Dopamine­iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease.

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

Intracellular delivery of Parkin-RING0-based fragments corrects Parkin-induced mitochondrial dysfunction through interaction with SLP-2.

BACKGROUND: Loss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson's disease (PD). We have previously identified mitoch ondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function. METHODS: In fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function. RESULTS: Using a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations. CONCLUSIONS: These findings place further emphasis on the importance of the protein-protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein-protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.

Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson's disease.

Biallelic mutations in PINK1/PRKN cause recessive Parkinson's disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA integrity and inflammation as disease modifiers in carriers of mutations in these genes. Mitochondrial DNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson's disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mitochondrial DNA variant load (area under the curve = 0.83, CI 0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbour more heteroplasmic mitochondrial DNA variants in blood (P = 0.0006, Z = 3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in induced pluripotent stem cell-derived (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and post-mortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Last, the heteroplasmic mitochondrial DNA variant load correlated with IL6 levels in PINK1/PRKN mutation carriers (r = 0.57, P = 0.0074). PINK1/PRKN mutations predispose individuals to mitochondrial DNA variant accumulation in a dose- and disease-dependent manner.

A genome on shaky ground: exploring the impact of mitochondrial DNA integrity on Parkinson's disease by highlighting the use of cybrid models.

Mitochondria play important roles in the regulation of key cellular processes, including energy metabolism, oxidative stress response, and signaling towards cell death or survival, and are distinguished by carrying their own genome (mtDNA). Mitochondrial dysfunction has emerged as a prominent cellular mechanism involved in neurodegeneration, including Parkinson's disease (PD), a neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons and the occurrence of proteinaceous Lewy body inclusions. The contribution of mtDNA variants to PD pathogenesis has long been debated and is still not clearly answered. Cytoplasmic hybrid (cybrid) cell models provided evidence for a contribution of mtDNA variants to the PD phenotype. However, conclusive evidence of mtDNA mutations as genetic cause of PD is still lacking. Several models have shown a role of somatic, rather than inherited mtDNA variants in the impairment of mitochondrial function and neurodegeneration. Accordingly, several nuclear genes driving inherited forms of PD are linked to mtDNA quality control mechanisms, and idiopathic as well as familial PD tissues present increased mtDNA damage. In this review, we highlight the use of cybrids in this PD research field and summarize various aspects of how and to what extent mtDNA variants may contribute to the etiology of PD.

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Parkin Interacts with Apoptosis-Inducing Factor and Interferes with Its Translocation to the Nucleus in Neuronal Cells.

Mutations in the PRKN gene (encoding parkin) have been linked to the most frequent known cause of recessive Parkinson's disease (PD), and parkin dysfunction represents a risk factor for sporadic PD. Parkin is widely neuroprotective through different cellular pathways, as it protects dopaminergic neurons from apoptosis in a series of cellular and animal models of PD. The mitochondrial protein apoptosis-inducing factor (AIF) is an important cell death effector, which, upon cellular stress in many paradigms, is redistributed from the mitochondria to the nucleus to function as a proapoptotic factor, mostly independent of caspase activity, while in normal mitochondria it functions as an antiapoptotic factor. AIF is known to participate in dopaminergic neuron loss in experimental PD models and in patients with PD. We, therefore, investigated possible crosstalk between parkin and AIF. By using immunoprecipitation and proximity ligation assays, we demonstrated a physical interaction between the two proteins. Nuclear AIF translocation was significantly reduced by parkin expression in neuroblastoma SH-SY5Y cells after exposure to an apoptogenic stimulus. These results were confirmed in primary murine cortical neurons, which showed a higher nuclear translocation of AIF in parkin-deficient neurons upon an excitotoxic stimulus. Our results indicate that the interaction of parkin with AIF interferes with the nuclear translocation of AIF, which might contribute to the neuroprotective activity of parkin.

Serum iron level and kidney function: a Mendelian randomization study.

