Common Variants Near ZIC1 and ZIC4 in Autopsy-Confirmed Multiple System Atrophy

Background: Multiple System Atrophy is a rare neurodegenerative disease with alpha-synuclein aggregation in glial cytoplasmic inclusions and either predominant olivopontocerebellar atrophy or striatonigral degeneration, leading to dysautonomia, parkinsonism, and cerebellar ataxia. One prior genome-wide association study in mainly clinically diagnosed patients with Multiple System Atrophy failed to identify genetic variants predisposing for the disease. Objective: Since the clinical diagnosis of Multiple System Atrophy yields a high rate of misdiagnosis when compared to the neuropathological gold standard, we studied only autopsy-confirmed cases. Methods: We studied common genetic variations in Multiple System Atrophy cases (N = 731) and controls (N = 2898). Results: The most strongly disease-associated markers were rs16859966 on chromosome 3, rs7013955 on chromosome 8, and rs116607983 on chromosome 4 with P-values below 5 × 10−6, all of which were supported by at least one additional genotyped and several imputed single nucleotide polymorphisms. The genes closest to the chromosome 3 locus are ZIC1 and ZIC4 encoding the zinc finger proteins of cerebellum 1 and 4 (ZIC1 and ZIC4). Interpretation: Since mutations of ZIC1 and ZIC4 and paraneoplastic autoantibodies directed against ZIC4 are associated with severe cerebellar dysfunction, we conducted immunohistochemical analyses in brain tissue of the frontal cortex and the cerebellum from 24 Multiple System Atrophy patients. Strong immunohistochemical expression of ZIC4 was detected in a subset of neurons of the dentate nucleus in all healthy controls and in patients with striatonigral degeneration, whereas ZIC4-immunoreactive neurons were significantly reduced inpatients with olivopontocerebellar atrophy. These findings point to a potential ZIC4-mediated vulnerability of neurons in Multiple System Atrophy.

Multiple system atrophy (MSA) is a rapidly progressive rare neurodegenerative disease presenting with variable combinations of dysautonomia, parkinsonism, and cerebellar ataxia. 1 Two forms of MSA can be clinically distinguished, characterized by either predominant parkinsonism or predominant cerebellar symptoms. 2Its estimated prevalence is 3.4-4.9cases per 100,000 individuals in the general population, and 7.8 cases per 100,000 in persons older than 40 years. 3The mean survival time from disease onset is 6-10 years. 4,5urrently, only limited symptomatic treatments and no disease-modifying therapies are available. 6e typical symptoms of MSA are caused by the progressive degeneration of neurons in different brain regions, particularly in the substantia nigra, striatum, inferior olivary nucleus, pons, and cerebellum, but also other parts of the central nervous systems, emphasizing the multisystem character of MSA. 2,7The histological hallmarks in brains of patients with MSA are glial cytoplasmic inclusions (Papp-Lantos bodies) in oligodendrocytes containing aggregated and misfolded α-synuclein. 8Neuropathologically, two subtypes can be distinguished, one with predominant olivopontocerebellar atrophy (OPCA), the other with mainly striatonigral degeneration (SND). 9,10In addition, a mixed phenotype displaying features of both OPCA and SND is found in the brains of some patients. 9,10e pathogenesis of MSA is unclear.MSA is considered a sporadic disease. 11−14 Hypothesis-driven candidate gene studies have been inconsistent with respect to variants that might be associated with MSA.6][17][18][19][20] One prior genome-wide association study (GWAS) did not identify hits of statistical significance at a genome-wide level, despite the analysis of 918 cases and 3864 controls. 21This GWAS had mainly included clinically diagnosed MSA cases.It needs to be stressed that clinical diagnosis is frequently not accurate in MSA.For example, a recent clinicopathological study demonstrated a false-positive diagnosis at autopsy in 38% of patients with clinically diagnosed MSA. 22 avoid inclusion of misdiagnosed patients in the GWAS described in this study, we included only autopsy-confirmed cases and appropriate ethnicity-matched controls.

