M.J.

OBJECTIVE: Recently, mutations in DCTN1 were found to cause Perry syndrome, a parkinsonian disorder with TDP-43-positive pathology. Previously, mutations in DCTN1 were identified in a family with lower motor neuron disease, in amyotrophic lateral sclerosis (ALS), and in a family with ALS/frontotemporal dementia (FTD), suggesting a central role for DCTN1 in neurodegeneration. METHODS: In this study we sequenced all DCTN1 exons and exon-intron boundaries in 286 samples diagnosed with Parkinson disease (PD), frontotemporal lobar degeneration (FTLD), or ALS. RESULTS: This analysis revealed 36 novel variants (9 missense, 5 silent, and 22 noncoding). Segregation analysis in families and association studies in PD, FTLD, and ALS case-control series did not identify any variants segregating with disease or associated with increased disease risk. CONCLUSIONS: This study suggests that pathogenic mutations in DCTN1 are rare and do not play a common role in the development of Parkinson disease, frontotemporal lobar degeneration, or amyotrophic lateral sclerosis.

Vilariño-Güell, C., Soto, A.I., Lincoln, S.J., Ben Yahmed, S., Kefi, M., Heckman, M.G., Hulihan, M.M., Chai, H., Diehl, N.N., Amouri, R., Rajput, A., Mash, D.C., Dickson, D.W., Middleton, L.T., Gibson, R.A., Hentati, F. & Farrer, M.J.

Recessively inherited mutations in ATP13A2 result in Kufor-Rakeb syndrome (KRS), whereas genetic variability and elevated ATP13A2 expression have been implicated in Parkinson disease (PD). Given this background, ATP13A2 was comprehensively assessed to support or refute its contribution to PD. Sequencing of ATP13A2 exons and intron-exon boundaries was performed in 89 probands with familial parkinsonism from Tunisia. The segregation of mutations with parkinsonism was subsequently assessed within pedigrees. The frequency of genetic variants and evidence for association was also examined in 240 patients with nonfamilial PD and 372 healthy controls. ATP13A2 mRNA expression was also quantified in brain tissues from 38 patients with nonfamilial PD and 38 healthy subjects from the United States. Sequencing analysis revealed 37 new variants; seven missense, six silent, and 24 that were noncoding. However, no single ATP13A2 mutation segregated with familial parkinsonism in either a dominant or recessive manner. Four markers showed marginal association with nonfamilial PD, prior to correction for multiple testing. ATP13A2 mRNA expression was marginally decreased in PD brains compared with tissue from control subjects. In conclusion, neither ATP13A2 genetic variability nor quantitative gene expression in brain appears to contribute to familial parkinsonism or nonfamilial PD.

Vilariño-Güell, C., Chai, H., Keeling, B.H., Young, J.E., Rajput, A., Lynch, T., Aasly, J.O., Uitti, R.J., Wszolek, Z.K., Farrer, M.J. & Lin, S.C

Restless legs syndrome (RLS) is a neurologic condition characterized by a distressing urge to move the legs, usually accompanied by an uncomfortable sensation described as a crawling, muscle ache, or tension. It is usually brought on by rest, worse in the evening or night, and relieved by movement. Recently, MEIS1 and BTBD9 have been identified and confirmed as RLS susceptibility genes; however, all the variants found to be associated with disease are located deep in intronic regions and are not thought to be functional. To date, sequencing of MEIS1 and BTBD9 in RLS samples has not been reported and therefore functional variants responsible for the increased disease risk have not been identified. Here we report sequencing of all MEIS1 and BTBD9 coding exons and exon-intron boundaries in RLS familial probands followed by assessment of segregation of novel variants with disease within families and evaluation of prevalence in patients with RLS and the general population.

Farrer, M.J., Hulihan, M.M., Kachergus, J.M., Dachsel, J.C., Stoessl, A.J., Grantier, L.L., Calne, S., Calne, D.B., Lechevalier, B., Chapon, F., Tsuboi, Y., Yamada, T., Gutmann, L., Elibol, B., Bhatia, K.P., Wider, C., Vilariño-Güell, C., Ross, O.A., Brown, L.A., Castanedes-Casey, M., Dickson, D.W. & Wszolek, Z.K.

Perry syndrome consists of early-onset parkinsonism, depression, severe weight loss and hypoventilation, with brain pathology characterized by TDP-43 immunostaining. We carried out genome-wide linkage analysis and identified five disease-segregating mutations affecting the CAP-Gly domain of dynactin (encoded by DCTN1) in eight families with Perry syndrome; these mutations diminish microtubule binding and lead to intracytoplasmic inclusions. Our findings show that DCTN1 mutations, previously associated with motor neuron disease, can underlie the selective vulnerability of other neuronal populations in distinct neurodegenerative disorders.