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Analysis of DNAJC13 mutations in French-Canadian/French cohort of Parkinson’s disease

Jay P. Ross,1,2, Nicolas Dupre,3 Yves Dauvilliers,4, Stephanie Strong,2,5, Amirthagowri Ambalavanan,1,2, Dan Spiegelman,2,5, Alexandre Dionne-Laporte,2,5, Emanuelle Pourcher,3 Melanie Langlois,3 Michel Boivin,6,7, Claire S. Leblond,1,2, Patrick A. Dion,2,5, Guy A. Rouleau,1,2,5, Ziv Gan-Or1,2,5,*.

Author affiliations 1 Department of Human Genetics, McGill University, Montréal, QC, H3A 0G4 Canada2 Montreal Neurological Institute, McGill University, Montréal, QC, H3A 0G4 Canada 3 Division of Neurology, CHU de Québec, and Faculty of Medicine, Laval University, Quebec City, QC, G1V 0A6 Canada4 Sleep Unit, National Reference Network for Narcolepsy, Department of Neurology Hôpital-Gui-de Chauliac, CHU Montpellier, INSERM U1061, France5 Department of Neurology and Neurosurgery, McGill University, Montréal, QC, H3A 0G4 Canada 6 GRIP, École de Psychologie, Université Laval, Québec city, QC, G1V 0A6 Canada7 Institute of Genetic, Neurobiological and Social Foundations of Child Development, Tomsk State University, Tomsk, Russia s* Corresponding Author:Ziv Gan-OrMontreal Neurological Institute and hospital,The Department of Human Genetics, McGill University1033 Pine Avenue West,Ludmer Pavilion, room 327Montreal, QC, H3A 1A1Tel: +1-514-398-6821Fax: +1-514 398-8248e-mail: ziv.gan-or@mail.mcgill.ca

Keywords: DNAJC13, Parkinson’s disease, MIPs

Word count: Main text: 1581, Abstract: 150, References: 26; Tables and Figures: 2

Conflict of interests: All authors report no conflict of interests.

Funding Sources: Michael J. Fox Foundation for Parkinson’s Research

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Abstract

DNAJC13 mutations have been suggested to cause Parkinson’s disease (PD), yet subsequent

studies reported conflicting results on this association. In the current study, we sequenced the

coding region of DNAJC13 in a French-Canadian/French cohort of 528 PD patients and 692

controls. A total of 62 (11.7%) carriers of rare DNAJC13 variants were identified among PD

patients compared to 82 (11.8%) among controls (p=1.0). Two variants that were previously

suggested to be associated with PD, p.R1516H and p.L2170W, were identified with similar

directions of association as previously reported. The p.R1516H was found in 2 (0.4%) patients

vs. 6 (0.9%, non-significant) controls (meta-analysis OR=0.32, 95%CI=0.15-0.68, p=0.0037)

and the p.L2170W variant was found in 9 (1.7%) patients and 5 (0.7%, non-significant) controls

(meta-analysis OR=2.68, 95%CI=1.32-5.42, p=0.007). Our results provide some support for the

possibility that specific DNAJC13 variants may play a minor role in PD susceptibility, although

studies in additional populations are necessary.

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Introduction

Recently, DNAJC13 mutations have been implicated in the pathogenesis of PD. The DNAJC13

gene is a possible source of risk factor variants as well as rare causal mutations.(Appel-

Cresswell, et al., 2014,Gustavsson, et al., 2015,Vilarino-Guell, et al., 2014) A single variant in

this gene, p.N855S, has been suggested to cause late-onset PD, and has been demonstrated to co-

segregate with PD within five families of Dutch–German–Russian Mennonite origin from

Saskatchewan and British Columbia, Canada, albeit with incomplete penetrance and with

phenocopies.(Vilarino-Guell, et al., 2014) An additional study suggested that other rare

DNAJC13 variants, such as p.R1516H, p.E1740Q, and p.L2170W, may be associated with

increased or decreased risk for developing PD in Caucasian Canadian and Norwegian

populations.(Gustavsson, et al., 2015) However, subsequent studies in large cohorts of Singapore

Chinese and non-Hispanic-Caucasian populations did not identify mutations in DNAJC13

associated with PD.(Foo, et al., 2014,Lorenzo-Betancor, et al., 2015)

While DNAJC13 is a possible candidate gene for PD, the exact nature of the association

remains inconclusive. To further examine the potential role of DNAJC13 mutations in PD, we

sequenced its coding regions in a cohort of French Canadian and French PD patients and

controls.

