17q21.31 sub-haplotypes underlying H1-associated risk for Parkinson’s disease are associated with LRRC37A/2 expression in astrocytes

Parkinson’s disease (PD) is genetically associated with the H1 haplotype of the MAPT 17q.21.31 locus, although the causal gene and variants underlying this association have not been identified. To better understand the genetic contribution of this region to PD, we fine-mapped the 17q21.31 locus in order to identify novel mechanisms conferring risk for the disease. We identified three novel H1 sub-haplotype blocks across the 17q21.31 locus associated with PD risk. Protective sub-haplotypes were associated with increased LRRC37A/2 copy number and expression in human brain tissue. We found that LRRC37A/2 is a membrane-associated protein that plays a role in cellular migration, chemotaxis and astroglial inflammation. In human substantia nigra, LRRC37A/2 was primarily expressed in astrocytes, interacted directly with soluble α-synuclein, and co-localized with Lewy bodies in PD brain tissue. These data indicate that a novel candidate gene, LRRC37A/2, contributes to the association between the 17q21.31 locus and PD via its interaction with α-synuclein and its effects on astrocytic function and inflammatory response. These data are the first to associate the genetic association at the 17q21.31 locus with PD pathology, and highlight the importance of variation at the 17q21.31 locus in the regulation of multiple genes other than MAPT and KANSL1, as well as its relevance to non-neuronal cell types.

