Search

Parkinson’s disease (PD) is a neurodegenerative disorder marked by the death of dopaminergic neurons in the midbrain, the accumulation of α-synuclein aggregates, and motor deficits. A major contributor to dopaminergic neuronal loss is neuroinflammation. The inflammasome is a multiprotein complex that perpetuates neuroinflammation in neurodegenerative disorders including PD. Increases in inflammasome proteins are associated with worsened pathology. Thus, the inhibition of inflammatory mediators has the potential to aid in PD treatment. Here, we investigated inflammasome signaling proteins as potential biomarkers of the inflammatory response in PD. Plasma from PD subjects and healthy age-matched controls were evaluated for levels of the inflammasome protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin (IL)-18. This was carried out using Simple Plex technology to identify changes in inflammasome proteins in the blood of PD subjects. The area under the curve (AUC) was obtained through calculation of the receiver operating characteristics (ROC) to obtain information on biomarker reliability and traits. Additionally, we completed a stepwise regression selected from the lowest Akaike information criterion (AIC) to assess how the inflammasome proteins caspase-1 and ASC contribute to IL-18 levels in people with PD. PD subjects demonstrated elevated caspase-1, ASC, and IL-18 levels when compared to controls; each of these proteins were found to be promising biomarkers of inflammation in PD. Furthermore, inflammasome proteins were determined to significantly contribute to and predict IL-18 levels in subjects with PD. Thus, we demonstrated that inflammasome proteins serve as reliable biomarkers of inflammation in PD and that inflammasome proteins provide significant contributions to IL-18 levels in PD.

BackgroundEuropean local ancestry (ELA) surroundingAPOE4is associated with a higher risk for Alzheimer Disease (AD) compared to African local ancestry (ALA). We previously demonstrated significantly higherAPOE4expression in ELA vs ALA in the frontal cortex ofAPOE4/4AD patients. Differences in chromatin accessibility could contribute to these differences inAPOE4expression.MethodsWe performed single nuclei Assays for Transposase Accessible Chromatin sequencing (snATAC-seq) and single nuclei RNA sequencing (snRNA-seq) from frozen frontal cortex of six ALA and six ELA AD patients, all homozygous for local ancestry andAPOE4.ResultsWe demonstrated thatAPOE4, including its promoter area, has greater chromatin accessibility in ELA vs ALA astrocytes. This increased accessibility in ELA astrocytes extended genome wide. Genes with increased accessibility and expression in ELA in astrocytes were enriched for synaptic function, cholesterol processing and astrocyte reactivity.ConclusionOur results suggest that increased chromatin accessibility ofAPOE4in astrocyte with the ELA contributes to the observed elevatedAPOE4expression, corresponding to the increased AD risk in ELA vs ALAAPOE4/4carriers.

Thrombotic storm is a rare clinical entity characterized by acute to subacute thrombosis developing at multiple sites over a few days to a few weeks. An 11-year-old boy presented with headache and facial nerve palsy. He was found to have cortical sinus venous thrombosis and was initiated on low molecular weight heparin, but rapidly progressed with thromboses involving the pulmonary arteries and deep veins of the legs. Thereafter managed on high-dose unfractionated heparin, he eventually stabilized after a hospital stay of 34 days. Genetic analysis showed potentially pathogenic variants in the factor V and stabilin-2 genes.

INTRODUCTION Apolipoprotein E (APOE) e4 confers less risk for Alzheimer's disease (AD) in carriers with African local genomic ancestry (ALA) than APOE e4 carriers with European local ancestry (ELA). Cell type specific transcriptional variation between the two local ancestries (LAs) could contribute to this disease risk differences. METHODS Single-nucleus RNA sequencing was performed on frozen frontal cortex of homozygous APOE e4/e4 AD patients: seven with ELA, four with ALA. RESULTS A total of 60,908 nuclei were sequenced. Within the LA region (chr19:44-46Mb), APOE was the gene most differentially expressed, with ELA carriers having significantly more expression (overall P