Patient Recruitment
Ethical approval had been obtained from all responsible ethics committees.All participants had given written consent.
Neuropathologists at each recruitment site (Table 1) based the definite neuropathological diagnosis of MSA on histopathological criteria, taking into account glial cytoplasmic inclusions immunoreactive for α-synuclein in characteristic anatomical distribution as a defining feature of MSA. 23Age, sex, disease history (including disease onset and duration), and neuropathological findings were recorded in a standardized manner for all cases.
Controls were ethnically matched to cases and either derived from biobanks KORA-gen 24 or popGen 25 (Europe sites) or from a North American site (Alzheimer's Disease Genetics Consortium). 26The Alzheimer's Disease Genetics Consortium assembled and genotyped DNA from subjects enrolled in the 29 NIA-Alzheimer's Disease Centers located across the United States.For this study, the Alzheimer's Disease Genetics Consortium provided a subset of mostly clinical, cognitively normal controls.Patients and controls were of North-Western European and African American ancestry.

DNA Extraction
We isolated DNA from 30 mg frozen cerebellar cortex using QIAamp DNA Mini Kit (Qiagen, Venlo, the Netherlands).DNA extraction was performed at German Center for Neurodegenerative Diseases (DZNE, Munich, Germany).DNA was stored at −80°C until use.DNA concentration was measured using a NanoDrop Spectro-photometer.DNA quality was determined by gel electrophoresis.

Genotyping
All samples were genotyped on Infinium Global Screening Arrays (Illumina, San Diego, CA, USA).The cases were genotyped at the Institute of Clinical Molecular Biology, Kiel University, Germany.The samples were genotyped in one batch on array version 2.0 for cases and version 1.0 for controls.Genotypes were called using Illumina Genome studio according to the manufacturer's instructions using in-house cluster files.

Quality Control and Imputation
We used PLINK (v.1.9) [1] and R (v. 3.6.3) 27for all analyses.Only variants successfully genotyped in both the patient and the control populations were included in the subsequent analyses.Variants with multicharacter allele codes, insertions, deletions, duplicated markers, and all A/T and G/C variants were excluded.We excluded all samples discordant between reported and genotypic sex.Missing sex was imputed, and samples with ambiguously imputed sex were discarded.After a first step of filtering out samples and variants with call rate of less than 85%, we excluded variants with an individual call rate of less than 98% in a second filtering step.Next, we removed variants with a minor allele frequency <0.01, a significant deviation from Hardy-Weinberg equilibrium (P < 1 × 10 −6 ) in controls, or informative missingness (P < 1 × 10 −5 ).Subsequently, we excluded individuals with a variant call rate of <98% or an outlying heterozygosity rate (mean ± 3 standard deviations).We used a pruned dataset containing only markers in low linkage-disequilibrium regions (pairwise r 2 < 0.2) to test for duplicated individuals and cryptic relatedness (Pihat > 0.125) using pairwise genome-wide estimates of the proportion of identity by descent.For each detected sample pair we excluded the individual with a lower call rate.Ethnical outliers were identified by a principal-component analysis (PCA) together with the publically available 1000 Genomes data with known ethnicities. 28Because the study population has genetically a mainly European ancestry, as ascertained by the PCA, we determined a European center and excluded samples more than 1.5 times the maximal European Euclidean distance away from this center.After a first association analysis of genotyped single nucleotide polymorphisms (SNPs) only, we inspected visually the cluster plots of all variants with a P value <1 × 10 −5 and discarded variants without adequate cluster separation.Imputation was carried out on the quality-assured dataset using the TOPMed Imputation Server, which employs Eagle2 for phasing and minimac4 for the imputation of genotypes. 29,30The most likely genotype is used in downstream analyses.Variants were again filtered for minor allele frequency and deviation from Hardy-Weinberg equilibrium in controls with the same thresholds as before.In addition, SNPs with an imputation quality score R 2 < 0.7 were excluded, leaving 8,131,900 variants for analyses.As a final step of the quality-control procedure, we used the R package PCAmatchR to ethnically match cases to controls with a 1:4 ratio to overcome possible difficulties with population stratification, leading to 3240 individuals for the analyses. 31

