dataesr/scientific-paragraphs-categorization huggingface dataset:
We therefore looked for potential genetic interactions with established PD mechanisms. Following RNAi-mediated knockdown, 15 of the genes modulated mitochondrial dynamics in human neuronal cultures and four candidates enhanced α-synucleininduced neurodegeneration in Drosophila. Based on complementary analyses in independent human datasets, five functionally validated genes-GPATCH2L, UHRF1BP1L, PTPRH, ARSB, and VPS13C-also showed evidence consistent with genetic replication. Conclusions: By integrating human genetic and functional evidence, we identify several PD susceptibility gene candidates for further investigation. Our approach highlights a powerful experimental strategy with broad applicability for future studies of disorders with complex genetic etiologies.
We further couple the human genetic studies with functional screening in mammalian cell culture and invertebrate animal models, successfully identifying those candidate genes showing interactions with established PD mechanisms, including mitochondrial dynamics and α-synuclein-mediated neurodegeneration. Although no sufficiently powered exome dataset was available for definitive replication, human genetic validation was undertaken in several independent datasets. Our integrated approach identifies five strong candidate PD susceptibility genes worthy of further investigation, and exemplifies a powerful strategy with potential broad applicability to the follow-up of future rare variant studies in PD and other neurologic disorders with complex genetic etiologies.
Discovery of recessive LoF variants from PD exomes A total of 920,896 variants (93. 2% single nucleotide variants and 6. 8% insertions and deletions) were called in a WES dataset of 1651 participants, including 1148 young-onset PD cases (average age of onset, 40. 6 years; range, 5-56 years) and 503 control participants with European ancestry. As our cohort has an average age at onset of less than 45 years, we focused our search on homozygous and putative compound heterozygous variants, consistent with a recessive inheritance model. Although most PD cases were prescreened for mutations in established PD genes, we identified two participants with homozygous exonic variants in parkin and PINK1 (Additional file 1: Table  S1 ). In order to identify novel PD gene candidates, we focused on variants that are rare in control populations.
Approximately 17% of the variants are absent in public allele frequency databases (1000 Genomes Project (1000G), Exome Sequencing Project v. 6500 (ESP6500), or Exome Aggregation Consortium (ExAC)) and therefore implicated to be novel. Except in the case of ARSB, the other 26 genes harbor LoF variants in only a single case, consistent with the hypothesis that novel recessive PD alleles may consist of many rare, "private" mutations. Four PD cases in our cohort were identified with a LoF variant in the ARSB gene, in which mutations have previously been linked with the recessive lysosomal storage disorder, MPS VI (also called Maroteaux-Lamy syndrome). All four individual cases, along with one control participant, were homozygous for a variant (rs138279020) predicted to disrupt splicing. Although this variant is neither reported in ExAC nor was frequency information available from dbSNP, the MAF was 0.
065 in our cohort (MAF CASES = 0. 073, MAF CONTROLS = 0. 052, p = 0. 054). Although relatively frequent in our control dataset (MAF > 1%), we have retained it among our results, based on three considerations. First, information was not present in dbSNP, ExAC, or ESP6500, which was the basis for applying this frequency filter in all other cases. Second, at least one of the homozygous individuals had clinical manifestations consistent with MPS VI, supporting potential pathogenicity of this allele (see "Discussion"). Lastly, as detailed below, our functional Fig. 1  Flowchart explaining multiple filtering steps to select LoF variants with assumed recessive inheritance pattern. Functional annotation was performed with transcripts of RefSeq and UCSC databases. MAF annotations were based on 1000 Genomes project, Exome variant Server, and the ExAC database.
Seventeen genes harbored homozygous variants causing stopgain or loss and one gene contained a homozygous splicing variant. For the putative compound heterozygous genes, six genes were selected based on the presence of two LoF variants, and three genes were based on the presence of one LoF variant and one missense variant (predicted to belong to the 1% most harmful variants of the genome)  studies identify links between manipulation of ARSB and cellular/organismal phenotypes consistent with a potential role in PD. Of note, while the analyses of the IPDGC WES dataset and subsequent work described here were in progress, an independent family-based sequencing study identified VPS13C as a cause of autosomal recessive parkinsonism  [21]. Although the single IPDGC subject with compound heterozygous VPS13C LoF alleles was published as a replicate case in that work, we retained it among the 27 candidates described here, since it was independently carried forward for all analyses detailed below.