We previously demonstrated that in Alzheimer's disease (AD) patients, European apolipoprotein E (APOE) ε4 carriers express significantly more APOE ε4 in their brains than African AD carriers. We examined single nucleotide polymorphisms near APOE with significant frequency differences between African and European/Japanese APOE ε4 haplotypes that could contribute to this difference in expression through regulation. Two enhancer massively parallel reporter assay (MPRA) approaches were performed, supplemented with single fragment reporter assays. We used Capture C analyses to support interactions with the APOE promoter. Introns within TOMM40 showed increased enhancer activity in the European/Japanese versus African haplotypes in astrocytes and microglia. This region overlaps with APOE promoter interactions as assessed by Capture C analysis. Single variant analyses pinpoints rs2075650/rs157581, and rs59007384 as functionally different on these haplotypes. Identification of the mechanisms for differential regulatory function for APOE expression between African and European/Japanese haplotypes could lead to therapeutic targets for APOE ε4 carriers.

Most Alzheimer’s disease (AD)-associated genetic variants do not change protein coding sequence and thus likely exert their effects through regulatory mechanisms. RNA editing, the post-transcriptional modification of RNA bases, is a regulatory feature that is altered in AD patients that differs across ancestral backgrounds. Editing QTLs (edQTLs) are DNA variants that influence the level of RNA editing at a specific site. To study the relationship of DNA variants genome-wide, and particularly in AD-associated loci, with RNA editing, we performed edQTL analyses in self-reported individuals of African American (AF) or White (EU) race with corresponding global genetic ancestry averaging 82.2% African ancestry (AF) and 96.8% European global ancestry (EU) in the two groups, respectively. We used whole-genome genotyping array and RNA sequencing data from peripheral blood of 216 AD cases and 212 age-matched, cognitively intact controls. We identified 2144 edQTLs in AF and 3579 in EU, of which 1236 were found in both groups. Among these, edQTLs in linkage disequilibrium (r2 > 0.5) with AD-associated genetic variants in the SORL1, SPI1 and HLA-DRB1 loci were associated with sites that were differentially edited between AD cases and controls. While there is some shared RNA editing regulatory architecture, most edQTLs had distinct effects on the rate of RNA editing in different ancestral populations suggesting a complex architecture of RNA editing regulation. Altered RNA editing may be one possible mechanism for the functional effect of AD-associated variants and may contribute to observed differences in the genetic etiology of AD between ancestries.

INTRODUCTIONThe difference in APOEε4 risk for Alzheimer disease (AD) between different populations is associated with APOEε4 local ancestry (LA). We examined LA SNPs with significant frequency differences between African and European/Japanese APOEε4 haplotypes for areas of differential regulation.METHODSWe performed two enhancer Massively Parallel Reporter Assay (MPRA) approaches, supplemented with single fragment reporter assays. We utilized Capture C analyses to support interactions with the APOE promoter.RESULTSThe TOMM40 intron 2 and 3 region showed increased enhancer activity in the European/Japanese versus African LA haplotypes in astrocytes and microglia. This region overlaps with APOE promoter interactions as assessed by Capture C analysis. Single variant analyses pinpoints rs2075650/rs157581, and rs59007384 as functionally different on these haplotypes.DISCUSSIONBoth differential regulatory function and Capture C data support an intronic region in TOMM40 as contributing to the differential APOE expression between African and European/Japanese LA.

Objective The aim of this study was to search for genes/variants that modify the effect of LRRK2 mutations in terms of penetrance and age-at-onset of Parkinson's disease. Methods We performed the first genome-wide association study of penetrance and age-at-onset of Parkinson's disease in LRRK2 mutation carriers (776 cases and 1,103 non-cases at their last evaluation). Cox proportional hazard models and linear mixed models were used to identify modifiers of penetrance and age-at-onset of LRRK2 mutations, respectively. We also investigated whether a polygenic risk score derived from a published genome-wide association study of Parkinson's disease was able to explain variability in penetrance and age-at-onset in LRRK2 mutation carriers. Results A variant located in the intronic region of CORO1C on chromosome 12 (rs77395454; P-value = 2.5E-08, beta = 1.27, SE = 0.23, risk allele: C) met genome-wide significance for the penetrance model. Co-immunoprecipitation analyses of LRRK2 and CORO1C supported an interaction between these two proteins. A region on chromosome 3, within a previously reported linkage peak for Parkinson's disease susceptibility, showed suggestive associations in both models (penetrance top variant: P-value = 1.1E-07; age-at-onset top variant: P-value = 9.3E-07). A polygenic risk score derived from publicly available Parkinson's disease summary statistics was a significant predictor of penetrance, but not of age-at-onset. Interpretation This study suggests that variants within or near CORO1C may modify the penetrance of LRRK2 mutations. In addition, common Parkinson's disease associated variants collectively increase the penetrance of LRRK2 mutations. This article is protected by copyright. All rights reserved.