Association Analysis
We used logistic regression to test the additive genetic model of each marker for association with disease status.Following scree plot analysis, we incorporated the first two dimensions of the PCA and sex as covariates.We used a genome-wide significance threshold of P < 5 × 10 −8 and the threshold of P < 5 × 10 −6 for suggestive association.Conditional analyses, including, in turn, each SNP with a suggestive association as additional covariate, were conducted to identify adjacent independent signals.Furthermore, we tested for clumps of correlated SNPs, ie, to assess how many independent loci had been associated, and determined the number of variants supporting the lead SNP at each locus, ie, variants with P values less than the clumping threshold of 5 × 10 −5 are in linkage disequilibrium (r 2 ≥ 0.4) and not farther than 250 kb away from the respective SNP.Visualization of the results was carried out with R and LocusZoom 32 for regional plots.Variant positions in this article are reported on human genome version 38 (GRCh38/hg38).

Immunohistochemistry on MSA Patients' Brain
Formalin-fixed and paraffin-embedded (FFPE) tissues from patients with MSA and controls without neurological or psychiatric diseases were obtained from the Neurobiobank Munich (Germany).All autopsy cases of the Neurobiobank Munich were collected on the basis of an informed consent according to the guidelines of the ethics commission of the Ludwig-Maximilians-University (Munich, Germany; #345-13).MSA cases had been diagnosed according to established histopathological diagnostic criteria. 10,23or ZIC4 immunohistochemistry, 5-μm-thick sections of FFPE tissues of the frontal cortex and the cerebellar hemisphere, including the dentate nucleus, were prepared.After deparaffinization, heat-induced epitope retrieval was performed in Tris/EDTA, pH 9, at 95°C for 30 minutes.For blocking of endogenous peroxidase and unspecific protein binding, the sections were incubated with 5% H 2 O 2 in methanol for 20 minutes and I-Block reagent (Applied Biosystems, Waltham, MA, USA) for 15 minutes.Subsequently, ZIC4 primary antibody (rabbit, polyclonal; Merck/Sigma-Aldrich, Darmstadt, Germany) was applied overnight at 4°C at a dilution of 1:100.Signal detection was performed using the DCS ChromoLine DAB kit (DCS, Hamburg, Germany) according to the manufacturer's instructions.Sections were counterstained for 1 minute with Mayer's hemalum solution (Waldeck, Münster, Germany).
To determine the fractions of ZIC4-positive neurons of all neurons in the dentate nucleus, we scanned stained slides using a slide scanner (Axio Scan.Z1; Zeiss, Oberkochen, Germany) and visualized using the free ZEN lite software (v.3.3; Zeiss).For statistical evaluation of the data, Student t test was used, and statistical significance was defined as P < 0.05.

Patient Sample
From the initial sample of 731 cases, 13 cases had to be excluded because of insufficient tissue quality.After thorough quality control and filtering, 648 cases and 2592 controls covering 8,131,900 variants were included in the association analysis (Fig. 1).The number of excluded samples and variants in each step of the quality-control procedure is shown in Tables S1 and S2.