Methods

Study population

A cohort of unrelated and consecutively recruited 528 PD patients and 692 controls were

included in this study. The cohort was composed of French-Canadian patients and controls from

Quebec, collected at the Centre Hospitalier Universitaire de (CHU) de Quebec and Montreal

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Neurological Institute, Montreal, Canada, and from French patients and controls recruited at

Montpellier, France. Average patient age was 65.7 ± 10.9 years, with 64.2% men. The control

population included two groups, elderly controls (n=105, average age at enrollment of 61.5 ± 7.6

years) and young controls (n=576, average age at enrollment of 34.6 ± 6.4 years, data on age was

not available for 11 controls). There was no significant difference in DNAJC13 variants

frequency between the two groups, therefore we chose to analyze them as a single control

population, with average age of 38.7 ± 11.8 years with 39.6% men. PD was diagnosed by

movement disorder specialists according to the UK Brain Bank Criteria,(Hughes, et al., 1992)

without excluding patients who have relatives with PD. All patients signed informed consent

before entering the study, and the institutional review boards approved the study protocols.

DNA extraction and DNAJC13 Sequencing

DNA was extracted using a standard salting out protocol. The coding sequence of DNAJC13 was

targeted using molecular inversion probes (MIPs), that were designed as described by O’Roak

and colleagues.(O'Roak, et al., 2012) MIPs were selected based on their predicted quality,

coverage and overlap, and Supplementary Table 1 includes the MIPs used to sequence

DNAJC13 in the current study. The library was sequenced using the Illumina HiSeq 2500

platform at the McGill University and Génome Québec Innovation Centre. Sequence processing

was done using Burrows-Wheeler Aligner (BWA),(Li and Durbin, 2009) the Genome Analysis

Toolkit (GATK v.2.6.4)(McKenna, et al., 2010) and ANNOVAR.(Wang, et al., 2010) Online

prediction and conservation tools (SIFT,(Kumar, et al., 2009) PolyPhen 2(Adzhubei, et al., 2010)

and GERP++(Davydov, et al., 2010)) were used to assess the deleteriousness of the variants.

Data on the frequency of each DNAJC13 variant was extracted from public databases (Exome

Aggregation Consortium (ExAC), 1000 Genomes Project, NHLBI GO Exome Sequencing

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Project – Exome Variant Server). Only variants with high coverage and read quality were

included in the analysis. All detected mutations were validated with Sanger sequencing in

samples identified as carriers by MIPs.

Statistical Analysis

The association between DNAJC13 variants and PD was analyzed using a binary logistic

regression with the status (patient or control) as a dependent variable, and age and sex as

covariates. Since there was no difference in DNAJC13 variant frequencies between the two

control groups (elderly and young) and between men and women, we also used Fisher’s Exact

Test to examine the association between all DNAJC13 mutations combined or private DNAJC13

variants and PD. To perform a combined analysis of the p.R1516H and p.L2170W variants, we

performed a meta-analysis by using an R package (Metafor).(Wolfgang, 2010) Cochran-Mantel-

Haenszel test was used to pool the studies and calculate the odds ratios (OR) with a fixed-effect

model. Tarone's test was applied to estimate heterogeneity. All other statistical analysis was

performed using the SPSS V2.1 software (IBM Inc.).