Purpose To clinically and molecularly investigate a new family with North Carolina macular dystrophy (NCMD) from Turkey, a previously unreported geographic origin for this phenotype. Methods Clinical ophthalmic examinations, including fundus imaging and spectral domain-optical coherence tomography (SD-OCT), were performed on eight members of a two-generation non-consanguineous family from southern Turkey. Whole genome sequencing (WGS) was performed on two affected subjects, followed by variant filtering and copy number variant (CNV) analysis. Junction PCR and Sanger sequencing were used to confirm and characterize the duplication involving PRDM13 at the nucleotide level. The underlying mechanism was assessed with in silico analyses. Results The proband presented with lifelong bilateral vision impairment and displayed large grade 3 coloboma-like central macular lesions. Five of her six children showed similar macular malformations, consistent with autosomal dominant NCMD. The severity grades in the six affected individuals from two generations are not evenly distributed. CNV analysis of WGS data of the two affected family members, followed by junction PCR and Sanger sequencing, revealed a novel 56.2 kb tandem duplication involving PRDM13 (chr6:99560265–99616492dup, hg38) at the MCDR1 locus. This duplication cosegregates with the NCMD phenotype in the five affected children. No other (likely) pathogenic variants in known inherited retinal disease genes were found in the WGS data. Bioinformatics analyses of the breakpoints suggest a replicative-based repair mechanism underlying the duplication. Conclusions We report a novel tandem duplication involving the PRDM13 gene in a family with NCMD from a previously unreported geographic region. The duplication size is the smallest that has been reported thus far and may correlate with the particular phenotype.

Objective: The aim of this study was to search for genes/variants that modify the effect of LRRK2 mutations in terms of penetrance and age-at-onset of Parkinson's disease. Methods: We performed the first genome-wide association study of penetrance and age-at-onset of Parkinson's disease in LRRK2 mutation carriers (776 cases and 1,103 non-cases at their last evaluation). Cox proportional hazard models and linear mixed models were used to identify modifiers of penetrance and age-at-onset of LRRK2 mutations, respectively. We also investigated whether a polygenic risk score derived from a published genome-wide association study of Parkinson's disease was able to explain variability in penetrance and age-at-onset in LRRK2 mutation carriers. Results: A variant located in the intronic region of CORO1C on chromosome 12 (rs77395454; P-value=2.5E-08, beta=1.27, SE=0.23, risk allele: C) met genome-wide significance for the penetrance model. A region on chromosome 3, within a previously reported linkage peak for Parkinson's disease susceptibility, showed suggestive associations in both models (penetrance top variant: P-value=1.1E-07; age-at-onset top variant: P-value=9.3E-07). A polygenic risk score derived from publicly available Parkinson's disease summary statistics was a significant predictor of penetrance, but not of age-at-onset. Interpretation: This study suggests that variants within or near CORO1C may modify the penetrance of LRRK2 mutations. In addition, common Parkinson's disease associated variants collectively increase the penetrance of LRRK2 mutations.