Association Results
We performed logistic regression incorporating sex and determined the first two dimensions of PCA as covariates using the scree plot method.The genomic inflation factor of λ = 1.01 (unimputed λ = 1.01;Fig. S1) indicates that no significant population stratification was present (Fig. 2A).We did not identify any disease-associated variants with a P value less than the genome-wide significance threshold of P < 5 × 10 −8 , but suggestive associations with P < 5 × 10 −6 at 10 different loci (Fig. 2B) with the leading SNP at each locus shown in Table 2. Conditional analyses, including, in turn, any SNP with P < 5 × 10 −6 , excluded the presence of multiple independent signals at each locus.All variants with suggestive associations are listed in Table S3.The most noteworthy hits were rs16859966 on chromosome 3 (P = 8.6 × 10 −7 ; odds ratio [OR], 1.58; 95% confidence interval [CI]: 1.32-1.89),rs7013955 on chromosome 8 (P = 3.7 × 10 −6 ; OR, 1.8; 95% CI: 1.40-2.31),and rs116607983 on chromosome 4 (P = 4.0 × 10 −6 ; OR, 2.93; 95% CI: 1.86-4.63),which were supported by at least one additional genotype, as well as several imputed SNPs with P values less than the clumping threshold of 5 × 10 −5 as discovered in the clumping analysis (Table 2).The genes closest to the chromosome 3 locus are the Long Intergenic Non-Protein Coding RNA 2032 (LINC02032) approximately 100 kb downstream and the zinc-finger proteins of cerebellum 1 and 4 genes (ZIC1, ZIC4), located roughly 600 kb upstream (Fig. 2E).The top SNP rs7013955 on chromosome 8 maps to the lysyl oxidaselike 2 gene (LOXL2; Fig. 2D).The association signal around SNP rs116607983 on chromosome 4 is located in a region devoid of protein-coding genes approximately 2000 kb to either side (Fig. 2E).A fourth locus on chromosome 5 (rs2279135) was also supported by multiple clumped SNPs, but all SNPs, including the lead SNP, were imputed (Table 2).Several variants clumped at the chromosome 5 locus were located in the ARHGEF37 gene, coding for Rho Guanine Nucleotide Exchange Factor 37 (Fig. 2F).None of the identified SNPs is an expression quantitative trait locus in brain tissues according the Genotype Tissue Expression project. 33At four of the six remaining loci with variants exhibiting suggestive associations, at most two supporting SNPs were present, which were all imputed; in the other two loci, no supporting SNPs could be found in the clumping analysis (Table 2, Fig. S2).We did not investigate these loci further because it is unlikely that they represent valid associations.No significant associations with Bonferroni-adjusted P values were detected with previously reported Parkinson's disease associations from a meta-analysis of 17 datasets from a Parkinson's disease GWAS (Table S4). 34

ZIC4 Immunohistochemistry on MSA Patients' Brain
ZIC4 and ZIC1 are known to play a critical role in the embryonal development of the cerebellum.Heterozygous deletions comprising the ZIC1 and ZIC4 locus have been associated with the Dandy-Walker malformation, a rare congenital condition characterized by a hypoplastic cerebellar vermis and an enlarged fourth ventricle. 35,36n mice, deletions of ZIC1 and ZIC4 lead to a striking phenotype similar to the Dandy-Walker malformation with cerebellar hypoplasia and foliation defects. 35,36In addition, paraneoplastic autoantibodies against ZIC4 protein are linked to severe cerebellar dysfunction and degeneration. 37,38cause cerebellar degeneration and corresponding symptoms are also a central hallmark of MSA, we decided to follow up on a potential role of ZIC4 in MSA patient brains by performing immunohistochemical stainings.For ZIC1, no primary antibody was appropriately sensitive and specific on human tissue in our hands.Thus, FFPE tissues of the cerebellum and, for comparison, the frontal cortex of patients with MSA (n = 10 SND, n = 14 OPCA/mixed phenotype) and healthy controls (b = 5) were stained with antibodies raised against ZIC4.
Nuclear and cytoplasmic staining of frontal cortex neurons was observed in all brains examined without differences between healthy controls and patients with MSA (Fig. 3A-C).In the cerebellar dentate nucleus, we found strong expression of ZIC4 in a subset of neurons in healthy controls, as well as patients with MSA with predominant SND (Fig. 3D,E,G,H).In contrast, patients with MSA with mixed subtype or OPCA showed reduced numbers of ZIC4-positive neurons, which were furthermore only weakly stained (Fig. 3F,I).Quantification of the proportions of ZIC4-positive neurons among the total number of dentate nucleus neurons depicted relatively constant proportions in healthy controls and patients with MSA-SND (33.2% ± 0.0% vs 32.6% 0.0%), whereas in patients with MSA-OPCA or MSA-mixed phenotype, we found significantly lower percentages of ZIC4positive neurons (15.5% ± 0.1%) (Fig. 3J).