Results

A total of 42 non-synonymous variants in DNAJC13 were found in patients and controls (Table

1), including four common variants (allele frequency > 0.01 in our controls or in the 1000

Genomes Project and ExAC databases) and 38 rare variants. The combined frequency of rare

variants was nearly identical in patients and controls (11.7% vs. 11.8%, respectively, OR=0.99,

95% CI = 0.70 – 1.41, p=1.0). The purported causal variant, p.N855S,(Vilarino-Guell, et al.,

2014) was not identified in any of our cases or controls. Two variants that were previously

reported to be associated with PD risk, p.R1516H and p.L2170W, were observed in 2 (0.4%)

patients and 6 (0.9%) controls, and 9 (1.7%) patients and 5 (0.7%) controls, respectively (Table

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1, p>0.05 for both variants). Carriers of the p.R1516H variant had an average age of 55 for

patients and 38.8 for controls, while carriers of the p.L2170W variant had an average age of 63

for patients and 41.8 for controls. A combined analysis of these two variants using both the

present study and the different populations in the first study(Gustavsson, et al., 2015) (Table 2)

showed an OR of 0.32 (95%CI=0.15-0.68, p=0.0037, p for heterogeneity = 0.16) for p.R1516H

and an OR of 2.68 (95%CI=1.32-5.42, p=0.007, p for heterogeneity = 0.83) for p.L2170W. We

further examined whether variants that are predicted to be deleterious by both SIFT and

PolyPhen2 are associated with PD. Among PD patients, 53 (10.0%) carried such mutations and

among controls 56 carried a predicted deleterious variant (8.1%, p>0.05).

Discussion

None of the variants found in our cohort was independently associated with PD. However, two

variants, p.L2170W and p.R1516H, had similar frequencies and risk direction as previously

reported, and meta-analyses of these variants suggested that they may have a role in PD

susceptibility. The meta-analysis included our population, and three populations that were

previously published (Gustavsson, et al., 2015). These populations included a discovery cohort

of 201 PD patients and 194 controls from Canada, an additional population of 1042 PD patients

and 497 controls from Canada, and 920 PD patients and 635 controls from Norway. The size of

the current control population (n=692) is larger than the published individual control

populations, and our patient population (n=528) is in-between the smallest and the largest PD

populations, thus adding a substantial amount of patients and controls for the meta-analysis.

Other studies in Caucasian (Lorenzo-Betancor, et al., 2015) and Chinese (Foo, et al., 2014)

populations did not identify these variants, since they genotyped specific exons or variants that

did not include p.L2170W and p.R1516H. The initially reported mutation, p.N855S, was not

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found in our cohort, suggesting that while this variant may be causative for PD in other

ethnicities,(Gustavsson, et al., 2015) it is probably not a an important cause of PD in French

Canadians/French. The original family in which the p.N855S variant was identified was of

Dutch–German–Russian Mennonite ancestry;(Vilarino-Guell, et al., 2014) it has been shown that

p.N855S mutation is not a cause of PD in Caucasians,(Lorenzo-Betancor, et al., 2015) suggesting

that even between similar ethnic groups there could be differences in genetic causes of PD. In

addition, our study identified several rare and novel variants in the DNAJC13 gene. However,

many of these were non-informative (only observed in one patient or control), or were not

significantly associated with PD. Therefore, these variants need to be studied in additional

populations in order to reach conclusions as to the role of DNAJC13 in PD pathogenesis. A

previous sequencing study of GBA mutations that included 212 patients and 190 controls from

the current study was performed, and none of the carriers of GBA mutations was a carrier of

either p.L2170W or p.R1516H. Therefore, it is unlikely that GBA mutations had an effect on the

current study results, and further studies are needed to determine if there is any association or

interaction between GBA and DNAJC13 in PD. The DNAJC13 gene has previously been shown

to be highly conserved across species.(Girard, et al., 2005,Gustavsson, et al., 2015,Vilarino-

Guell, et al., 2014) While this may suggest that variations in the gene result in non-tolerated

functional changes, it is possible that these mutations are exceedingly rare and difficult to

associate with pathological changes.