Background: The Latino population is greatly understudied in biomedical research, including genetics. Very little information is available on presence of known variants originally identified in non-Hispanic white patients or novel variants in the Latino population. The Latino population is admixed, with contributions of European, African, and Amerindian ancestries. Therefore, the ancestry surrounding a gene (local ancestry, LA) can be any of the three contributing ancestries and thus can determine the presence or risk effect of variants detected. Methods: We sequenced the major exons and exons of reported Latino-specific variants in GBA and LRRK2 and performed genome-wide genotyping for LA assessments in 79 Latino Parkinson disease (PD) patients, of which ~80% identified as Caribbean Latino. Results: We observed five carriers of LRRK2 p.G2019S, one GBA p.T408M, and three GBA p.N409S on European as well as three GBA p.L13R on African LA backgrounds. Previous Latino variant GBA p.K237E was not observed in this dataset. A novel highly conserved and predicted damaging variant LRRK2 p.D734N was identified in two unrelated individuals with African LA. Additionally, we identified rare, functional variants LRRK2 p.P1480L and GBA p.S310G in one individual each heterozygous for European/Amerindian LA. Discussion: Additional functional analysis will be needed to determine the pathogenicity of the novel variants in PD. However, the identification of novel disease variants in the Latino cohort potentially contributing to PD supports to importance of inclusion of Latinos in genetics research to provide insight in PD genetics in Latinos specifically as well as other populations with the same ancestral contributions.

APOEε4African local genomic ancestry (LA) confers less risk for Alzheimer disease (AD) relative to European LA (LA) carriers. Single nucleus RNA sequencing from AD-APOEε4/4frontal cortex found European LA carriers have a 1.45-fold greaterAPOEε4expression (p< 1.8 E10−313) and are associated with a unique A1 reactive astrocyte cluster. This suggests a potential mechanism for the increased risk for AD seen in European LA carriers ofAPOEε4.

Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However, most iPSC lines are derived in advance of, and without the benefit of, neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants, these methods require outgrowth of cadaver tissue, which require additional time and is often only successful ∼50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue, iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases.

The UMi028-A-2 human induced pluripotent stem cell line carries a homozygous mutation (rs377155188, C>G, p.S1038C) in the tetratricopeptide repeat domain 3 (TTC3) gene that was introduced via CRISPR/Cas9 genome editing. The line was originally derived from a neurologically normal male and has been thoroughly characterized following editing. The p.S1038C variant has been shown to potentially contribute to the risk of late onset Alzheimer’s disease and is a resource to further investigate the consequences of TTC3 and this alteration in disease pathology.

Objective: To minimize pathologic heterogeneity in genetic studies of Parkinson disease (PD), the Autopsy-Confirmed Parkinson Disease Genetics Consortium conducted a genome-wide association study using both patients with neuropathologically confirmed PD and controls. Methods: Four hundred eighty-four cases and 1,145 controls met neuropathologic diagnostic criteria, were genotyped, and then imputed to 3,922,209 variants for genome-wide association study analysis. Results: A small region on chromosome 1 was strongly associated with PD (rs10788972; p = 6.2 × 10 −8 ). The association peak lies within and very close to the maximum linkage peaks of 2 prior positive linkage studies defining the PARK10 locus. We demonstrate that rs10788972 is in strong linkage disequilibrium with rs914722, the single nucleotide polymorphism defining the PARK10 haplotype previously shown to be significantly associated with age at onset in PD. The region containing the PARK10 locus was significantly reduced from 10.6 megabases to 100 kilobases and contains 4 known genes: TCEANC2 , TMEM59 , miR-4781 , and LDLRAD1 . Conclusions: We confirm the association of a PARK10 haplotype with the risk of developing idiopathic PD. Furthermore, we significantly reduce the size of the PARK10 region. None of the candidate genes in the new PARK10 region have been previously implicated in the biology of PD, suggesting new areas of potential research. This study strongly suggests that reducing pathologic heterogeneity may enhance the application of genetic association studies to PD.

Traumatic knee injuries often result in damage to articular cartilage and other joint structures. Such trauma is a strong risk factor for the future development and progression of osteoarthritis (OA). The molecular mechanisms and signaling pathways modulating response to knee joint trauma remain unclear. Moreover, investigations of biomarkers influencing responses have been targeted rather than broad, unbiased discovery studies. Herein, we characterize the complete complement of extracellular RNA (exRNA) in the synovial fluid of 14 subjects following knee injury. Fluid was collected during surgery from the injured knees, and from the contralateral knee in a subset, undergoing surgical repair of the ACL and/or meniscal repair/debridement. Arthroscopic grading of chondral damage in four knee compartments was performed using the Outerbridge classification. exRNA was extracted and subjected to massively parallel total RNA sequencing. Differential abundance of RNA was calculated between the subject cohorts of injured and non-injured knee, average Outerbridge score ≥0.5 and less, and chronic and acute injury duration defined as ≤4 months till surgery or longer. Overall, expression of several thousand genes was identified in the synovial fluid. Furthermore, differential expression analysis suggests a role of exRNA fragments of matrix metalloproteinases and skeletal muscle fiber genes in the response to traumatic injury. Together, these data suggest that high-throughput approaches can indicate exRNA molecular signatures following knee trauma. Future studies are required to more fully characterize the biological roles of these exRNA and the cadence of their respective release that may lead to translational treatment options for post-traumatic OA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1659-1665, 2018.

