Early-onset Parkinson's disease (PD) has been associated with different mutations in the Parkin gene (PARK2). To study distribution and type of Parkin mutations, we carried out a comprehensive literature review that demonstrated two prominent types of mutations among 379 unrelated mutation carriers: exon rearrangements involving exon 3, 4, or both, and alterations in exons 2 and 7, suggesting mutational hot spots or founders. To elucidate the origin of 14 recurrent Parkin mutations in our samples, we carried out a detailed haplotype analysis at the PARK2 locus. Thirty-eight mutation-positive individuals, available family members, and 62 mutation-negative individuals were genotyped. We determined allele frequencies and linkage disequilibrium (LD) to evaluate the significance of shared haplotypes. We observed no LD between markers at PARK2. Our data support a common founder for the most frequent Parkin point mutation (924C>T; exon 7) and indicate a mutational hot spot as cause of a common small deletion (255/256delA; exon 2). Furthermore, the most frequent Parkin exon deletion (Ex4del) arose independently in 2 of our subjects. However, it also occurred as the result of a founder mutation in 2 cases that shared identical deletion break points. This study provides evidence for both mutational hot spots and founder mutations as a source of recurrent mutations in Parkin, regardless of the mutation type.

Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown. We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli. Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations. Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

Early-onset parkinsonism (EOP) may be associated with different mutations in the parkin gene, including exon deletions and duplications. To test for gene dosage alterations, we developed a new method of quantitative duplex PCR using the fluorescence resonance energy transfer technique on the LightCycler (Roche Diagnostics). In 21 patients with EOP, three mutations (a single base pair substitution in exon 3 and small deletions in exon 9) were detected by conventional mutational screening (single-strand conformation polymorphism and sequence analysis), while alterations of gene dosage were found in seven patients. We identified heterozygous and compound heterozygous deletions of exons 2, 3, 5 and 7. The latter was also found in the homozygous state. In addition, two heterozygous duplications of exon 4 were observed. Remarkably, two patients carried more than two parkin mutations. This is the first study systematically screening all 12 exons of parkin by real-time, kinetic quantification and clearly shows that mutational analysis of the parkin gene should include gene dosage studies. Furthermore, our method of quantitative PCR is easily applicable to any other gene to be screened for deletions or duplications of whole exons.

The authors systematically screened all 12 Parkin exons by quantitative PCR and conventional methods in 50 probands. Eight mutations were newly reported, 2 of which are localized in exon 1, and 38% of the mutations were gene dosage alterations. These results underline the need to screen all exons and to undertake gene dosage studies. Furthermore, this study reveals a frequency of heterozygous mutation carriers that may signify a unique mode of inheritance and expression of the Parkin gene.

All Mendelian hypertension syndromes described to date involve increased sodium reabsorption in the distal nephron. 5 The sole exception is autosomal-dominant hypertension with BDE (HTNB, OMIM #112410), first reported in a Turkish kindred. 2,6 HTNB was linked to chromosome 12p in six unrelated families. 2,7,8 The locus accounts for a ~50 mm Hg mean blood pressure difference at age 50 years. 2 The penetrance is 100% (Fig. 1a). Previously, we reported a rearrangement on chromosome 12p common to all families. 8,9 A linkage study in Chinese hypertensive families without BDE coincided with the HTNB locus, supporting relevance to essential hypertension. 10 Whole-genome sequencing of Turkish family members revealed a heterozygous missense mutation in PDE3A (Gene ID: 5139), a gene encoding a cGMP/cAMP phosphodiesterase with a prominent role in the heart, VSMC, oocytes and platelets. 11 Resequencing of all 48 affected persons in six unrelated families identified six independently clustered heterozygous missense mutations in exon 4 (Fig. 1a, b Supplementary Fig. 1).We detected none of the previously described chromosomal breakpoints on chromosome 12p12.2-12.1, perhaps due to high repetitive content in the breakpoint regions Fig. 2a-c). 4 A haplotype analysis identified a novel recombination that reduced the linkage interval and eliminated an inversion common to all affected individuals in the six families (Fig. 2c). 9 In contrast, the affected mother's haplotype showed co-segregation with the more severe brachydactyly phenotype.PDEs are involved during early stages of osteogenesis. 12 PDE4D mutations have been associated with severe brachydactyly in acrodysostosis. 13,14 In mice, Pde3a was expressed in the developing limbs, consistent with a role during chondrogenesis (Fig. 2d, Supplementary Fig. 3a, b). Chondrogenic downregulation of PTHLH encoding PTHrP was associated with BDE. 15 We also observed PTHLH downregulation in chondrogenically induced fibroblasts from affected persons (Fig. 2e, Supplementary Fig. 3c).We addressed the functional consequences of the identified PDE3A mutations in HeLa cells expressing the six mutations. Forskolin or L-arginine stimulated the adenylate or guanylate cyclases to enhance cellular cAMP or cGMP levels, respectively. 16,17 We detected significantly reduced cAMP levels, consistent with gain-of-function mutations with no change in cGMP levels for the PDE3A mutations ( Supplementary Fig. 4a, b). Three PDE3A isoforms, PDE3A1 (microsomal), PDE3A2 and PDE3A3 (microsomal and cytosolic), have been identified in human myocardium. 18,19 PDE3A3 does not contain the sequence harboring the detected mutations. The predominant isoform in VSMC is PDE3A2. 18,20 To directly elucidate the mutations' effects, we compared the Michaelis-Menten kinetics of cAMPhydrolytic activity for recombinant T445N FLAG-tagged PDE3A1 and PDE3A1-WT and the tagged A2 isoforms purified from transfected cells (Fig. 3a, b, Supplementary Fig. 4d-k). The T445N mutation increased the affinity of both enzyme's isoforms for cAM...