DNAJC13 is a component of the retromer complex involved in the endosomal-lysosomal

retrograde pathway,(Girard, et al., 2005,Seaman and Freeman, 2014) and aids in localization of

the WASH complex in the endosomal pathway for substrate recognition.(Perrett, et al., 2015)

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Several components of the retromer endosomal recycling pathway were associated with

Parkinson’s disease, in addition to LRRK2, VPS35, and RAB29, that were previously

mentioned. Variants in the promoter of RAB7L1 (RAB29), were demonstrated to reduce the risk

for PD.(Gan-Or, et al., 2012,Guo, et al., 2014,Pihlstrom, et al., 2015) In addition, it was

demonstrated that LRRK2 mutations or knockdown of either VPS35 or RAB7L1 leads to the

impairment of this pathway and to lysosomal dysfunction.(MacLeod, et al., 2013,Miura, et al.,

2014) Genome wide association studies identified variants in a locus that includes the GAK gene,

(Pankratz, et al., 2009,Rhodes, et al., 2011) encoding for a G-associated kinase that recruits

chaperones to clathrin-coated vesicles in the retromer endosomal recycling pathway (Park, et al.,

2015). Similarly, DNAJC6, implicated in atypical juvenile Parkinsonism and encoding the

auxilin protein,(Edvardson, et al., 2012) is also involved in chaperone recruitment to clathrin-

coated vesicles. DNAJC13 (RME-8) localizes to endosomal membranes and is involved in

regulation of vesicles in the retromer endosomal recycling pathway.(Girard, et al., 2005) The

importance of this pathway in PD pathology is further supported by the possible association of

DNAJC13 variants with PD risk.

Our study has several limitations. The control population is not age and sex matched,

which may reduce our ability to detect genetic variants that are associated with PD. However,

since DNAJC13 mutations are mostly rare, these differences are less likely to affect the results.

Furthermore, the regression model was adjusted for age and sex when analyzing the cumulative

burden of DNAJC13 mutations in patients and controls. The age of carriers for both p.R1516H

and p.L2170W variants was younger in controls than in patients, which may impact the

penetrance of the variants; however, in the case of p.L2170W, if controls will develop PD in the

future, it will strengthen the association. If controls with the p.R1516H variant will develop PD,

it will reduce the association. Therefore, follow-up studies will be needed to determine the exact

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risk associated with these variants. Haplotype analysis was not performed on our samples, but

given the difference in ethnicity and that several haplotypes exist for DNAJC13 mutation

carriers,(Gustavsson, et al., 2015,Vilarino-Guell, et al., 2014) it is likely that our cohort does not

overlap with those previously reported.

Overall, the results presented here provide additional support, although minor, to the

hypothesis that DNAJC13 may be associated with PD. It is clear, however, that if indeed

DNAJC13 has a role in PD it is probably only relevant to a very small number of PD patients. On

the other hand, the fact that numerous PD-associated genes converge into the autophagy

lysosomal and retromer endosomal recycling pathways highlights their importance in PD, which

may lead to the development of novel treatments for PD.

Acknowledgements

We thank the patients and controls for their participation in the study. This work was financially

supported by the Michael J. Fox Foundation. ZGO is supported by a postdoctoral fellowship

from the Canadian Institutes for Health Research (CIHR). GAR holds a Canada Research Chair

in Genetics of the Nervous System and the Wilder Penfield Chair in Neurosciences. We thank

Daniel Rochefort, Pascale Hince, Helene Catoire, Cynthia Bourassa, Pierre Provencher, Cathy

Mirarchi and Vessela Zaharieva for their assistance. We thank the Quebec Parkinson’s Network

and its members (http://rpq-qpn.ca/) for their collaboration.

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Table 1. Rare and common DNAJC13 Variants in PD patients and controls