Background We have reported that intermediate repeat lengths of the C9ORF72 repeat are a risk factor for Parkinson's disease (PD) in a clinically diagnosed data set. Because 10% to 25% of clinically diagnosed PD have different diagnoses upon autopsy, we hypothesized that this may reflect phenotypic heterogeneity or concomitant pathology of other neurodegenerative disorders. Methods We screened 488 autopsy-confirmed PD cases for expansion haplotype tag rs3849942T. In 196 identified haplotype carriers, the C9ORF72 repeat was genotyped using the repeat-primed polymerase chain reaction assay. Results No larger (intermediate or expanded) repeats were found in these autopsy-confirmed PD samples. This absence of larger repeats is significantly different from the frequency in clinically diagnosed datasets (P = 0.002). Conclusions Our results suggest that expanded or intermediate C9ORF72 repeats in clinically diagnosed PD or parkinsonism might be an indication of heterogeneity in clinically diagnosed PD cases. Further studies are needed to elucidate the potential contribution of the C9ORF72 repeat to autopsy-confirmed PD. © 2014 International Parkinson and Movement Disorder Society

Recently, vacuolar protein sorting 35 (VPS35) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) have been identified as 2 causal Parkinson disease (PD) genes. We used whole exome sequencing for rapid, parallel analysis of variations in these 2 genes.We performed whole exome sequencing in 213 patients with PD and 272 control individuals. Those rare variants (RVs) with5% frequency in the exome variant server database and our own control data were considered for analysis. We performed joint gene-based tests for association using RVASSOC and SKAT (Sequence Kernel Association Test) as well as single-variant test statistics.We identified 3 novel VPS35 variations that changed the coded amino acid (nonsynonymous) in 3 cases. Two variations were in multiplex families and neither segregated with PD. In EIF4G1, we identified 11 (9 nonsynonymous and 2 small indels) RVs including the reported pathogenic mutation p.R1205H, which segregated in all affected members of a large family, but also in 1 unaffected 86-year-old family member. Two additional RVs were found in isolated patients only. Whereas initial association studies suggested an association (p = 0.04) with all RVs in EIF4G1, subsequent testing in a second dataset for the driving variant (p.F1461) suggested no association between RVs in the gene and PD.We confirm that the specific EIF4G1 variation p.R1205H seems to be a strong PD risk factor, but is nonpenetrant in at least one 86-year-old. A few other select RVs in both genes could not be ruled out as causal. However, there was no evidence for an overall contribution of genetic variability in VPS35 or EIF4G1 to PD development in our dataset.

Parkinson’s disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson’s disease or parkinsonian disorders. The pathogenesis of Parkinson’s disease remain largely elusive. Here, we report a novel genetic locus for an autosomal dominant, clinically typical and Lewy body confirmed Parkinson’s disease on the short arm of chromosome 20 (20pter-p12) and TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. The disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide the first genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson’s disease, with novel implications in understanding the pathogenic mechanism of Parkinson’s disease and for developing rational therapies.

Background: Autosomal dominant dopa-responsive dystonia (AD-DRD) is caused by a biochemical defect primarily resulting from guanosine triphosphate cyclohydrolase 1 gene (GCH1) mutations. Few families have been reported without mutations in GCH1. Methods: Genome-wide linkage analysis and positional cloning to identify the genetic defect in a Belgian AD-DRD family was carried out. Results and Conclusion: In this study, we report on the identification and characterization of a novel 24-kb deletion spanning exon 1 and the 5 0 regulatory region of GCH1 causing a wide spectrum of motor and nonmotor symptoms in a large Belgian AD-DRD family. This largescale deletion of regulatory sequences leads to decreased GCH1 activity in all carriers, most probably resulting from allelic loss of transcription. We mapped the breakpoints of this deletion to the nucleotide level, allowing the development of a straightforward polymerase chain reaction assay for fast, efficient detection of this large deletion, which will prove valuable for preimplantation genetic diagnosis. V C 2012 Movement Disorder Society