Discussion
As part of the study, brain banks were contacted worldwide, and all available white MSA brains were included.As in the prior GWAS with 918 predominantly clinically diagnosed MSA patients, our current GWAS of 648 patients with autopsy-confirmed MSA did not identify disease-associated common variants less than the genome-wide significance threshold.Previously, hypothesis-driven candidate gene studies found inconsistent results for genetic variants and genes potentially associated with MSA.18][19][20]39 However, these genes have not been convincingly confirmed in other candidate gene studies and have not been associated in a previous MSA GWAS. 21This preceding GWAS analyzed 918 mostly clinical cases and 3864 controls.Overall, this GWAS did not identify any genomewide significant hits.Because our prior GWAS of 219 patients with autopsy-confirmed corticobasal degeneration did identify significant disease-associated common variants, our current findings strongly suggest that the genetic contribution to disease risk is smaller in MSA. 40vertheless, our study demonstrates several suggestive associations at different loci, which may provide relevant hypotheses for follow-up investigations into the pathogenesis of MSA.Specifically, we identified a variant on chromosome 3 (rs16859966; P = 8.6 × 10 −7 ; OR, 1.58; 95% CI: 1.32-1.89)located upstream of ZIC1 and ZIC4.ZIC1 and ZIC4 are located in close genomic proximity to each other and encode transcription factors highly expressed in different brain areas. 41,42oper function of these proteins is critical for the development of the CNS, particularly the cerebellum.36 Although no effect of rs16859966 on ZIC1 or ZIC4 expression is recorded in the Genotype Tissue Expression database, rare genetic variants or deletions in ZIC1 or ZIC4 result in congenital cerebellar defects. 35,36,43A heterozygous deletion of ZIC1 and ZIC4 causes the Dandy-Walker malformation, a developmental disorder of the cerebellum. 35,44emarkably, two recent epigenomic analyses in brain tissue of MSA point to ZIC4. 45,46oreover, paraneoplastic autoantibodies against ZIC4 induce cerebellar degeneration. 38Due to the pronounced cerebellar degeneration in MSA, we followed up on a possible role of ZIC4 In MSA.
Although we could detect a relatively constant proportion of approximately one-third ZIC4positive neurons among all neurons in the cerebellar dentate nucleus in healthy controls and patients with MSASND, cases with MSA-OPCA or the mixed MSA phenotype showed significantly lower fractions of ZIC4-positive neurons.This finding suggests that ZIC4 may be involved in the neurodegeneration in MSA.The involvement of ZIC4 mutations in the Dandy-Walker cerebellar malformation and the paraneoplastic ZIC4 autoantibodyassociated cerebellar degeneration could suggest a pathomechanism in MSA, by which altered ZIC4 expression could increase neuronal vulnerability.Further analyses of a potential functional interaction of α-synuclein and ZIC4 are currently ongoing.
Explorative analysis of PD-related associations identified by GWAS yielded no significant association in MSA when adjusting for multiple testing.However, for unadjusted P values, five SNPs reached a significance threshold of P< 0.05,which might be interesting to study further.
This study has a major limitation.Typically, a GWAS is conceptualized as a two-stage design with a discovery stage and a replication stage and supposedly achieving "genomewide significance" in the discovery stage.The P values in the replication stage should remain significant after Bonferroni correction.Due to the limited number of autopsyconfirmed MSA cases worldwide, we could not conduct a two-stage procedure, let alone a further independent replication.In view of the aforementioned diagnostic uncertainty in clinical cases, a replication in predominantly clinically diagnosed MSA cases did not seem desirable.
