Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.

Parkinson's disease (PD; OMIM #168600) is the second most common neurodegenerative disorder in the Western world and presents as a progressive movement disorder. The hallmark pathological features of PD are loss of dopaminergic neurons from the substantia nigra and neuronal intracellular Lewy body inclusions. Parkinsonism is typically sporadic in nature; however, several rare familial forms are linked to genetic loci, and the identification of causal mutations has provided insight into the disease process. PARK8, identified in 2002 by Funayama and colleagues, appears to be a common cause of familial PD. We describe here the cloning of a novel gene that contains missense mutations segregating with PARK8-linked PD in five families from England and Spain. Because of the tremor observed in PD and because a number of the families are of Basque descent, we have named this protein dardarin, derived from the Basque word dardara, meaning tremor.

No abstract

We have established that the frequency of LRRK2 mutations in a series of 118 cases of familial Parkinson's disease is 5.1%. In the largest family with autosomal dominant, late-onset Parkinson's disease where affected subjects share a Y1699C missense mutation we provide a detailed clinical, pathological and imaging report. The phenotype in this large British kindred included asymmetrical, levodopa-responsive parkinsonism where unilateral leg tremor at onset and foot dystonia were prominent features. There was no significant abnormality of cognition but there was prominent behavioural disorder. We observed a lower age of onset in successive generations. Histopathology in one patient showed substantia nigra cell loss and Lewy body formation, with small numbers of cortical Lewy bodies. 18F-dopa positron emission tomography (PET) in another patient showed a pattern of nigrostriatal dysfunction typical of idiopathic Parkinson's disease. 18F-dopa-PET scans in unaffected family members prior to identifying the disease locus did not detect subclinical nigrostriatal dysfunction. Olfaction was assessed in affected subjects and Lewy bodies were identified in the olfactory bulb as well as cortex and brainstem of one deceased patient. In order to assess the role of mutations in this gene in other familial cases we undertook a mutation screen of all 51 exons of LRRK2 in 117 other smaller British kindreds with familial Parkinson's disease. The commonest mutation was G2019S and we also identified two novel mutations, R1941H and T2356I, in the coding sequence. These data suggest that parkinsonism caused by mutations in LRRK2 is likely to represent the commonest locus for autosomal dominant Parkinson's disease with a phenotype, pathology and in vivo imaging similar to idiopathic, late-onset Parkinson's disease.

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis. Keywords: autosomal-recessive juvenile parkinsonism, Lewy body, microtubule organizing centre, mitochondrial processing peptidase, Parkinson's disease, ubiquitin-proteasome system J. Neurochem. E-mail: d.latchman@bbk.ac.uk or n.wood@ion.ucl.ac.uk Abbreviations used: AR-JP, autosomal-recessive juvenile parkinsonism; BCA, bicinchoninic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate; DMSO, dimethylsulphoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; hsp70, heat-shock protein 70; LB, Lewy body; MnSOD, manganese superoxide dismutase; MTOC, microtubule organizing centre; PBS, phosphate-buffered saline; PD, Parkinson's disease; PINK1, PTENinduced putative kinase 1; RIPA, radioimmunoprecipitation assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; Tim, translocase of the inner membrane; UPS, ubiquitin-proteasome system; VDAC1, voltage dependent anion channel 1.

Understanding the genetic architecture of disease is an enormous challenge, and should be guided by evolutionary principles. Recent studies in evolutionary genetics show that sexual selection can have a profound influence on the genetic architecture of complex traits. Here, we summarise data from heritability studies and genome-wide association studies (GWASs) showing that common genetic variation influences many diseases and medically relevant traits in a sex-dependent manner. In addition, we discuss how the discovery of sex-dependent effects in population samples is improved by joint interaction analysis (rather than separate-sex), as well as by recently developed software. Finally, we argue that although genetic variation that has sex-dependent effects on disease risk could be maintained by mutation-selection balance and genetic drift, recent evidence indicates that intra-locus sexual conflict could be a powerful influence on complex trait architecture, and maintain sex-dependent disease risk alleles in a population because they are beneficial to the opposite sex.

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.

The divergent reproductive roles of males and females favour different phenotypes 1,2 . However, responses to these selective pressures are constrained by a shared genome, leading to 'sexual antagonism' where different alleles at given loci are favoured in the two sexes 1,3-5 . Sexual antagonism is taxonomically widespread and imposes an important evolutionary constraint on adaptation 1,4 . It also maintains genetic variation with sexspecific deleterious effects 6,7 , contributing to disease susceptibility in humans 8,9 . Yet, despite its prevalence and importance, we know virtually nothing about the evolutionary dynamics of sexually antagonistic variation [10][11][12] or the general properties of the biological processes underlying antagonistic loci 13 . Here we report the first genomewide identification and characterisation of sexually antagonistic SNPs in a population of D. melanogaster. We identify 4,899 antagonistic SNPs, distributed in 1,462 local clusters across the genome. Contrary to longstanding predictions 3 , we show that these loci are significantly underrepresented on the X chromosome. Comparative analyses reveal that sexual antagonism exerts widespread and evolutionarily persistent constraints on the species' genome. Thus, antagonistic loci generate a detectable signature of balancing selection in populations across the distribution range of D. melanogaster, and often segregate as extended haplotypes with homogeneous sex-specific fitness effects. In functional terms, we find that sexual antagonism is rooted in the regulation of development and associated primarily with cis-regulatory elements. We further detect multiple associations with the sexual differentiation cascade, including the gene fruitless, a major driver of neuronal and behavioural differentiation between males and females. This reveals that sexual antagonism resides at the core of sex-specific development. Collectively, these results provide unprecedented insights into the biology and evolution of sexual antagonism, and open opportunities for more detailed studies of constraints on sex-specific adaptation and mechanisms that resolve them.A wealth of quantitative genetic studies has established sexual antagonism as near ubiquitous across a wide range of taxa, including mammals 14 birds 15 , reptiles 16 , insects 17,18 , fish 13,19 and plants 20 . Accordingly, sexual antagonism can be considered a major constraint on adaption and an important mechanism for the maintenance of fitness variation within populations 6 . However, despite its evolutionary importance, we have little understanding of the biological mechanisms underlying this conflict and virtually no empirical data on the identity and evolutionary dynamics of antagonistic alleles 13 . While a small number of individual antagonistic loci have been identified 13,19 , these are of limited use for elucidating general properties of loci experiencing sexual antagonism. On a genome-wide scale, previous transcriptomic work has associated antagonistic fitness effects with patterns o...