The rate of DNA variation discovery has accelerated the need to collate, store and interpret the data in a standardised coherent way and is becoming a critical step in maximising the impact of discovery on the understanding and treatment of human disease. This particularly applies to the field of neurology as neurological function is impaired in many human disorders. Furthermore, the field of neurogenetics has been proven to show remarkably complex genotype-to-phenotype relationships. To facilitate the collection of DNA sequence variation pertaining to neurogenetic disorders, we have initiated the Neurogenetics Consortium under the umbrella of the Human Variome Project. The Consortiums founding group consisted of basic researchers, clinicians, informaticians and database creators. This report outlines the strategic aims established at the preliminary meetings of the Neurogenetics Consortium and calls for the involvement of the wider neurogenetic community in enabling the development of this important resource.

To date, molecular genetic analyses have identified over 500 distinct DNA variants in five disease genes associated with familial Parkinson disease; α-synuclein (SNCA), parkin (PARK2), PTEN-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and Leucine-rich repeat kinase 2 (LRRK2). These genetic variants include ∼82% simple mutations and ∼18% copy number variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database (http://www.molgen.ua.ac.be/PDmutDB). In addition, we address the biological relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of observed genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiology of other Lewy Body Diseases and Parkinson-plus syndromes, indicating that mutation screening is recommendable in these patient groups. Hum Mutat 31:763–780, 2010. © 2010 Wiley-Liss, Inc.

The relative contribution of simple mutations and copy number variations (CNVs) in SNCA, PARK2, PINK1, PARK7, and LRRK2 to the genetic etiology of Parkinson disease (PD) is still unclear because most studies did not completely analyze each gene. In a large group of Belgian PD patients (N=310) and control individuals (N=270), we determined the mutation frequency of both simple mutations and CNVs in these five PD genes, using direct sequencing, multiplex amplicon quantification (MAQ), and real-time PCR assays. Overall, we identified 14 novel heterozygous variants, of which 11 were absent in control individuals. We observed eight PARK2 (multiple) exon multiplications in PD patients and one exon deletion in a control individual. Furthermore, we identified one SNCA whole-gene duplication. The PARK2 and LRRK2 mutation frequencies in Belgian PD patients were similar to those reported in other studies. However, at this stage the true pathogenic nature of some heterozygous mutations in recessive genes remains elusive. Furthermore, though mutations is SNCA, PINK1, and PARK7 are rare, our identification of a SNCA duplication confirmed that screening of these genes remains meaningful. Hum Mutat 30:1–8, 2009. © 2009 Wiley-Liss, Inc.

In one genetic study, the high temperature requirement A2 (HTRA2) mitochondrial protein has been associated with increased risk for sporadic Parkinson disease (PD). One missense mutation, p.Gly399Ser, in its C-terminal PDZ domain (from the initial letters of the postsynaptic density 95, PSD-95; discs large; and zonula occludens-1, ZO-1 proteins [Kennedy, 1995]) resulted in defective protease activation, and induced mitochondrial dysfunction when overexpressed in stably transfected cells. Here we examined the contribution of genetic variability in HTRA2 to PD risk in an extended series of 266 Belgian PD patients and 273 control individuals. Mutation analysis identified a novel p.Arg404Trp mutation within the PDZ domain predicted to freeze HTRA2 in an inactive form. Moreover, we identified six patient-specific variants in 5′ and 3′ regulatory regions that might affect HTRA2 expression as supported by data of luciferase reporter gene analyses. Our study confirms a role of the HTRA2 mitochondrial protein in PD susceptibility through mutations in its functional PDZ domain. In addition, it extends the HTRA2 mutation spectrum to functional variants possibly affecting transcriptional activity. The latter underpins a previously unrecognized role for altered HTRA2 expression as a risk factor relevant to parkinsonian neurodegeneration. Hum Mutat 29(6), 832–840, 2008. © 2008 Wiley-Liss, Inc.

