Brain DNA methylomic analysis of frontotemporal lobar degeneration reveals OTUD4 in shared dysregulated signatures across pathological subtypes.

Frontotemporal lobar degeneration (FTLD) is an umbrella term describing the neuropathology of a clinically, genetically and pathologically heterogeneous group of diseases, including frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP). Among the major FTLD pathological subgroups, FTLD with TDP-43 positive inclusions (FTLD-TDP) and FTLD with tau-positive inclusions (FTLD-tau) are the most common, representing about 90% of the cases. Although alterations in DNA methylation have been consistently associated with neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, little is known for FTLD and its heterogeneous subgroups and subtypes. The main goal of this study was to investigate DNA methylation variation in FTLD-TDP and FTLD-tau. We used frontal cortex genome-wide DNA methylation profiles from three FTLD cohorts (142 FTLD cases and 92 controls), generated using the Illumina 450K or EPIC microarrays. We performed epigenome-wide association studies (EWAS) for each cohort followed by meta-analysis to identify shared differentially methylated loci across FTLD subgroups/subtypes. In addition, we used weighted gene correlation network analysis to identify co-methylation signatures associated with FTLD and other disease-related traits. Wherever possible, we also incorporated relevant gene/protein expression data. After accounting for a conservative Bonferroni multiple testing correction, the EWAS meta-analysis revealed two differentially methylated loci in FTLD, one annotated to OTUD4 (5'UTR-shore) and the other to NFATC1 (gene body-island). Of these loci, OTUD4 showed consistent upregulation of mRNA and protein expression in FTLD. In addition, in the three independent co-methylation networks, OTUD4-containing modules were enriched for EWAS meta-analysis top loci and were strongly associated with the FTLD status. These co-methylation modules were enriched for genes implicated in the ubiquitin system, RNA/stress granule formation and glutamatergic synaptic signalling. Altogether, our findings identified novel FTLD-associated loci, and support a role for DNA methylation as a mechanism involved in the dysregulation of biological processes relevant to FTLD, highlighting novel potential avenues for therapeutic development.

A Pentanucleotide ATTTC Repeat Insertion in the Non-coding Region of DAB1, Mapping to SCA37, Causes Spinocerebellar Ataxia.

Advances in human genetics in recent years have largely been driven by next-generation sequencing (NGS); however, the discovery of disease-related gene mutations has been biased toward the exome because the large and very repetitive regions that characterize the non-coding genome remain difficult to reach by that technology. For autosomal-dominant spinocerebellar ataxias (SCAs), 28 genes have been identified, but only five SCAs originate from non-coding mutations. Over half of SCA-affected families, however, remain without a genetic diagnosis. We used genome-wide linkage analysis, NGS, and repeat analysis to identify an (ATTTC)n insertion in a polymorphic ATTTT repeat in DAB1 in chromosomal region 1p32.2 as the cause of autosomal-dominant SCA; this region has been previously linked to SCA37. The non-pathogenic and pathogenic alleles have the configurations [(ATTTT)7-400] and [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90], respectively. (ATTTC)n insertions are present on a distinct haplotype and show an inverse correlation between size and age of onset. In the DAB1-oriented strand, (ATTTC)n is located in 5' UTR introns of cerebellar-specific transcripts arising mostly during human fetal brain development from the usage of alternative promoters, but it is maintained in the adult cerebellum. Overexpression of the transfected (ATTTC)58 insertion, but not (ATTTT)n, leads to abnormal nuclear RNA accumulation. Zebrafish embryos injected with RNA of the (AUUUC)58 insertion, but not (AUUUU)n, showed lethal developmental malformations. Together, these results establish an unstable repeat insertion in DAB1 as a cause of cerebellar degeneration; on the basis of the genetic and phenotypic evidence, we propose this mutation as the molecular basis for SCA37.

Genome-wide analyses as part of the international FTLD-TDP whole-genome sequencing consortium reveals novel disease risk factors and increases support for immune dysfunction in FTLD.

Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) represents the most common pathological subtype of FTLD. We established the international FTLD-TDP whole-genome sequencing consortium to thoroughly characterize the known genetic causes of FTLD-TDP and identify novel genetic risk factors. Through the study of 1131 unrelated Caucasian patients, we estimated that C9orf72 repeat expansions and GRN loss-of-function mutations account for 25.5% and 13.9% of FTLD-TDP patients, respectively. Mutations in TBK1 (1.5%) and other known FTLD genes (1.4%) were rare, and the disease in 57.7% of FTLD-TDP patients was unexplained by the known FTLD genes. To unravel the contribution of common genetic factors to the FTLD-TDP etiology in these patients, we conducted a two-stage association study comprising the analysis of whole-genome sequencing data from 517 FTLD-TDP patients and 838 controls, followed by targeted genotyping of the most associated genomic loci in 119 additional FTLD-TDP patients and 1653 controls. We identified three genome-wide significant FTLD-TDP risk loci: one new locus at chromosome 7q36 within the DPP6 gene led by rs118113626 (p value = 4.82e - 08, OR = 2.12), and two known loci: UNC13A, led by rs1297319 (p value = 1.27e - 08, OR = 1.50) and HLA-DQA2 led by rs17219281 (p value = 3.22e - 08, OR = 1.98). While HLA represents a locus previously implicated in clinical FTLD and related neurodegenerative disorders, the association signal in our study is independent from previously reported associations. Through inspection of our whole-genome sequence data for genes with an excess of rare loss-of-function variants in FTLD-TDP patients (n ≥ 3) as compared to controls (n = 0), we further discovered a possible role for genes functioning within the TBK1-related immune pathway (e.g., DHX58, TRIM21, IRF7) in the genetic etiology of FTLD-TDP. Together, our study based on the largest cohort of unrelated FTLD-TDP patients assembled to date provides a comprehensive view of the genetic landscape of FTLD-TDP, nominates novel FTLD-TDP risk loci, and strongly implicates the immune pathway in FTLD-TDP pathogenesis.

The wide genetic landscape of clinical frontotemporal dementia: systematic combined sequencing of 121 consecutive subjects.

PurposeTo define the genetic spectrum and relative gene frequencies underlying clinical frontotemporal dementia (FTD).MethodsWe investigated the frequencies and mutations in neurodegenerative disease genes in 121 consecutive FTD subjects using an unbiased, combined sequencing approach, complemented by cerebrospinal fluid Aß1-42 and serum progranulin measurements. Subjects were screened for C9orf72 repeat expansions, GRN and MAPT mutations, and, if negative, mutations in other neurodegenerative disease genes, by whole-exome sequencing (WES) (n = 108), including WES-based copy-number variant (CNV) analysis.ResultsPathogenic and likely pathogenic mutations were identified in 19% of the subjects, including mutations in C9orf72 (n = 8), GRN (n = 7, one 11-exon macro-deletion) and, more rarely, CHCHD10, TARDBP, SQSTM1 and UBQLN2 (each n = 1), but not in MAPT or TBK1. WES also unraveled pathogenic mutations in genes not commonly linked to FTD, including mutations in Alzheimer (PSEN1, PSEN2), lysosomal (CTSF, 7-exon macro-deletion) and cholesterol homeostasis pathways (CYP27A1).ConclusionOur unbiased approach reveals a wide genetic spectrum underlying clinical FTD, including 11% of seemingly sporadic FTD. It unravels several mutations and CNVs in genes and pathways hitherto not linked to FTD. This suggests that clinical FTD might be the converging downstream result of a delicate susceptibility of frontotemporal brain networks to insults in various pathways.

Somatic retrotransposition alters the genetic landscape of the human brain.

Retrotransposons are mobile genetic elements that use a germline 'copy-and-paste' mechanism to spread throughout metazoan genomes. At least 50 per cent of the human genome is derived from retrotransposons, with three active families (L1, Alu and SVA) associated with insertional mutagenesis and disease. Epigenetic and post-transcriptional suppression block retrotransposition in somatic cells, excluding early embryo development and some malignancies. Recent reports of L1 expression and copy number variation in the human brain suggest that L1 mobilization may also occur during later development. However, the corresponding integration sites have not been mapped. Here we apply a high-throughput method to identify numerous L1, Alu and SVA germline mutations, as well as 7,743 putative somatic L1 insertions, in the hippocampus and caudate nucleus of three individuals. Surprisingly, we also found 13,692 somatic Alu insertions and 1,350 SVA insertions. Our results demonstrate that retrotransposons mobilize to protein-coding genes differentially expressed and active in the brain. Thus, somatic genome mosaicism driven by retrotransposition may reshape the genetic circuitry that underpins normal and abnormal neurobiological processes.

