P arkinson's disease (PD) is the second most prevalent neurodegenerative disorder, but the etiology remains poorly understood (1, 2). Patients with PD suffer from rigidity, slowness of movement, tremor, and disturbances of balance (1, 2). PD is characterized by the progressive loss of dopamine-containing neurons in the substantia nigra pars compacta (3, 4) and the accumulation of Lewy bodies, proteinaceous intracytoplasmic accumulations of eosinophilic material that stain for ubiquitin (5). Novel insights into the molecular mechanisms of the pathogenesis of PD have come from the identification of genes associated with rare forms of familial PD (6). Mutations in ␣-synuclein (A53T and A30P) are linked to autosomal dominant PD (7,8). This led to the discovery that ␣-synuclein is a major component of Lewy bodies and suggests that derangements in ␣-synuclein could be a major cause or contributor to the pathogenesis of sporadic PD (9, 10). Consistent with this notion are observations that overexpression of ␣-synuclein in transgenic fruit flies and mice causes a Parkinsonian phenotype and replicates many of the pathological features of PD (11-13).Other autosomal dominant genes or loci linked to PD have been described and include a mutation (I93M) in ubiquitin carboxyl-terminal-hydrolase-L1 (14), a member of the ubiquitin C-terminal hydrolase family that hydrolyzes small C-terminal adducts of ubiquitin to generate ubiquitin monomers and is involved in facilitating the degradation and processing of proteins through the 26 S proteasome. Thus, derangements in ubiquitin processing may be linked to the pathogenesis of PD. Linkages to chromosome 2P and 4P have been described, as well as yet to be identified loci, but the genes await identification (15-17).Mutations in the Parkin gene are responsible for autosomal recessive PD (18). Several Parkin-associated pedigrees have been described with both deletions and point mutations, as well as compound heterozygosity causing autosomal recessive PD (19)(20)(21). Recent studies suggest that mutations in Parkin are the major cause of autosomal recessive familial PD (19); thus, understanding the function of Parkin and how mutations interfere with the function of Parkin may provide novel insights into the pathogenesis of PD. The function of the Parkin protein remains unknown. However, Parkin shows mild homology to ubiquitin at the N terminus and contains two ring-finger motifs and an in-between ring-finger (IBR) domain at the C terminus (22). Recently, a few proteins with ring-finger motifs similar to Parkin were shown to be involved in E2-dependent ubiquitination (23-26). Ubiquitination requires the ATP-dependent activation of ubiquitin by the ubiquitin-activating enzyme E1. Ubiquitin is transferred to an E2 ubiquitin-conjugating enzyme, which works in conjunction with an E3 ubiquitin-protein ligase to ubiquitinate substrate proteins (23,24). The existence of two ring-finger motifs and the N-terminal homology to ubiquitin suggests that Parkin may be involved in the ubiquitination pathw...

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.

OBJECTIVES: Sequencing of cell-free fetal DNA from maternal plasma, also known as non-invasive prenatal testing (NIPT), has enabled accurate prenatal diagnosis of aneuploidy. This approach is gaining clinical acceptance, as it significantly reduces the necessity of invasive diagnostic procedures such as amniocentesis that carry a significant risk of fetal loss. Recent studies have demonstrated the potential for NIPT to detect subchromosomal abnormalities. We investigated whether NIPT using semiconductor sequencing could reliably detect subchromosomal deletions and duplications in a large population of women carrying high-risk fetuses. METHODS: We applied a sliding window method to reduce required sequencing depth. First, we demonstrated that increasing concentration of abnormal DNA as well as increasing number of sequencing reads improved detection of chromosomal abnormalities. We then analyzed plasma from 1456 pregnant women to develop a method to predict the fraction of fetal DNA based on the size distribution of DNA fragments (r = 0.818, p<2.2e À 16, linear regression model). Finally, we collected plasma from 938 of pregnant women having a fetus with structural abnormalities detected on ultrasound who also underwent amniocentesis, chorionic villous sampling, or umbilical cord blood sampling. We sequenced these samples using from 3M up to 15M reads to detect subchromosomal abnormalities, in parallel with array comparative genomic hybridization performed on each invasively-derived sample as a comparison. RESULTS: In total, 100% (57/57) instances of aneuploidy and 36/40 (90%) of samples with subchromosomal abnormalities greater than 5MB in size were detected using only 3 million (3 M) reads, while 5M reads was required to detect two more samples subchromosomal abnormalities greater than 5MB in size and 12/19 samples with chromosomal abnormalities between 1.5 and 5 MB. While increasing sequencing depth up to 15M reads increased accuracy for larger deletions/duplications, 88.1% (52/59) abnormalities larger than 1.5M could be detected using this method, while 5 abnormalities under 1.5MB were not reliably detected. CONCLUSIONS: This study demonstrates the viability of NIPT using semiconductor sequencing to accurately detect subchromosomal abnormalities greater than 1.5MB in a high-risk population.