We have reported that intermediate repeat lengths of the C9ORF72 repeat are a risk factor for Parkinson's disease (PD) in a clinically diagnosed data set. Because 10% to 25% of clinically diagnosed PD have different diagnoses upon autopsy, we hypothesized that this may reflect phenotypic heterogeneity or concomitant pathology of other neurodegenerative disorders.We screened 488 autopsy-confirmed PD cases for expansion haplotype tag rs3849942T. In 196 identified haplotype carriers, the C9ORF72 repeat was genotyped using the repeat-primed polymerase chain reaction assay.No larger (intermediate or expanded) repeats were found in these autopsy-confirmed PD samples. This absence of larger repeats is significantly different from the frequency in clinically diagnosed datasets (P = 0.002).Our results suggest that expanded or intermediate C9ORF72 repeats in clinically diagnosed PD or parkinsonism might be an indication of heterogeneity in clinically diagnosed PD cases. Further studies are needed to elucidate the potential contribution of the C9ORF72 repeat to autopsy-confirmed PD.

Objective: Given their reported function in phagocytosis and clearance of protein aggregates in Alzheimer disease (AD), we hypothesized that variants in ATP-binding cassette transporter A7 ( ABCA7 ) might be involved in Parkinson disease (PD). Methods: ABCA7 variants were identified using whole-exome sequencing (WES) on 396 unrelated patients with PD and 222 healthy controls. In addition, we used the publicly available WES data from the Parkinson9s Progression Markers Initiative (444 patients and 153 healthy controls) as a second, independent data set. Results: We observed a higher frequency of loss-of-function (LOF) variants and rare putative highly functional variants (Combined Annotation Dependent Depletion [CADD] >20) in clinically diagnosed patients with PD than in healthy controls in both data sets. Overall, we identified LOF variants in 11 patients and 1 healthy control (odds ratio [OR] 4.94, Fisher exact p = 0.07). Four of these variants have been previously implicated in AD risk (p.E709AfsX86, p.W1214X, p.L1403RfsX7, and rs113809142). In addition, rare variants with CADD >20 were observed in 19 patients vs 3 healthy controls (OR 2.85, Fisher exact p = 0.06). Conclusion: The presence of ABCA7 LOF variants in clinically defined PD suggests that they might be risk factors for neurodegeneration in general, especially those variants hallmarked by protein aggregation. More studies will be needed to evaluate the overall impact of this transporter in neurodegenerative disease.

We set out to determine whether expansions in the C9ORF72 repeat found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) families are associated with Parkinson disease (PD). We determined the repeat size in a total of 889 clinically ascertained patients (including PD and essential tremor plus Parkinsonism (ETP)) and 1144 controls using a repeat-primed PCR assay. We found that large C9ORF72 repeat expansions (>30 repeats) were not contributing to PD risk. However, PD and ETP cases had a significant increase in intermediate (>20 to 30+) repeat copies compared to controls. Overall, 14 cases (13 PD, 1 ETP) and three controls had >20 repeat copies (Fisher's exact test p= 0.002). Further, seven cases and no controls had >23 repeat copies (p= 0.003). Our results suggest that intermediate copy numbers of the C9ORF72 repeat contribute to risk for PD and ETP. This also suggests that PD, ALS and FTD share some pathophysiological mechanisms of disease. Further studies are needed to elucidate the contribution of the C9ORF72 repeat in the overall PD population and to determine whether other common genetic risk factors exist between these neurodegenerative disorders.

A high density comparative genomic hybridization array was designed to evaluate CNVs in the genomic region of six familial PD genes in 181 PD cases and 67 controls. No CNV was found in PARK7, ATP13A2, PINK1, and LRRK2. Intronic-only CNVs were found in SNCA and PARK2 but were not associated with PD risk. A whole-gene duplication of SNCA was found in one case. The allele frequency of PARK2 exonic CNV is significantly higher in cases than in controls (P = 0.02), higher in early-onset (AAO ≤ 40) than in late-onset cases (P = 0.001), and higher in familial than in sporadic cases (P = 0.005). Except for single exon 2 duplications, all PARK2 exonic CNVs have different breakpoints, even when the same exon(s) were involved. In conclusion, except for SNCA and PARK2, CNVs are not a major contributing mechanism for the familial PD genes examined. The majority of PARK2 exonic CNVs are not recurrent.