The MAPT 17q21.31 locus lies within a 1.5Mb inversion region of high linkage 8 4 disequilibrium (LD), conferring two distinct haplotypes; H1, which has a frequency of ~0.8 in 8 5 European ancestry populations, and the less common, inverted H2 haplotype (frequency ~0.2), 8 6 which is absent or lower frequency in East and South Asian populations (frequency 0 -0.09) (Fig   8  7 1A). The major haplotype, H1, has been genetically associated with increased risk for multiple 8 8 neurodegenerative disorders, including APOE 4-negative Alzheimer's disease (AD) 1 , MAPT. However, a causal role for astrocytes in PD pathogenesis has been recently proposed [22][23][24][25] . Indeed, many PD-associated genes are expressed in astrocytes and have known functional roles 1 1 0 in astrocyte biology 24 . Furthermore, α -synuclein-positive aggregates have been identified in the  Although PD, like PSP and CBD is associated with the H1 haplotype, the odds ratio for 1 1 5 risk is substantially lower (1.5 vs 4.5) in PD [2][3][4][5][6][7][8][9][10] . In contrast to PSP and CBD, PD is not generally 1 1 6 associated with the accumulation of tau. Together these observations suggest that the PD genetic 1 1 7 association with the 17q21.31 locus may be driven by genes other than MAPT. Here, we report 1 1 8 three sub-haplotype blocks associated with PD risk within the 17q21.31 H1 haplotype clade, 1 1 9 with both protective and risk-associated sub-haplotypes within each block. We show that 1 2 0 protective sub-haplotypes are associated with increased expression and copy number of LRRC37A/2, which we demonstrate is an astrocyte-enriched membrane-associated protein with a synuclein and Lewy bodies in human substantia nigra. These findings link the genetic association 1 2 4 at the 17q21.31 locus with PD pathology, and support the hypothesis of astroglial dysfunction as 1 2 5 a key contributing factor to PD disease pathogenesis. Figure S1B-C, F). Both SNPs were associated with odds ratios (ORs) ~0.8 (95% CI ± 0.1; Figure   1 3 7 S1B-C, Table S1), consistent with previously reported effect sizes 6,8 . Due to a smaller cohort size 1 3 8 and consequent lack of power, the association with 17q21.31 was less prominent in Stage 2 data 1 3 9 (Figure S1D-E, Table S1), and the top SNP did not tag the H2 haplotype. However, meta- analysis of both cohorts confirmed a significant association between PD risk and the 17q21.31  0.76-0.89)) and the H1 haplotype therefore associated with increased risk. As the major H1 haplotype was associated with increased risk for PD, we repeated the 1 4 4 association analysis in H1 homozygotes alone in order to identify variants of H1 that may confer 1 4 5 additional risk for PD ( Figure S1G-J, Table S1). While association across the 17q21.31 locus 1 4 6 was weaker in H1 homozygotes compared to the full data set, we observed a distinct signal 1 4 7 spanning MAPT and KANSL1 in Stage 1 and Stage 2 analyses ( Figure S1G-H). The Stage 1 top a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1  1  7  q  2  1  .  3  1  s  u  b  -h  a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1  1  7  q  2  1  .  3  1  s  u  b  -h  a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1  1  7  q  2  1  .  3  1  s  u  b  -h  a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1  1  7  q  2  1  .  3  1  s  u  b  -h  a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1   1  1 As PD sub-haplotypes converge on the expression and/or copy number of LRRC37A/2, we explored the likely function of this gene. We carried out RNA-seq analysis in HEK293T cells overexpressing LRRC37A/2 in order to mimic increased copy number and to elucidate a potential 2 4 2 function for this gene. The number of significantly differentially expressed protein-coding genes 2 4 3 (fold change ± 1.5, adjusted p<0.05) in the context of LRRC37A/2 overexpression was minimal 2 4 4 (28 upregulated, 21 downregulated), suggesting that LRRC37A/2 is unlikely to play a major 2 4 5 regulatory role. In order to confirm that we were not observing spurious changes in gene  3  1  s  u  b  -h  a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1  1  7  q  2  1  .  3  1  s  u  b  -h  a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1 1 3 In order to assess whether LRRC37A/2 was expressed in mature astrocytes in human 2 8 4 brain tissue, we carried out multiplex immunofluorescence staining in human substantia nigra 2 8 5 from PD, PSP and aged controls ( Figure 4D). We found that in all cases, LRRC37A/2 co-2 8 6 localized with the astrocyte marker GFAP, but not the microglia marker IBA1 ( Figure 4D, 2 8 7 Figure S6H). In contrast, α -synuclein positivity was observed only in PD substantia nigra, and 2 8 8 hyperphosphorylated tau (labeled with AT8) was present only in PSP brain ( Figure 4D). Interestingly, in regions with Lewy body pathology there was reduced staining intensity of  