Variant SIFT PolyPhen2GERP+

+ Freq. 1K Freq. ExAC Patients (n=528) Controls (n=692)Rare variants

p.T66M 0.27 0.993 4.52 0.0000569 1 (0.2%) 1 (0.1%)p.R73C 0.02 0.999 5.4 0.00013 1 (0.2%) 0 (0.0%)p.N141I 0 0.25 4.64 0.0000569 1 (0.2%) 0 (0.0%)p.K225R 0.36 0.982 5.6 0.00000813 1 (0.2%) 0 (0.0%)p.L318F 0.01 0.994 5.98 1 (0.2%) 0 (0.0%)p.G416A 0.4 0.006 5.86 0 (0.0%) 1 (0.1%)p.A423V 0.41 0.848 5.86 0.000599 0.00022 1 (0.2%) 0 (0.0%)p.H521D 0.03 0.044 5.29 0 (0.0%) 1 (0.1%)p.R654C 0 1 5.53 0.0000163 0 (0.0%) 1 (0.1%)p.D674A 0.11 0.888 5.76 0.0002 0.000277 0 (0.0%) 1 (0.1%)p.N695S 0.47 0.009 2.07 0.00000813 0 (0.0%) 1 (0.1%)p.I738V 0.55 0 -1.5 1 (0.2%) 1 (0.1%)p.E789Q 0.12 0.998 5.59 0.0000163 1 (0.2%) 0 (0.0%)p.S790Y 0.01 0.553 5.59 0.004393 0.00513 10 (1.9%) 7 (1.0%)p.T944S 0.12 0.956 5.78 1 (0.2%) 0 (0.0%)

p.R1118C 0 1 5.78 0.00000813 0 (0.0%) 1 (0.1%)p.G1133D 0.01 1 5.65 0.0000651 0 (0.0%) 2 (0.3%)p.I1272V 0.06 0.734 6.07 0.0002 0.0000813 1 (0.2%) 0 (0.0%)p.E1291G 0.03 0.995 5.64 0.002995 0.00444 8 (1.5%) 10 (1.4%)p.N1307Y 0.01 0.272 3.18 0 (0.0%) 2 (0.3%)p.Q1312R 0.12 0.383 5.64 0.00000813 1 (0.2%) 0 (0.0%)p.P1366S 0.27 1 5.26 0 (0.0%) 2 (0.3%)p.R1400Q 0.16 0.103 5.32 0.000399 0.000179 1 (0.2%) 0 (0.0%)p.R1462H 0.22 0 3.37 0.005591 0.00913 5 (1.0%) 13 (1.9%)p.S1482G 0.16 0.329 5.86 0.00000813 1 (0.2%) 0 (0.0%)p.V1483A 0.2 0.214 5.86 0 (0.0%) 1 (0.1%)p.R1516H 0.07 0.521 3.6 0.001398 0.00189 2 (0.4%) 6 (0.9%)p.T1895M 0.23 0.007 5.2 0.000599 0.00157 2 (0.4%) 3 (0.4%)p.N1952T 0.51 0.008 3.46 0.005591 0.00163 0 (0.0%) 1 (0.1%)p.Q1954R 0.72 0 -3.12 1 (0.2%) 0 (0.0%)p.V1995L 0.06 0.977 5.25 0.001597 0.00116 3 (0.6%) 5 (0.7%)p.L2027F 0.03 0.006 3.9 0 (0.0%) 2 (0.3%)p.A2057S 0.5 0.126 5.98 0.000399 0.000805 3 (0.6%) 4 (0.6%)p.V2125L 0.11 0.196 5.34 1 (0.2%) 0 (0.0%)p.L2170W 0.03 0.999 5.45 0.00234 9 (1.7%) 5 (0.7%)p.Y2206H 0.01 1 5.96 1 (0.2%) 2 (0.3%)p.M2225I 0.39 0 3.29 0.009984 0.00912 3 (0.6%) 9 (1.3%)p.S2226A 1 0.003 4.27 1 (0.2%) 0 (0.0%)

Common variantsp.A1421V 0.01 0.998 5.42 0.01278 0.018 23 (4.4%) 26 (3.8%)p.A1463S 1 0 5.17 0.739018 0.569 365 (69.5%) 489 (70.8%)

2324

307

13

p.P1515S 0.16 0.044 5.81 0.011981 0.021 36 (6.9%) 32 (4.6%)p.Y1673C 0.06 0.992 5.75 0.024161 0.045 68 (13.0%) 91 (12.2%)

Threshold values for deleteriousness: SIFT – less than 0.05, Polyphen2 – greater than 0.86. GERP++ is a score for the conservation of the amino acid, scores > 3 can be considered as highly conserved. Freq. 1K, frequency in the 1000 genomes project; Freq. ExAC, frequency in the ExAC database.

2526

308

309310311312

313

314