Robert Kleta

Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

Epilepsy, Ataxia, Sensorineural Deafness, Tubulopathy, and KCNJ10 Mutations

BACKGROUND Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy). METHODS Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice. RESULTS Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting. CONCLUSIONS Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.

Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy.

BACKGROUND Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population. METHODS We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method). RESULTS In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). CONCLUSIONS An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.

Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-associated mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Mutations in SLC6A19 , encoding B 0 AT1, cause Hartnup disorder

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B0AT1 (ref. 4). We isolated the human homolog of B0AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is characterized by large platelets that lack α-granules. Here we show that mutations in NBEAL2 (neurobeachin-like 2), which encodes a BEACH/ARM/WD40 domain protein, cause GPS and that megakaryocytes and platelets from individuals with GPS express a unique combination of NBEAL2 transcripts. Proteomic analysis of sucrose-gradient subcellular fractions of platelets indicated that NBEAL2 localizes to the dense tubular system (endoplasmic reticulum) in platelets.

A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.

Type III 3-methylglutaconic aciduria (optic atrophy plus syndrome, or Costeff optic atrophy syndrome): Identification of the OPA3 gene and its founder mutation in Iraqi Jews

Type III 3-methylglutaconic aciduria (MGA) (MIM 258501) is a neuro-ophthalmologic syndrome that consists of early-onset bilateral optic atrophy and later-onset spasticity, extrapyramidal dysfunction, and cognitive deficit. Urinary excretion of 3-methylglutaconic acid and of 3-methylglutaric acid is increased. The disorder has been reported in approximately 40 patients of Iraqi Jewish origin, allowing the mapping of the disease to chromosome 19q13.2-q13.3, by linkage analysis. To isolate the causative gene, OPA3, we sequenced four genes within the critical interval and identified, in the intronic sequence of a gene corresponding to cDNA clone FLJ22187, a point mutation that segregated with the type III MGA phenotype. The FLJ22187-cDNA clone, which we identified as the OPA3 gene, consists of two exons and encodes a peptide of 179 amino acid residues. Northern blot analysis revealed a primary transcript of approximately 5.0 kb that was ubiquitously expressed, most prominently in skeletal muscle and kidney. Within the brain, the cerebral cortex, the medulla, the cerebellum, and the frontal lobe, compared to other parts of the brain, had slightly increased expression. The intronic G-->C mutation abolished mRNA expression in fibroblasts from affected patients and was detected in 8 of 85 anonymous Israeli individuals of Iraqi Jewish origin. Milder mutations in OPA3 should be sought in patients with optic atrophy with later onset, even in the absence of additional neurological abnormalities.

ADCK4 mutations promote steroid-Resistant nephrotic syndrome through CoQ10 biosynthesis disruption

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.

Antiphospholipase A2 Receptor Antibody Titer and Subclass in Idiopathic Membranous Nephropathy

The phospholipase A(2) receptor (PLA(2)R) is the major target antigen in idiopathic membranous nephropathy. The technique for measuring antibodies against PLA(2)R and the relationship between antibody titer and clinical characteristics are not well established. Here, we measured anti-PLA(2)R (aPLA(2)R) antibody titer and subclass in a well defined cohort of 117 Caucasian patients with idiopathic membranous nephropathy and nephrotic-range proteinuria using both indirect immunofluorescence testing (IIFT) and ELISA. We assessed agreement between tests and correlated antibody titer with clinical baseline parameters and outcome. In this cohort, aPLA(2)R antibodies were positive in 74% and 72% of patients using IIFT and ELISA, respectively. Concordance between both tests was excellent (94% agreement, κ=0.85). Among 82 aPLA(2)R-positive patients, antibody titer significantly correlated with baseline proteinuria (P=0.02). Spontaneous remissions occurred significantly less frequently among patients with high antibody titers (38% versus 4% in the lowest and highest tertiles, respectively; P<0.01). IgG4 was the dominant subclass in the majority of patients. Titers of IgG4, but not IgG1 or IgG3, significantly correlated with the occurrence of spontaneous remission (P=0.03). In summary, these data show high agreement between IIFT and ELISA assessments of aPLA(2)R antibody titer and highlight the pathogenetic role of these antibodies, especially the IgG4 subclass, given the observed relationships between aPLA(2)R titer, baseline proteinuria, and outcome.