However, there was no association between tau AT8 positivity and LRRC37A/2 expression in PSP substantia nigra ( Figure 4D), indicating that LRR37A/2 accumulation is specific to PD pathology. To validate the association of LRRC37A/2 with α -synuclein, we performed co- immunoprecipitation from control, PSP and PD substantia nigra tissue ( Figure 4E). We found indicating that LRRC37A/2 and α -synuclein likely form a normal complex in human brain that 2 9 7 becomes disordered in the context of PD pathogenesis. These data are not only the first to 2 9 8 identify a role for LRRC37A/2 in astrocytes, but are the first to link the genetic association at the 2 9 9 17q21.31 locus with PD pathology. By constructing discrete sub-haplotype blocks across the 17q21.31 H1 locus, we have identified multiple novel H1 sub-haplotypes associated with variable levels of PD risk. The MAPT gene, encoding the microtubule associated protein tau, is central to this locus and is likely a  p  l  o  t  y  p  e  s  u  n  d  e  r  l  y  i  n  g  H  1  -a  s  s  o  c  i  a  t  e  d  r  i  s  k  f  o  r  P  D  B  o  w  l  e  s  e  t  a  l  2  0  2  1   1  4   the causal gene for other neurodegenerative disorders genetically associated with the H1   3  0  6 haplotype, such as PSP and CBD 2,3 , given that they are characterized neuropathologically by tau has therefore been unclear, although a recent study proposed variants in KANSL1 as underlying this association by altering mitophagy 32 . In contrast, we did not find any effect of our PD- associated H1 sub-haplotypes on either KANSL1 expression or copy number, but did observe a these data do not rule out that KANSL1 variants may alter disease risk independently of 3 1 5 LRRC37A/2 expression between the major haplotype clades and H1 sub-haplotypes. We found that protective H1 sub-haplotypes were associated with increased expression of LRRC37A/2. This is consistent with increased expression of these genes in the protective H2 haplotype, suggesting that there is likely a shared mechanism of protection from PD between H2 and specific sub-haplotypes of H1. Furthermore, analysis of CNVs in the 3' distal end of the 17q21.31 locus suggested that protective sub-haplotypes were tagging structural variants defined by increased gamma region 20 and LRRC37A/2 copy number, which likely underlies the increased expression of these genes. LRRC37A is a core duplicon on chromosome 17 33 and is 3 2 3 present at the inversion breakpoint of the 17q21.31 locus; it has been hypothesized that its 3 2 4 propensity for CNVs is responsible for the evolutionary toggling of this region that resulted in the distinct H1 and H2 haplotypes 34 . Due to the complex structural variation surrounding 3 2 6 LRRC37A and the presence of its paralog LRRC37A2, it is challenging to genotype or sequence 3 2 7 this region of the genome. As such, our analyses have not been able to separate the contribution  of each gene and have considered them together. As a consequence of the CNVs in this region, 3 2 9 genotyping data across LRRC37A and LRRC37A2 is of low confidence and quality, and as such 3 3 0 is excluded from GWAS analyses, as is visible in any association plot. Therefore, the association between LRRC37A/2 variants and any disease or phenotype has never been tested, and it is likely that additional variation within LRRC37A/2 itself is contributing to its altered expression and 3 3 3 function that may also impact PD risk. Alternatively, differential epigenetic modifications or 3 3 4 chromatin accessibility and looping may also be contributing to variable LRRC37A/2 expression between sub-haplotypes. Unfortunately due to the relatively low frequency of different H1 sub- haplotypes, we do not yet have the brain tissues or iPSC lines available to test this possibility. Little is known about the function of LRRC37A/2, although increased copy number has 3 3 8 been associated with an increased antibody response to an Anthrax vaccine 35 , and overexpression 3 3 9 in HeLa cells appeared to promote the formation of filopodia 33 , indicating that LRRC37A/2 may that several immune phenotypes are significantly associated with H1/H2 haplotypes across the cells, we observe enrichment of pathways consistent with these data; we found that increased 3 4 6 LRRC37A/2 expression upregulated cellular migration and chemotaxis pathways, which are both 3 4 7 essential mechanisms involved in wound healing and inflammation. Within these pathways we 3 4 8 observe increased expression of pro-inflammatory cytokines IL17 and IL32, as well as the 3 4 9 chemoattractant IL16, each of which are involved in the astrocytic inflammatory response 30,31 .