Human genes harboring homozygous LoF variants-especially those observed recurrently in large population-based datasets-potentially identify genes that are dispensable for fetal and subsequent child development. Given the limited human phenotypic information available, we further investigated the potential tolerability for the implicated genes using a cross-species approach, performing systematic LoF analysis in the nematode, C. elegans. Out of the 27 candidate genes identified in our WES analysis, ten were well conserved in the C. elegans genome and nine had readily available RNA-interference (RNAi) reagents for LoF screening (see "Methods"). Each gene was targeted for knockdown using RNAi and we assessed for developmental lethality and survival. The results of these studies, along with other LoF data from public databases, are available in Additional file 1: Table  S3.
Genetic replication of candidate PD genes from WES We next evaluated our 27 gene candidates in additional available genetic datasets including: (1) an independent exome sequencing dataset from the Parkinson Progression Markers Initiative (PPMI) project  [51] ; (2) a wholegenome sequencing dataset including PD index cases of a Dutch genetic isolate belonging to the Genetic Research in Isolated Population (GRIP) program  [52] ; (3) an independent NeuroX exome array dataset  [7, 53] ; and (4) a large PD GWAS dataset  [53]. Within the PPMI exome dataset, including 462 PD cases and 183 controls, evidence supporting replication was discovered for two genes, in which we identified the identical variants from the IPDGC discovery exome dataset (Additional file 1: Table  S9 ). A PD case from PPMI carries the same homozygous stopgain variant (p.
The second gene harboring an identical LoF variant is FAM83A. The p. G86X variant in FAM83A, detected within an IPDGC participant with sporadic PD diagnosed at the age of 28 years, was also observed in a single sporadic PD case from PPMI with an age of onset of 62 years. These FAM83A carriers presented with similar symptoms, including bradykinesa, rigidity, and resting tremor. In both datasets, the p. G86X allele is predicted to be in trans with another variant: p. R347X or p. V137G in PPMI and IPDGC, respectively. The second genetic independent dataset that was investigated included a whole-genome sequencing study (39 PD index cases and 19 controls) of a genetic GRIP isolate from the Netherlands, focusing on variants within our candidate genes that were present in at least two PD index cases and absent in controls.
We identified a heterozygous missense variant (NM_001127444:c. 1176G > T:p. L392F) in CD36 for three PD index cases. Although not consistent with a recessive inheritance model, this variant has not been observed in the 60,706 unrelated individuals of the ExAC database, suggesting potential enrichment in PD cases. These heterozygote variant carriers have a substantial higher age of onset (range, 61-79 years) in comparison to the PD patient (age of onset, 38 years) with the putative compound heterozygous variant within the discovery WES dataset. This observation supports an additive model of pathogenicity, implying more severe disease onset when two alleles are affected. Further, CD36 (p. L392F) is predicted to represent the top 1% most harmful variants within the genome (CADD score = 23. 3).
In the IPDGC discovery dataset, the discovered compound heterozygous variants, p. Q74X and p. P412S (Table  1 ), are also predicted to be strongly deleterious (CADD scores of 26. 5 and 25. 9, respectively). We next interrogated the independent IPDGC NeuroX dataset, including genotypes from 6801 individuals with PD and 5970 neurologically healthy controls. NeuroX is a genotyping array that includes pre-selected exonic variants and is therefore not suitable to search for the identical recessive LoF variants implicated by our WES analyses. Instead, we examined the burden of multiple variant classes within the 27 candidate genes, following the same variant categories as for the original IPDGC WES dataset (Additional file 1: Table  S10 ). When only considering variants predicted to be deleterious (CADD > 20), an association is detected for UHRF1BP1L with PD risk (p = 0.