Background: Iron depletion is a known consequence of chronic kidney disease (CKD), but there is contradicting epidemiological evidence on whether iron itself affects kidney function and whether its effect is protective or detrimental in the general population. While epidemiological studies tend to be affected by confounding and reverse causation, Mendelian randomization (MR) can provide unconfounded estimates of causal effects by using genes as instruments. Methods: We performed an MR study of the effect of serum iron levels on estimated glomerular filtration rate (eGFR), using genetic variants known to be associated with iron. MR estimates of the effect of iron on eGFR were derived based on the association of each variant with iron and eGFR from two large genome-wide meta-analyses on 48 978 and 74 354 individuals. We performed a similar MR analysis for ferritin, which measures iron stored in the body, using variants associated with ferritin. Results: A combined MR estimate across all variants showed a 1.3% increase in eGFR per standard deviation increase in iron (95% confidence interval 0.4­2.1%; P = 0.004). The results for ferritin were consistent with those for iron. Secondary MR analyses of the effects of iron and ferritin on CKD did not show significant associations but had very low statistical power. Conclusions: Our study suggests a protective effect of iron on kidney function in the general population. Further research is required to confirm this causal association, investigate it in study populations at higher risk of CKD and explore its underlying mechanism of action.

Meta-analysis of genome-wide association studies identifies two loci associated with circulating osteoprotegerin levels.

Osteoprotegerin (OPG) is involved in bone homeostasis and tumor cell survival. Circulating OPG levels are also important biomarkers of various clinical traits, such as cancers and atherosclerosis. OPG levels were measured in serum or in plasma. In a meta-analysis of genome-wide association studies in up to 10 336 individuals from European and Asian origin, we discovered that variants >100 kb upstream of the TNFRSF11B gene encoding OPG and another new locus on chromosome 17q11.2 were significantly associated with OPG variation. We also identified a suggestive locus on chromosome 14q21.2 associated with the trait. Moreover, we estimated that over half of the heritability of OPG levels could be explained by all variants examined in our study. Our findings provide further insight into the genetic regulation of circulating OPG levels.

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations.

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10(-204)) and 10 loci for sphingolipids (smallest P-value = 3.10×10(-57)). After a correction for multiple comparisons (P-value<2.2×10(-9)), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.

Haplotype analysis of the 4p16.3 region in Portuguese families with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary choreic movements, cognitive impairment, and behavioral changes, caused by the expansion of an unstable CAG repeat in HTT. We characterized the genetic diversity of the HD mutation by performing an extensive haplotype analysis of ∼1Mb region flanking HTT in over 300 HD families of Portuguese origin. We observed that haplotype A, marked by HTT delta2642, was enriched in HD chromosomes and carried the two largest expansions reported in the Portuguese population. However, the most frequent HD haplotype B carried one of the largest (+12 CAGs) expansions, which resulted in an allele class change to full penetrance. Despite having a normal CAG distribution skewed to the higher end of the range, these two core haplotypes had similar expanded CAG repeat sizes compared to the other major core haplotypes (C and D) and there was no statistical difference in transmitted repeat instability across haplotypes. We observed a diversity of HTT region haplotypes in both normal and expanded chromosomes, representative of more than one ancestral chromosome underlying HD in Portugal, where multiple independent events on distinct chromosome 4 haplotypes have given rise to expansion into the pathogenic range.

Parkin R274W mutation affects muscle and mitochondrial physiology.

Recessive mutations in the Parkin gene (PRKN) are the most common cause of young-onset inherited parkinsonism. Parkin is a multifunctional E3 ubiquitin ligase that plays a variety of roles in the cell including the degradation of proteins and the maintenance of mitochondrial homeostasis, integrity, and biogenesis. In 2001, the R275W mutation in the PRKN gene was identified in two unrelated families with a multigenerational history of postural tremor, dystonia and parkinsonism. Drosophila models of Parkin R275W showed selective and progressive degeneration of dopaminergic neuronal clusters, mitochondrial abnormalities, and prominent climbing defects. In the Prkn mouse orthologue, the amino acid R274 corresponds to human R275. Here we described an age-related motor impairment and a muscle phenotype in R274W +/+ mice. In vitro, Parkin R274W mutation correlates with abnormal myoblast differentiation, mitochondrial defects, and alteration in mitochondrial mRNA and protein levels. Our data suggest that the Parkin R274W mutation may impact mitochondrial physiology and eventually myoblast proliferation and differentiation.

Serum iron levels and the risk of Parkinson disease: a Mendelian randomization study.