Neuroinflammation of the substantia nigra is considered a characteristic feature of PD in addition  to neuronal loss 36,37 , and many genes associated with PD, such as GBA, LRRK2 and PINK1 are 3 5 2 thought to have a role in the inflammatory response in astrocytes 24,38 . Furthermore, the most 3 5 3 significantly enriched pathway in our analysis, Neuroactive-ligand receptor interaction, is also 3 5 4 involved in the inflammatory response and has previously been associated with PD; this pathway 3 5 5 was significantly enriched in a functional assessment of PD GWAS signals 39 , and is targeted by 3 5 6 microRNAs that are upregulated in a Drosophila model of PD 40 . We also observe upregulation 3 5 7 of TGFA, the infusion of which into the forebrain of a rat model of PD increased the proliferation nigra 41 , indicating that this may be a protective growth factor against neuronal loss in PD.
LRRC37A/2 overexpression in HEK293T cells was therefore able to recapitulate pathways associated with PD, despite being a cell type with limited relevance to PD pathogenesis. As these expression data were indicative of pathways relevant to astrocyte biology, and 3 6 3 we found that LRRC37A/2-associated gene expression changes were apparent in iPSC-astrocytes 3 6 4 but not iPSC-neurons, we hypothesized that this was likely the most relevant cell type for LRRC37A/2 expression. Indeed, in human substantia nigra tissue we observe localization of LRRC37A/2 specifically in astrocytes. The contribution of astrocytic dysfunction to PD 3 6 7 pathogenesis has gained attention in recent years, and is hypothesized to be a causal mechanism 3 6 8 for the initiation and progression of PD [22][23][24][25] . Many genes associated with PD risk are expressed 3 6 9 in astrocytes, the functions of which converge on the inflammatory response, lipid handling, 3 7 0 mitochondrial health and lysosomal function 24 . Our finding that LRRC37A/2 is expressed in astrocytes and plays a role in the inflammatory response is therefore consistent with known pathogenic mechanisms of PD.
The role of astroglial inflammation in PD is unclear; it has been reported as being both 3 7 4 absent and severe in PD substantia nigra 22 . As a further complication, in vitro studies of human 3 7 5 astrocyte cultures indicate that α -synuclein induces the release of pro-inflammatory 3 7 6 cytokines 22,25 , but these cells also release protective molecules such as GDNF in response to 3 7 7 dopaminergic neuronal damage, and such trophic support may benefit neuronal survival 22 . In 3 7 8 addition, the substantia nigra is considered to be particularly susceptible in PD as dopaminergic 3 7 9 neurons in this region are surrounded by the lowest proportion of astrocytes in the brain 42 .
Whether an inflammatory response in this context would be protective or exacerbate neuronal death is therefore unknown. As our data suggest increased LRRC37A/2 expression is protective and associated with increased expression of pro-inflammatory cytokines, astroglial inflammation 3 8 3 in response to α -synuclein may therefore be protective. However, our use of HEK293T cells 3 8 4 limits our interpretation of these data, and further investigation of LRRC37A/2 function in 3 8 5 astrocytes is required. Interestingly, we observe an interaction between LRRC37A/2 and soluble α -synuclein, as 3 8 7 well as co-localization of LRRC37A/2 with Lewy bodies in PD substantia nigra. The function 3 8 8 and mechanism of this interaction is untested, although it is likely that a complex is formed in 3 8 9 astrocytes and propagated to neurons. iPSC-astrocytes have been reported as expressing low synuclein released from neuronal axon terminals is taken up by astrocytes 28 , which can be further 3 9 2 transferred to neurons 23,29 . This raises the possibility that LRRC37A/2 may influence α -synuclein release, aggregation and/or propagation. In conclusion, we have identified novel sub-haplotype variants of the 17q21.31 H1 clade 3 9 5 significantly associated with protection against PD. While the genetic association across this locus is typically ascribed to MAPT or KANSL1, we find evidence for the involvement of a novel 3 9 7 gene, LRRC37A/2, in PD risk. We propose that in a similar mechanism to other PD-associated 3 9 8 genes, LRRC37A/2 is expressed in astrocytes and plays a role in the regulation of astroglial 3 9 9 inflammation, specifically in the release of pro-inflammatory cytokines, chemotaxis and cellular    Pre-imputation QC Each dataset was obtained with different QC filters already applied, and so were all subsequently passed through the same, more stringent QC pipeline to ensure consistency. Plink  screened for strand mismatches. Filtered chromosome 17 data from each cohort was submitted individually to the 4 2 7 Michigan imputation server 46 (https://imputationserver.sph.umich.edu) and imputed against the were removed, and remaining SNPs were filtered for a 99% call rate. Genotyping call rates for merged, and finally filtered once more with a SNP call rate of 99%. Prior to analysis, variants 4 3 3 were filtered to exclude SNPs with a MAF < 0.01.