Exonic deletions in AUTS2 cause a syndromic form of intellectual disability and suggest a critical role for the C terminus.

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.

DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway.

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H(2)O(2) in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.

Epigenetic Age Acceleration in Frontotemporal Lobar Degeneration: A Comprehensive Analysis in the Blood and Brain.

Frontotemporal lobar degeneration (FTLD) includes a heterogeneous group of disorders pathologically characterized by the degeneration of the frontal and temporal lobes. In addition to major genetic contributors of FTLD such as mutations in MAPT, GRN, and C9orf72, recent work has identified several epigenetic modifications including significant differential DNA methylation in DLX1, and OTUD4 loci. As aging remains one of the major risk factors for FTLD, we investigated the presence of accelerated epigenetic aging in FTLD compared to controls. We calculated epigenetic age in both peripheral blood and brain tissues of multiple FTLD subtypes using several DNA methylation clocks, i.e., DNAmClockMulti, DNAmClockHannum, DNAmClockCortical, GrimAge, and PhenoAge, and determined age acceleration and its association with different cellular proportions and clinical traits. Significant epigenetic age acceleration was observed in the peripheral blood of both frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP) patients compared to controls with DNAmClockHannum, even after accounting for confounding factors. A similar trend was observed with both DNAmClockMulti and DNAmClockCortical in post-mortem frontal cortex tissue of PSP patients and in FTLD cases harboring GRN mutations. Our findings support that increased epigenetic age acceleration in the peripheral blood could be an indicator for PSP and to a smaller extent, FTD.

A multi-omics dataset for the analysis of frontotemporal dementia genetic subtypes.

Understanding the molecular mechanisms underlying frontotemporal dementia (FTD) is essential for the development of successful therapies. Systematic studies on human post-mortem brain tissue of patients with genetic subtypes of FTD are currently lacking. The Risk and Modyfing Factors of Frontotemporal Dementia (RiMod-FTD) consortium therefore has generated a multi-omics dataset for genetic subtypes of FTD to identify common and distinct molecular mechanisms disturbed in disease. Here, we present multi-omics datasets generated from the frontal lobe of post-mortem human brain tissue from patients with mutations in MAPT, GRN and C9orf72 and healthy controls. This data resource consists of four datasets generated with different technologies to capture the transcriptome by RNA-seq, small RNA-seq, CAGE-seq, and methylation profiling. We show concrete examples on how to use the resulting data and confirm current knowledge about FTD and identify new processes for further investigation. This extensive multi-omics dataset holds great value to reveal new research avenues for this devastating disease.

The Foundational Data Initiative for Parkinson Disease: Enabling efficient translation from genetic maps to mechanism.

The Foundational Data Initiative for Parkinson Disease (FOUNDIN-PD) is an international collaboration producing fundamental resources for Parkinson disease (PD). FOUNDIN-PD generated a multi-layered molecular dataset in a cohort of induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons, a major affected cell type in PD. The lines were derived from the Parkinson's Progression Markers Initiative study, which included participants with PD carrying monogenic PD variants, variants with intermediate effects, and variants identified by genome-wide association studies and unaffected individuals. We generated genetic, epigenetic, regulatory, transcriptomic, and longitudinal cellular imaging data from iPSC-derived DA neurons to understand molecular relationships between disease-associated genetic variation and proximate molecular events. These data reveal that iPSC-derived DA neurons provide a valuable cellular context and foundational atlas for modeling PD genetic risk. We have integrated these data into a FOUNDIN-PD data browser as a resource for understanding the molecular pathogenesis of PD.

Drug screen in iPSC-Neurons identifies nucleoside analogs as inhibitors of (G4C2)n expression in C9orf72 ALS/FTD.

An intronic (G4C2)n expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G4C2)n foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders.

Distinct cell type-specific protein signatures in GRN and MAPT genetic subtypes of frontotemporal dementia.