Autosomal dominant dopa-responsive dystonia (AD-DRD) is caused by a biochemical defect primarily resulting from guanosine triphosphate cyclohydrolase 1 gene (GCH1) mutations. Few families have been reported without mutations in GCH1.Genome-wide linkage analysis and positional cloning to identify the genetic defect in a Belgian AD-DRD family was carried out.In this study, we report on the identification and characterization of a novel 24-kb deletion spanning exon 1 and the 5' regulatory region of GCH1 causing a wide spectrum of motor and nonmotor symptoms in a large Belgian AD-DRD family. This large-scale deletion of regulatory sequences leads to decreased GCH1 activity in all carriers, most probably resulting from allelic loss of transcription. We mapped the breakpoints of this deletion to the nucleotide level, allowing the development of a straightforward polymerase chain reaction assay for fast, efficient detection of this large deletion, which will prove valuable for preimplantation genetic diagnosis.

Over the years there have been many different approaches and techniques that have been utilized to gather genetic information on family and patient data. Early on these focused on using family information and the pattern of inheritance of the disease in the family. The general location of the disease gene on the chromosomes could be determined using linkage analysis. But this required large families and good family histories. Association studies moved away from family data, but still required large numbers of patients sharing the same disease. Over the past several decades we have had continuing growth in the number of genetic “markers” that we could use in linkage and association analyses, the most recent work utilizing the specific sequence changes called single nucleotide polymorphisms or SNPs. But the use of these markers did not eliminate the need for large families or large population sizes. Thus, despite advances in genotyping technology, the parameters of the applications in which these techniques could be used have not changed much in 30 years or so. This prevented many of the most interesting cases and families with Mendelian inheritance (due to a mutation in a single gene) from being studied, as they were too small for analysis, or too rare. Sequencing genes provided more information, but the previous existing technology was too expensive and labor intensive to do a large number of genes. Thus investigators were dependent on choosing candidate genes for study, which historically has proven to be fairly inefficient. But this paradigm of needing large families to investigate a disease, or choosing a few candidate genes to test a hypothesis is now changing. The rapidly emerging sequencing technology has now allowed the first practical medical application of the most useful and enlightening measure of genetic information, the DNA sequence itself. The output of sequencing has been increasing at a rapid pace over the past several years, passing even the growth of the computer chip made famous by “Moore’s law” (Moore, G. E. 1965). During the 1990’s and early in this century, capillary sequencers, which produced the data for the human genome project, could identify 500 to 1,000 base pairs of sequence at a time. While impressive relative to previous slab gel techniques, the Next Generation Sequencers (NGS) of today have increased that sequencing output a million fold or more (Fig. 1). This also means that the cost of sequencing per base pair has dropped precipitously. The human genome is approximately 3.4 billion base pairs. The cost of sequencing was approximately 10 million dollars in 2003. This dropped to around a million dollars in 2007, less than a $100,000 by 2009, and is expected to be close to the $1,000 goal in 2011-2012. How has this remarkable increase been accomplished? Sanger sequencing has been the mainstay of sequencing for many years. The Sanger sequencing method that was used for the human genome project utilizes a small primer (usually 20 base pairs) , that allows the sequencing reaction to

Missense mutations were identified in the Grb10-Interacting GYF Protein-2 gene ( GIGYF2 ), located in the chromosomal region 2q36-q37, in familial Parkinson disease (PD) patients of European descent. To determine the contribution of GIGYF2 mutations in an extended ( N = 305) Belgian series of both familial and sporadic PD patients, we sequenced all 32 coding and non-coding exons of GIGYF2 . In three sporadic PD patients we identified two novel heterozygous missense mutations (c.1907A>G, p.Tyr636Cys and c.2501G>A, p.Arg834Gln), that were absent from control individuals ( N = 360). However, since we lack genetic as well as functional data supporting their pathogenic nature, we cannot exclude that these variants are benign polymorphisms. Together, our results do not support a role for GIGYF2 in the genetic etiology of Belgian PD.