BACKGROUND: Although levels of iron are known to be increased in the brains of patients with Parkinson disease (PD), epidemiological evidence on a possible effect of iron blood levels on PD risk is inconclusive, with effects reported in opposite directions. Epidemiological studies suffer from problems of confounding and reverse causation, and mendelian randomization (MR) represents an alternative approach to provide unconfounded estimates of the effects of biomarkers on disease. We performed a MR study where genes known to modify iron levels were used as instruments to estimate the effect of iron on PD risk, based on estimates of the genetic effects on both iron and PD obtained from the largest sample meta-analyzed to date. METHODS AND FINDINGS: We used as instrumental variables three genetic variants influencing iron levels, HFE rs1800562, HFE rs1799945, and TMPRSS6 rs855791. Estimates of their effect on serum iron were based on a recent genome-wide meta-analysis of 21,567 individuals, while estimates of their effect on PD risk were obtained through meta-analysis of genome-wide and candidate gene studies with 20,809 PD cases and 88,892 controls. Separate MR estimates of the effect of iron on PD were obtained for each variant and pooled by meta-analysis. We investigated heterogeneity across the three estimates as an indication of possible pleiotropy and found no evidence of it. The combined MR estimate showed a statistically significant protective effect of iron, with a relative risk reduction for PD of 3% (95% CI 1%-6%; p = 0.001) per 10 µg/dl increase in serum iron. CONCLUSIONS: Our study suggests that increased iron levels are causally associated with a decreased risk of developing PD. Further studies are needed to understand the pathophysiological mechanism of action of serum iron on PD risk before recommendations can be made.

Genome-wide association analysis and fine mapping of NT-proBNP level provide novel insight into the role of the MTHFR-CLCN6-NPPA-NPPB gene cluster.

High blood concentration of the N-terminal cleavage product of the B-type natriuretic peptide (NT-proBNP) is strongly associated with cardiac dysfunction and is increasingly used for heart failure diagnosis. To identify genetic variants associated with NT-proBNP level, we performed a genome-wide association analysis in 1325 individuals from South Tyrol, Italy, and followed up the most significant results in 1746 individuals from two German population-based studies. A genome-wide significant signal in the MTHFR-CLCN6-NPPA-NPPB gene cluster was replicated, after correction for multiple testing (replication one-sided P-value = 8.4 × 10(-10)). A conditional regression analysis of 128 single-nucleotide polymorphisms in the region of interest identified novel variants in the CLCN6 gene as independently associated with NT-proBNP. In this locus, four haplotypes were associated with increased NT-proBNP levels (haplotype-specific combined P-values from 8.3 × 10(-03) to 9.3 × 10(-11)). The observed increase in the NT-proBNP level was proportional to the number of haplotype copies present (i.e. dosage effect), with an increase associated with two copies that varied between 20 and 100 pg/ml across populations. The identification of novel variants in the MTHFR-CLCN6-NPPA-NPPB cluster provides new insights into the biological mechanisms of cardiac dysfunction.

Novel association to the proprotein convertase PCSK7 gene locus revealed by analysing soluble transferrin receptor (sTfR) levels.

The level of body iron storage and the erythropoietic need for iron are indicated by the serum levels of ferritin and soluble transferrin receptor (sTfR), respectively. A meta-analysis of five genome-wide association studies on sTfR and ferritin revealed novel association to the PCSK7 and TMPRSS6 loci for sTfR and the HFE locus for both parameters. The PCSK7 association was the most significant (rs236918, P = 1.1 × 10E-27) suggesting that proprotein convertase 7, the gene product of PCSK7, may be involved in sTfR generation and/or iron homeostasis. Conditioning the sTfR analyses on transferrin saturation abolished the HFE signal and substantially diminished the TMPRSS6 signal while the PCSK7 association was unaffected, suggesting that the former may be mediated by transferrin saturation whereas the PCSK7-associated effect on sTfR generation appears to be more direct.

Identification of a common variant in the TFR2 gene implicated in the physiological regulation of serum iron levels.