005). This gene also shows an association with PD in the IPDGC WES dataset when performing a similar burden analysis considering missense variants (see above, p = 0. 016). Using the NeuroX dataset, we additionally confirmed the enrichment of rare PTPRH variants in participants with PD (WES: p = 0. 034, NeuroX: p = 0. 045). Furthermore, VPS13C and ARSB show significant associations to PD when considering the joint effect of all variants, both common and rare (Additional file 1: Table  S10 ). Leveraging available IPDGC GWAS data (13,708 cases/ 95,282 controls), we next assessed for potential common variant association signals (p < 1 × 10 -4 ) using a 1-Mb genomic window centered on each of the 27 candidate genes. Three loci (VPS13C, PCDHA9, and TCHHL1) showed evidence consistent with an association peak (Additional file 2: Figure  S6 ).
We therefore coupled our WES analyses with functional studies in both mammalian cells and experimental animal models, including Drosophila and C. elegans, in order to prioritize genes for future study. Our results highlight 15 out of the 27 gene candidates that interact with mitochondrial dynamics and five loci that enhance αsynuclein-mediated neurodegeneration. As discussed below, while these results highlight a promising subset of genes with potential links to PD-relevant mechanisms, we cannot exclude contributions from other implicated genes/variants. All of these data, including promising variants from the human genetic analyses and results of functional studies, will be a valuable resource for future investigations of PD genomics. Analyses of several other WES and complementary large-scale, genetic datasets provide additional evidence supporting replication for 7 out of 27 genes.
Mitochondrial mechanisms have been strongly implicated in PD risk and pathogenesis  [28, 30]. Following shRNA-mediated knockdown, 15 candidate recessive loci identified in our WES dataset showed effects on mitochondrial morphology and Parkin translocation to mitochondria in cell culture. We focus our initial discussion on three genes, GPATCH2L, UHRF1BP1L, and VPS13C, for which we discovered additional genetic evidence consistent with replication in independent cohorts. In the IPDGC cohort, a single PD case was identified with a homozygous stopgain variant (p. R362X) in GPATCH2L and a second individual with the identical, rare genotype was discovered in PPMI. This variant is reported with a low frequency of 0. 003% in ExAC. Although minimal clinical or demographic information is available within ExAC, this finding is compatible with population prevalence estimates for PD  [20].
Another promising gene, UHRF1BP1L, harbored a homozygous stopgain variant (p. K1376X) in a single IPDGC case. This is a novel variant, based on its absence from the ExAC cohort. Additional support for UHRF1BP1L as a bona fide PD locus comes from complementary analyses in both the IPDGC WES and Neu-roX datasets, documenting a burden of rare missense and LoF variants in association with disease risk. In the UKBEC, UHRF1BP1L was associated with a substantia nigra co-expression module including both SNCA and PINK1, reinforcing potential links with established PD genetic mechanisms. Indeed, UHRF1BP1L knockdown cause sharply reduced mitochondrial numbers and altered morphology. Interestingly, UHRF1BP1L encodes a protein bearing an amino terminal homologous to yeast VPS13 and studies in cell culture provide support for a role in retrograde transport from the endosome to the trans-Golgi network  [60].
Thus, VPS13C and associated endosomal sorting pathways (including VPS35) may represent a point of convergence for mitochondrial and α-synuclein-mediated PD mechanisms. Consistent with this, evidence for the impact of α-synuclein toxicity on mitochondria has recently emerged  [28] , including from studies in mammals  [64]. In the IPDGC WES cohort, a single PD case was discovered with compound heterozygous LoF variants in PTPRH (p. Q887X and p. E200X). Both variants were also observed at low frequencies in the ExAC database (0. 039% and 0. 003%, respectively); however, they each met our prespecified threshold of < 1% based on the population prevalence of PD. Encoding a receptor protein tyrosine phosphatase, PTPRH (also called SAP-1) was first discovered for its potential association with gastrointestinal cancers  [65, 66]  and remains poorly studied in the nervous system context.