Frontotemporal dementia is characterized by progressive atrophy of frontal and/or temporal cortices at an early age of onset. The disorder shows considerable clinical, pathological, and genetic heterogeneity. Here we investigated the proteomic signatures of frontal and temporal cortex from brains with frontotemporal dementia due to GRN and MAPT mutations to identify the key cell types and molecular pathways in their pathophysiology. We compared patients with mutations in the GRN gene (n = 9) or with mutations in the MAPT gene (n = 13) with non-demented controls (n = 11). Using quantitative proteomic analysis on laser-dissected tissues we identified brain region-specific protein signatures for both genetic subtypes. Using published single cell RNA expression data resources we deduced the involvement of major brain cell types in driving these different protein signatures. Subsequent gene ontology analysis identified distinct genetic subtype- and cell type-specific biological processes. For the GRN subtype, we observed a distinct role for immune processes related to endothelial cells and for mitochondrial dysregulation in neurons. For the MAPT subtype, we observed distinct involvement of dysregulated RNA processing, oligodendrocyte dysfunction, and axonal impairments. Comparison with an in-house protein signature of Alzheimer's disease brains indicated that the observed alterations in RNA processing and oligodendrocyte function are distinct for the frontotemporal dementia MAPT subtype. Taken together, our results indicate the involvement of different brain cell types and biological mechanisms in genetic subtypes of frontotemporal dementia. Furthermore, we demonstrate that comparison of proteomic profiles of different disease entities can separate general neurodegenerative processes from disease-specific pathways, which may aid the development of disease subtype-specific treatment strategies.

Parkinsonian phenotype in Machado-Joseph disease (MJD/SCA3): a two-case report.

BACKGROUND: Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder of late onset, which is caused by a CAG repeat expansion in the coding region of the ATXN3 gene. This disease presents clinical heterogeneity, which cannot be completely explained by the size of the repeat tract. MJD presents extrapyramidal motor signs, namely parkinsonism, more frequently than the other subtypes of autosomal dominant cerebellar ataxias. Although parkinsonism seems to segregate within MJD families, only a few MJD patients develop parkinsonian features and, therefore, the clinical and genetic aspects of these rare presentations remain poorly investigated. The main goal of this work was to describe two MJD patients displaying the parkinsonian triad (tremor, bradykinesia and rigidity), namely on what concerns genetic variation in Parkinson's disease (PD) associated loci (PARK2, LRRK2, PINK1, DJ-1, SNCA, MAPT, APOE, and mtDNA tRNA(Gln) T4336C). CASE PRESENTATION: Patient 1 is a 40 year-old female (onset at 30 years of age), initially with a pure parkinsonian phenotype (similar to the phenotype previously reported for her mother). Patient 2 is a 38 year-old male (onset at 33 years of age), presenting an ataxic phenotype with parkinsonian features (not seen either in other affected siblings or in his father). Both patients presented an expanded ATXN3 allele with 72 CAG repeats. No PD mutations were found in the analyzed loci. However, allelic variants previously associated with PD were observed in DJ-1 and APOE genes, for both patients. CONCLUSIONS: The present report adds clinical and genetic information on this particular and rare MJD presentation, and raises the hypothesis that DJ-1 and APOE polymorphisms may confer susceptibility to the parkinsonian phenotype in MJD.

The Netherlands Twin Register biobank: a resource for genetic epidemiological studies.

In 2004 the Netherlands Twin Register (NTR) started a large scale biological sample collection in twin families to create a resource for genetic studies on health, lifestyle and personality. Between January 2004 and July 2008, adult participants from NTR research projects were invited into the study. During a home visit between 7:00 and 10:00 am, fasting blood and morning urine samples were collected. Fertile women were bled on day 2-4 of the menstrual cycle, or in their pill-free week. Biological samples were collected for DNA isolation, gene expression studies, creation of cell lines and for biomarker assessment. At the time of blood sampling, additional phenotypic information concerning health, medication use, body composition and smoking was collected. Of the participants contacted, 69% participated. Blood and urine samples were collected in 9,530 participants (63% female, average age 44.4 (SD 15.5) years) from 3,477 families. Lipid profile, glucose, insulin, HbA1c, haematology, CRP, fibrinogen, liver enzymes and creatinine have been assessed. Longitudinal survey data on health, personality and lifestyle are currently available for 90% of all participants. Genome-wide SNP data are available for 3,524 participants, with additional genotyping ongoing. The NTR biobank, combined with the extensive phenotypic information available within the NTR, provides a valuable resource for the study of genetic determinants of individual differences in mental and physical health. It offers opportunities for DNA-based and gene expression studies as well as for future metabolomic and proteomic projects.