3 4
Single SNP association analyses 4 3 5 Logistic regression association analyses using an additive model were carried out in SNP www.goldenhelix.com). As all potential covariate information was not available, the model was 4 3 8 corrected using the first 10 principal components as calculated by SVS8. Associations were 4 3 9 initially carried out on the entire cohort in order to confirm the 17q21.31 H1/H2 haplotype the association analysis was repeated with the same parameters.

2
Meta-analysis of SNP effects across multiple datasets was carried out using the R Haenszel) approaches. Calculation and visualization of linkage disequilibrium (LD) over large 4 4 5 genomic ranges was carried out in SVS8 using both r2 and D'. Inspection of LD between 4 4 6 individual SNPs of interest was carried out using Haploview 48 .

7
Haplotype block construction and association 4 4 8 Haplotype blocks were constructed in SVS8 using the D' measure of LD. Blocks were 4 4 9 defined using guidelines as described by Gabriel et al (2002) 49 . Each block contained a  haplotypes with a frequency < 0.01 were excluded from further analysis. Case-control 4 5 4 association analyses were carried out per block using a logistic regression model. Odds ratios 4 5 5 and associated Fisher's exact p-values were calculated for each sub-haplotype within each block 4 5 6 using the R package epitools 50 .

5 7
Human brain expression analysis 4 5 8 Publicly available RNA-seq expression data from human postmortem dorsolateral prefrontal (PFC) and temporal (TCX) cortices (Table S3) and associated genotype data were   SNP rs8070723. For sub-haplotype analysis, blocks previously defined in the PD analysis were 4 6 5 applied to the genotype data and haplotypes were estimated in the same manner. Statistical to the adjusted and combined z-scores. Human genomic DNA and accompanying genotype data was kindly provided by Drs. Research Center (ADRC ; Table S3). Sub-haplotypes were called from these genotype data in the 4 7 7 same manner as described above. To examine copy number variation in the 17q21.31 locus, 4 7 8 digital PCR was carried out using the ThermoFisher QuantStudio 3D digital PCR chip system. Taqman dPCR probes for loci within the alpha, beta and gamma CNV regions 20 , as well as  Schizophrenia study 53 , and the UCI ADRC (Table S6). The Icahn School of Medicine at Mount 4 8 5 Sinai IRB reviewed the relevant operating protocols as well as this specific study and determined 4 8 6 it was exempt from approval. 4 8 7 Cell culture 4 8 8 Unless otherwise specified, all cell culture materials were obtained from ThermoFisher Scientific. Human embryonic kidney cells (HEK293T) were cultured in Dulbecco's Modified Eagle Medium/F-12 with HEPES, supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. Cells were passaged every 3-4 days using Trypsin-EDTA (0.25%). For  For qRTPCR and protein biochemistry experiments, iPSC lines (Table S6) were 4 9 7 maintained in complete StemFlex media supplemented with 1% penicillin/streptomycin on 4 9 8 Matrigel (BD biosciences), and were differentiated to neural progenitor cells (NPCs) as 4 9 9 previously described 54 . Forebrain neuron-enriched cultures and astrocyte cultures were 5 0 0 differentiated from NPCs as previously described 54,55 . Neuronal and astrocytic identity was (Abcam), Tuj1 (Cell Signaling Technologies), S100β (Sigma Aldrich) and EAAT1 (Abcam).

0 3
Genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) and to confirm 17q21.31 haplotype. RNA was extracted from HEK293T cells, iPSC-derived neurons, astrocytes and human 5 0 8 brain tissue using the RNeasy Mini kit (Qiagen) and reverse transcribed using the High-Capacity RNA-to-cDNA kit (ThermoFisher Scientific). Gene expression was measured by commercially available Taqman qRTPCR assays.

2 7
Protein biochemistry 5  Membrane and cytosolic proteins were isolated from HEK293T cells, iPSC-derived 5 2 9 neurons and iPSC-derived astrocytes using the MEM-PER Plus Membrane Protein Extraction Kit (ThermoFisher Scientific), and protein concentrations were determined by bicinchoninic acid 5 3 1 (BCA) assay (ThermoFisher Scientific). For western blotting, protein fractions were subject to 5 3 2 SDS-PAGE electrophoresis through BOLT Bis-Tris gels (ThermoFisher Scientific) and were anti-pan-Cadherin antibody (Cell Signaling Technology), and cytosolic fractions were confirmed Restore plus western blot stripping buffer and re-probed with an anti-LRRC37A antibody 5 3 7 (ThermoFisher Scientific).