Besides our discovery of homozygous LoF in PTPRH, further analyses of the IPDGC WES dataset, and the substantially larger, independent NeuroX cohort, implicate a burden of rare variants at this locus in association with PD susceptibility. α-synuclein-induced neurodegeneration was also enhanced by knockdown of CG32191, a Drosophila homolog of ARSB. RNAi transgenic lines targeting three other conserved fly ARSB homologs showed consistent interactions with α-synuclein (Additional file 1: Table  S7  and Table  S8 ). In the IPDGC cohort, we discovered four PD cases homozygous for a variant predicted to disrupt splicing of exons 1 and 2 in ARSB. Although the identified variant has not previously been documented in ExAC, we identified a single IPDGC control homozygote. Additional evidence supporting association of the ARSB gene with PD susceptibility comes from burden analysis in the independent NeuroX cohort.
Importantly, Maroteaux-Lamy can be characterized by minimal or even absent clinical signs, leading to incidental discovery or diagnosis in adulthood, and such mild phenotypes have been suggested to accompany partial LoF with preserved low-level ARSB enzymatic activity  [70, 73, 74]. Similar genotype-phenotype relationships have been documented for other lysosomal-storage disorders, including Gaucher's disease, which has established links with PD risk  [75, 76]. While a full accounting is outside the scope of this study, at least one of the three IPDGC cases for which records were available revealed clinical features potentially overlapping with MPS VI. The strengths of our study include the largest PD WES discovery dataset assembled to date, complementary analyses in independent available cohorts to establish replication, and integration of promising human genetic findings with multiple functional assays relevant to PD mechanisms.
Improved confidence for the discovery of PD causal variants will likely come from PD WES cohorts with significantly enhanced sample sizes, as well as increased numbers of adult controls, including those with careful neurological assessments to exclude mild PD symptoms. Indeed, most of the variants implicated by the IPDGC WES cohort were represented at low frequencies within the largest available public database, ExAC  [77, 78] ; however, we have no information about potential PD manifestations in such individuals or even participant age. Since no single cellular or animal experimental model is expected to universally recapitulate all potential facets of disease biology, we note that the employed functional screening assays are potentially liable to false-negative or false-positive findings.
Conclusions We have identified five excellent PD gene candidates (GPATCH2L, UHRF1BP1L, PTPRH, ARSB, and VPS13C), harboring homozygous or compound heterozygous LoF variants in PD exomes, demonstrating functional interactions with mitochondrial and/or α-synuclein-mediated mechanisms, and supported by evidence of replication in independent human datasets. The recent report  [21]  of additional PD families segregating LoF mutations in VPS13C along with other experiments supporting a role in mitochondrial mechanisms significantly strengthens the evidence in support of this gene in PD and validates our overall approach. These loci are well-suited for future efforts directed at human genetic replication and in-depth functional dissection. We also make available results, including findings from human genetic analyses and functional studies in most cases, on 22 other promising loci.
Variant identification in IPDGC WES dataset Considering the worldwide prevalence of 0. 041% for PD in the age range of 40-49 years  [20] , we selected rare variants with a MAF < 1% (corresponding to a homozygous frequency of 0. 01%) in the European population. Because the specified 0. 041% of the population with young-onset Parkinson's disease (YOPD) is not caused by one shared genetic factor, we expect a homozygous frequency of 0. 01% to be an adequate cutoff, which would be able to determine variants present in approximately 25% of the YOPD population. As a comparison to the most common genetic cause of YOPD, parkin  [85] , the most frequent mutation is an exon 3 deletion, which has been identified in 16. 4% of YOPD patients  [86]. Using ANNOVAR  [83] , all variants were annotated with MAF information of ESP6500si (European American population)  [87] , 1000 Genomes Project (European population of April 2012 version)  [88] , and the ExAC browser (non-Finish European population)  [77, 78].
When no public allele frequency was available for homozygous variants, the in-house control dataset of 503 individuals was used as a reference for the general population. Homozygous variants were excluded when being common (>1%) in controls or having a relative higher frequency in controls than in cases. KGGseq  [89]  was used to count the number of homozygous variants for the cases versus controls. In addition to the population allele frequency filters, we only selected SNVs and indels affecting the position of the stop codon or located at a splice site (within 2 bp of splicing junction), which are variants expected to result in a loss of gene function. As the aim of this study was to validate our approach to identify high promising PD candidate genes, rather than discovering all putative PD genes present within our WES dataset, we set a conservative selection criteria by only including frameshifts that caused an immediate stopcodon at the position of the indel.