Clinical and pathologic phenotype of a large family with heterozygous STUB1 mutation.

OBJECTIVE: To describe the clinical and pathologic features of a novel pedigree with heterozygous STUB1 mutation causing SCA48. METHODS: We report a large pedigree of Dutch decent. Clinical and pathologic data were reviewed, and genetic analyses (whole-exome sequencing, whole-genome sequencing, and linkage analysis) were performed on multiple family members. RESULTS: Patients presented with adult-onset gait disturbance (ataxia or parkinsonism), combined with prominent cognitive decline and behavioral changes. Whole-exome sequencing identified a novel heterozygous frameshift variant c.731_732delGC (p.C244Yfs*24) in STUB1 segregating with the disease. This variant was present in a linkage peak on chromosome 16p13.3. Neuropathologic examination of 3 cases revealed a consistent pattern of ubiquitin/p62-positive neuronal inclusions in the cerebellum, neocortex, and brainstem. In addition, tau pathology was present in 1 case. CONCLUSIONS: This study confirms previous findings of heterozygous STUB1 mutations as the cause of SCA48 and highlights its prominent cognitive involvement, besides cerebellar ataxia and movement disorders as cardinal features. The presence of intranuclear inclusions is a pathologic hallmark of the disease. Future studies will provide more insight into its pathologic heterogeneity.

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6‒8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition.

BACKGROUND: The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. METHODS: Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. RESULTS: The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. CONCLUSIONS: Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

Genotypic and phenotypic characteristics of Dutch patients with early onset Parkinson's disease.

Early onset Parkinson's disease (EOPD) has been associated with mutations in the Parkin, DJ-1, PINK1, LRRK2, and SNCA genes. The aim of this study is to assess the contribution of these genes in a Dutch EOPD cohort and the phenotypic characteristics of the mutation carriers. A total of 187 unrelated Dutch EOPD patients (age at onset < or = 50 years) were phenotyped and screened for mutations in all exons of Parkin, DJ-1, and PINK1 by direct sequencing and gene dosage analysis. Additionally, analysis of the A30P mutation and exon dosage of SNCA and sequencing of exons 19,31,35,38,41, and 48 of LRRK2 was performed. Pathogenic variations could explain disease in 4% (7 of 187) of the patients including five patients carrying homozygous or compound heterozygous mutations in Parkin, one with a novel homozygous deletion in DJ-1 (P158Del) and one with a heterozygous mutation in LRRK2 (T2356I). We found seven novel mutations. The phenotypic characteristics of mutation carriers varied widely, comparable to the variability seen in sporadic EOPD. Parkin is the most frequently mutated gene in this EOPD cohort, followed by DJ-1, PINK1 and LRRK2. The low overall mutation frequency indicates that the extrapolation of mutation frequencies from other populations should be applied with caution.

A Functional polymorphism under positive evolutionary selection in ADRB2 is associated with human intelligence with opposite effects in the young and the elderly.

Comparative genomics offers a novel approach to unravel the genetic basis of complex traits. We performed a two stage analysis where genes ascertained for enhanced protein evolution in primates are subsequently searched for the presence of non-synonymous coding SNPs in the current human population at amino acid sites that differ between humans and chimpanzee. Positively selected genes among primates are generally presumed to determine phenotypic differences between humans and chimpanzee, such as the enhanced cognitive ability of our species. Amino acid substitutions segregating in humans at positively selected amino acid sites are expected to affect phenotypic differences among humans. Therefore we conducted an association study in two family based cohorts and one population based cohort between cognitive ability and the most likely candidate gene among the five that harbored more than one such polymorphism. The derived, human-specific allele of the beta-2 adrenergic receptor Arg16Gly polymorphism was found to be the increaser allele for performance IQ in the young, family based cohort but the decreaser allele for two different measures of cognition in the large Scottish cohort of unrelated individuals. The polymorphism is known to affect signaling activity and modulation of beta-2 adrenergic signaling has been shown to adjust memory consolidation, a trait related to cognition. The opposite effect of the polymorphism on cognition in the two age classes observed in the different cohorts resembles the effect of ADRB2 on hypertension, which also has been reported to be age dependent. This result illustrates the relevance of comparative genomics to detect genes that are involved in human behavior.