3 8
OPAL multiplex labelling 5 3 9 Formalin fixed paraffin embedded substantia nigra sections from human controls (N=4), with xylene and rehydrated with a graded series of ethanol concentrations. For epitope retrieval, 5 4 7 slides were microwaved in AR buffer for 45s at 100% power, followed by an additional 15 5 4 8 minutes at 20% power. After cooling, slides were blocked for 10 minutes in blocking buffer then 5 4 9 incubated with the first primary antibody at room temperature for 30 minutes. Slides were rinsed 5 5 0 three times in TBS-T, then incubated with the secondary polymer HRP for 1 hour at room  temperature. After additional washes, the first Opal fluorophore was incubated with the slides for 5 5 2 10 minutes at room temperature, followed by further washes in TBS-T. This process was 5 5 3 repeated from the microwave treatment step for each additional primary antibody, followed by 5 5 4 one final repetition of the microwave treatment to strip the primary-secondary antibody complex 5 5 5 from the tissue. Once all primary antibodies had been introduced, slides were counterstained 5 5 6 with DAPI for 5 minutes at room temperature, washed with TBS-T and coverslips were mounted 5 5 7 using ProLong Diamond Antifade mounting reagent (ThermoFisher Scientific). Multispectral imaging was carried out using the Vectra Quantitative Pathology Imaging system, applying 5 5 9 quantitative unmixing of fluorophores and removal of tissue autofluorescence. Images were 5 6 0 visualized using the HALO image analysis platform (Indica Labs).

6 1
Co-Immunoprecipitation 5 6 2 Frozen substantia nigra tissue was selected from the same Control (N=3), PSP (N=3) and PD (N=3) cases used for OPAL multiplex immunofluorescence. Protein lysates were generated 5 6 4 using cell lysis buffer (NEB) and brief sonication on ice, followed by centrifugation to pellet 5 6 5 insoluble material. Co-immunoprecipitation was carried out using the Dynabeads Protein G immunoprecipitation kit (ThermoFisher Scientific), with an anti-α-synuclein antibody (Abcam) 5 6 7 as bait. Proteins bound to beads were eluted and assayed by western blot (as described above) 5 6 8 and probed with an anti-LRRC37A antibody (ThermoFisher Scientific). Whole protein lysate 5 6 9 and IgG only controls were run on the same blots.  All aligned read counts and FASTQ files for LRRC37A-overexpressing HEK293T cells 5 7 2 will be deposited to the Gene Expression Omnibus once the manuscript is accepted for 5 7 3 publication. This work was supported by funding from the BrightFocus Foundation (KRB), 5 7 6 Association for Frontotemporal Degeneration (KRB) and CurePSP (KRB). The recruitment and 5 7 7 clinical characterization of research participants at Washington University were supported by Charitable Organization (CMK), NIH AG046374 (CMK). The McGill cohort was supported by   Health and Human Services: project numbers 1ZIA-NS003154, Z01-AG000949-02 and Z01-   rectangle between H1 and H2 to aid visualization of altered gene position between haplotypes.  genome wide suggestive significance p-value of 1x10 -5 .   Figure 2. PD-associated sub-haplotypes are associated with LRRC37A/2 expression and 6 1 7 copy number. 6 1 8 A. LRRC37A, B. LRRC37A2 and C. MAPT in human brain tissue, measured 6 1 9 by RNA-seq across three different cohorts, split by sub-haplotype in blocks H1.1, H1.2 and  to right) beta, alpha, LRRC37A and gamma.   All statistical comparisons are against the most common sub-haplotype. ns = not significant, *p 6 2 7

1
A. Enriched GO terms for significantly differentially expressed genes following LRRC37A/2 6 3 2 overexpression in HEK293T cells. Paler node colors indicate less significant enrichment p- 6 3 3 values, and edge thickness indicates the proportion of shared genes between GO terms. B. -log 10 adjusted p-values for top 12 functionally enriched gene ontology terms for significantly 6 3 5 differentially expressed genes in LRRC37A/2-overexpressing cells.  astrocytes. N=3 in duplicate. ns = not significant, *p < 0.05. HSP90 antibody, and membrane fractions were confirmed by labelling with an anti-Pan- 6 4 7 Cadherin antibody, N=6. in substantia nigra from control (C), PSP and PD brain, N=3. Whole protein lysates and IgG only    relative to the most common haplotype) and B. related ORs and 95% confidence intervals in 6 8 2 Stage 1 and Stage 2 data (black), random effects meta-analysis (blue) and fixed effects meta- 6 8 3 analysis (red). C-D. Block H1.2 C. Sub-haplotype SNP genotypes (green squares denote minor allele variation 6 8 5 relative to the most common haplotype) and D. related ORs and 95% confidence intervals in 6 8 6 Stage 1 and Stage 2 data (black), random effects meta-analysis (blue) and fixed effects meta- analysis (red). 6 8 8