Splice-site variants were only considered when being adjacently located to an exon that is coding for amino acids. As a final filter for the homozygous variants, we manually excluded variants that failed GATKVQSR and hard filtering. Quality predictions based on the ExAC database are more adequate, as it includes ~37× more samples than our dataset. For the putative compound heterozygous mutations, both variants should be located within the same transcript and at least one allele should contain a LoF variant. The second variant could be: (1) a LoF variant; or (2) a missense variant that is absent in dbSNP137  [90]  database and with a CADD score > 20 (predicted to belong to the 1% most deleterious variants of the total genome), indicating a pathogenic effect. The latter two filter criteria should decrease the chance of including benign missense variants.
Variant aggregation analyses in the IPDGC WES dataset SKAT-c  [92]  was used to analyze the burden of coding variants for each identified gene. Both rare variants only and the joint effect of common and rare variants were tested. Because variant aggregation tests are prone to coverage differences, capture usage and population stratification, we performed a more stringent individual and variant QC, resulting in a reduced dataset of 1540 samples (1062 cases and 478 controls) covering 268,038 variants. Individuals were excluded when failing gender test, showing evidence of relatedness, having dubious heterozygosity/genotype calls, or being a population outlier. Variants were removed when having a genotype missingness > 5%, a Hardy-Weinberg equilibrium p value < 1e -6 or a p value for non-random missingness by phenotype < 1e -5.
Genetic replication 1: variant identification in PPMI WES dataset We obtained permission to access WES data generated by the PPMI  [51]. After standard variant and individual QC, the dataset includes 477,512 variants for 462 PD cases and 183 neurologically healthy controls. A similar search for homozygous and putative compound heterozygous LoF variants, as described for the original IPDGC WES dataset, was applied for this second independent PPMI WES dataset by using ANNOVAR  [83]  and KGGSeq  [89].
Genetic replication 2: GRIP genetic isolate The southwest of the Netherlands contains a recently isolated population which is part of the GRIP program  [52]. A total of 39 PD index cases and 19 controls of this isolate were subjected to whole-genome sequencing to explore the genetic factors underlying PD within this geographic region. Missense and LoF variants which were present in at least two index cases and a MAF < 0. 1% in public databases (ExAC, 1000G dbSNP138, and ESP6500) were considered as potential PD variants. Genes harboring such variants were surveyed for overlap with our list of candidate genes.
Genetic replication 3: variant aggregation analyses in NeuroX We investigated the genetic burden of common and rare variants in these genes by using the independent Neu-roX dataset, which is generated by a custom-made genotype array  [53]  using a backbone of ~240,000 standard Illumina Exome content as a basis with an additional ~24,000 variants that are suggested to be involved neurological diseases. The same procedures as described for the burden test in the IPDGC WES dataset were applied. After QC, a total of 6801 PD cases and 5970 neurologically healthy controls remained with highquality genotype data for 178,779 variants. Based on the sample size, the MAF cutoff was 0. 0063.
Gene co-expression analyses We constructed gene co-expression networks (GCN) from two different substantia nigra datasets using the R software package, WGCNA (weighted gene coexpression network analysis)  [97]. This was followed by the same post-processing of WGCNA gene modules based on k-means: a heuristic to rearrange misplaced genes between modules using the number of modules detected by the standard WGCNA as k and the eigengenes as centroids. The first GCN is based on 19,152 genes from 65 substantia nigra control brains from the UKBEC consortium. The gene expression profiles are based on Affymetrix Exon 1. 0 ST Arrays  [98]. The second GCN is based on 63 samples from the same tissue, GTEx  [56]  V6 gene RPKM values. Genes were filtered with a RPKM based cutoff of 0. 2 and missingness < 30% resulting in the analysis of 18,363 Ensembl genes.