Therefore, we strongly encourage bringing MSA cases to autopsy and conducting a further independent replication study to confirm or refute the hypotheses provided by our study.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Authors
Franziska Hopfner, MD 1, † , Anja K. Tietz, MSc 2 , Viktoria C. Ruf, MD 3 , Owen A. Ross, PhD 4,5 , Shunsuke Koga, MD, PhD 6 , Dennis Dickson, MD, PhD 6 , Adriano Aguzzi, MD, PhD 7 , Johannes Attems, MD, PhD 8 , Thomas Beach, MD, PhD 9 , Allison Beller, MD, PhD 10 , William P. Cheshire, MD, PhD 11 , Vivianna van Deerlin, PhD 12 , Paula Desplats, MD, PhD 13,14 , Günther Deuschl, MD 2 , Charles Duyckaerts, MD 15,16 , David Ellinghaus, PhD 17 , Valentin Evsyukov, PhD 1 , Margaret Ellen Flanagan, MD, PhD 18,19 , Andre Franke, PhD 17 , Matthew P. Frosch, MD, PhD 20,21,22 , Marla Gearing, MD, PhD 23 , Ellen Gelpi, MD, PhD 24,25 , Jay A. van Gerpen, MD, PhD 11 , Bernardino Ghetti, MD, PhD 26 , Jonathan D. Glass, MD, PhD 27 , Lea T. Grinberg, MD, PhD 28,29,30,31 , Glenda Halliday, PhD 32 , Ingo Helbig, MD 33,34,35,36,37 , Matthias Höllerhage, MD 1 , Inge Huitinga, MD, PhD 38,39 , David John Irwin, MD, PhD 40 , Dirk C. Keene, MD, PhD 10 , Gabor G. Kovacs, MD, PhD 24,41,42 , Edward B. Lee, PhD 43 , Johannes Levin, MD 44,45,46   ZIC4 immunohistochemical staining of multiple system atrophy (MSA) patients and control brains.Representative ZIC4 immunohistochemical stainings of different brain regions (antibodies binding specifically to antigens in biological tissues, eg, brain tissue) of a control without neurodegenerative disease (A, D, G) and two MSA patients with striatonigral degeneration (SND) (B, E, H) and mixed subtype (C, F, I), respectively.(A-C) Nuclear and cytoplasmic expression of ZIC4 (brown staining) was detected in a comparable manner in the frontal cortex of healthy controls and patients with MSA.In the cerebellar dentate nucleus (dotted lines in D-I) of healthy controls and patients with SND, a constant subset of neurons stained strongly positive for ZIC4, whereas in patients with olivopontocerebellar atrophy (OPCA) or mixed subtype, only weak staining could be observed, and the number of ZIC4-positive neurons was clearly reduced (D-I, with higher magnification in G-I).(J) Quantification of ZIC4-immunoreactive neurons in relation to the total number of neurons of the dentate nucleus depicted on the entire slide showed significantly reduced fractions of ZIC4-immunoreactive neurons in patients with either mixed subtype (light blue) or OPCA (dark blue) compared with SND or controls without neurodegenerative disease, while no difference was seen between patients with SND and healthy controls.Results from an association analysis with logistic regression including sex and the first two dimensions of principal-component analysis (PCA) as covariates in 648 cases with MSA and 2898 controls.For an OR < 1, the minor allele has a protective effect, whereas an OR > 1 indicates that the minor allele is associated with an increased risk for development of the disease.Only the leading SNP at each locus with a suggestive association between the disease status and a variant is reported.Table S3 lists all suggestive associations.SNP, single-nucleotide polymorphism; CHR, chromosome; dbSNP, database of single-nucleotide polymorphism; BP, base-pair coordinates according to human reference genome GRCh38; MAF, minor allele frequency; OR, odds ratio; CI, confidence interval; IM, imputed; GT, genotyped.