The genetic determinants of variation in iron status are actively sought, but remain incompletely understood. Meta-analysis of two genome-wide association (GWA) studies and replication in three independent cohorts was performed to identify genetic loci associated in the general population with serum levels of iron and markers of iron status, including transferrin, ferritin, soluble transferrin receptor (sTfR) and sTfR-ferritin index. We identified and replicated a novel association of a common variant in the type-2 transferrin receptor (TFR2) gene with iron levels, with effect sizes highly consistent across samples. In addition, we identified and replicated an association between the HFE locus and ferritin and confirmed previously reported associations with the TF, TMPRSS6 and HFE genes. The five replicated variants were tested for association with expression levels of the corresponding genes in a publicly available data set of human liver samples, and nominally statistically significant expression differences by genotype were observed for all genes, although only rs3811647 in the TF gene survived the Bonferroni correction for multiple testing. In addition, we measured for the first time the effects of the common variant in TMPRSS6, rs4820268, on hepcidin mRNA in peripheral blood (n = 83 individuals) and on hepcidin levels in urine (n = 529) and observed an association in the same direction, though only borderline significant. These functional findings require confirmation in further studies with larger sample sizes, but they suggest that common variants in TMPRSS6 could modify the hepcidin-iron feedback loop in clinically unaffected individuals, thus making them more susceptible to imbalances of iron homeostasis.

A multi-centre clinico-genetic analysis of the VPS35 gene in Parkinson disease indicates reduced penetrance for disease-associated variants.

BACKGROUND: Two recent studies identified a mutation (p.Asp620Asn) in the vacuolar protein sorting 35 gene as a cause for an autosomal dominant form of Parkinson disease . Although additional missense variants were described, their pathogenic role yet remains inconclusive. METHODS AND RESULTS: We performed the largest multi-center study to ascertain the frequency and pathogenicity of the reported vacuolar protein sorting 35 gene variants in more than 15,000 individuals worldwide. p.Asp620Asn was detected in 5 familial and 2 sporadic PD cases and not in healthy controls, p.Leu774Met in 6 cases and 1 control, p.Gly51Ser in 3 cases and 2 controls. Overall analyses did not reveal any significant increased risk for p.Leu774Met and p.Gly51Ser in our cohort. CONCLUSIONS: Our study apart from identifying the p.Asp620Asn variant in familial cases also identified it in idiopathic Parkinson disease cases, and thus provides genetic evidence for a role of p.Asp620Asn in Parkinson disease in different populations worldwide.

Molecular phenotypes of mitochondrial dysfunction in clinically non-manifesting heterozygous PRKN variant carriers.

Homozygous or compound heterozygous (biallelic) variants in PRKN are causal for PD with highly penetrant symptom expression, while the much more common heterozygous variants may predispose to PD with highly reduced penetrance, through altered mitochondrial function. In the presence of pathogenic heterozygous variants, it is therefore important to test for mitochondrial alteration in cells derived from variant carriers to establish potential presymptomatic molecular markers. We generated lymphoblasts (LCLs) and human induced pluripotent stem cell (hiPSC)-derived neurons from non-manifesting heterozygous PRKN variant carriers and tested them for mitochondrial functionality. In LCLs, we detected hyperactive mitochondrial respiration, and, although milder compared to a biallelic PRKN-PD patient, hiPSC-derived neurons of non-manifesting heterozygous variant carriers also displayed several phenotypes of altered mitochondrial function. Overall, we identified molecular phenotypes that might be used to monitor heterozygous PRKN variant carriers during the prodromal phase. Such markers might also be useful to identify individuals at greater risk of eventual disease development and for testing potential mitochondrial function-based neuroprotective therapies before neurodegeneration advances.

Exome sequencing in a family with restless legs syndrome.

BACKGROUND: Restless legs syndrome (RLS) has a high familial aggregation. To date, several loci and genetic risk factors have been identified, but no causative gene mutation has been found. METHODS: We evaluated a German family with autosomal dominantly inherited RLS in 7 definitely and 2 possibly affected members by genome-wide linkage analysis and exome sequencing. RESULTS: We identified three novel missense and one splice site variant in the PCDHA3, WWC2, ATRN, and FAT2 genes that segregated with RLS in the family. All four exons of the PCDHA3 gene, the most plausible candidate, were sequenced in 64 unrelated RLS cases and 250 controls. This revealed three additional rare missense variants (frequency <1%) of unknown pathogenicity in 2 patients and 1 control. CONCLUSIONS: We present the first next-generation sequencing study on RLS and suggest PCDHA3 as a candidate gene for RLS.

Genetic determinants of circulating sphingolipid concentrations in European populations.

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08x10(-66). The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4) or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases.