RESUMO
Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7-2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.
RESUMO
BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.
Assuntos
Surdez , Peixe-Zebra , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Surdez/genética , Endocitose , Humanos , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Mutação , Proteinúria/metabolismo , Proteínas de Transporte Vesicular/genética , Adulto Jovem , Peixe-Zebra/metabolismoRESUMO
People exposed to liver ischaemia reperfusion (IR) injury often develop acute kidney injury and the combination is associated with significant morbidity and mortality. Molecular mediators released by the liver in response to IR injury are the likely cause of acute kidney injury (AKI) in this setting, but the mediators have not yet been identified. Identifying the mechanism of injury will allow the identification of therapeutic targets which may modulate both liver IR injury and AKI following liver IR injury.
RESUMO
BACKGROUND: FAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel-renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low-abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism. METHOD: This study used mass spectrometry-driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions. RESULTS: Shotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC-MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism. CONCLUSION: Using mass spectrometry-driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk.
Assuntos
Cálcio/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Redes Reguladoras de Genes/fisiologia , Lactação/metabolismo , Leite Humano/metabolismo , Proteômica/métodos , Cálcio/análise , Proteínas do Esmalte Dentário/análise , Proteínas do Esmalte Dentário/genética , Feminino , Humanos , Lactação/genética , Leite Humano/químicaRESUMO
HNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). Here, we find that expression of the conserved fly ortholog dHNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type dHNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. Our data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Síndrome de Fanconi/genética , Genes Dominantes , Fator 4 Nuclear de Hepatócito/genética , Animais , Morte Celular , Linhagem Celular , Núcleo Celular/metabolismo , Reprogramação Celular/genética , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mutação/genética , Néfrons/metabolismo , Néfrons/patologia , Fenótipo , Proteólise , Transdução de SinaisRESUMO
BACKGROUND: Steroid-sensitive nephrotic syndrome (SSNS), the most common form of nephrotic syndrome in childhood, is considered an autoimmune disease with an established classic HLA association. However, the precise etiology of the disease is unclear. In other autoimmune diseases, the identification of loci outside the classic HLA region by genome-wide association studies (GWAS) has provided critical insights into disease pathogenesis. Previously conducted GWAS of SSNS have not identified non-HLA loci achieving genome-wide significance. METHODS: In an attempt to identify additional loci associated with SSNS, we conducted a GWAS of a large cohort of European ancestry comprising 422 ethnically homogeneous pediatric patients and 5642 ethnically matched controls. RESULTS: The GWAS found three loci that achieved genome-wide significance, which explain approximately 14% of the genetic risk for SSNS. It confirmed the previously reported association with the HLA-DR/DQ region (lead single-nucleotide polymorphism [SNP] rs9273542, P=1.59×10-43; odds ratio [OR], 3.39; 95% confidence interval [95% CI], 2.86 to 4.03) and identified two additional loci outside the HLA region on chromosomes 4q13.3 and 6q22.1. The latter contains the calcium homeostasis modulator family member 6 gene CALHM6 (previously called FAM26F). CALHM6 is implicated in immune response modulation; the lead SNP (rs2637678, P=1.27×10-17; OR, 0.51; 95% CI, 0.44 to 0.60) exhibits strong expression quantitative trait loci effects, the risk allele being associated with lower lymphocytic expression of CALHM6. CONCLUSIONS: Because CALHM6 is implicated in regulating the immune response to infection, this may provide an explanation for the typical triggering of SSNS onset by infections. Our results suggest that a genetically conferred risk of immune dysregulation may be a key component in the pathogenesis of SSNS.
Assuntos
Canais de Cálcio/genética , Glicoproteínas de Membrana/genética , Síndrome Nefrótica/genética , Esteroides/uso terapêutico , Alelos , Proteína de Ligação a Androgênios/genética , Criança , Bases de Dados Factuais , Epitopos/química , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Cadeias alfa de HLA-DQ/genética , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Humanos , Sistema Imunitário , Masculino , Síndrome Nefrótica/tratamento farmacológico , Razão de Chances , Peptídeos/química , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.
Assuntos
Amidinotransferases/genética , Síndrome de Fanconi/genética , Falência Renal Crônica/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Idoso , Amidinotransferases/metabolismo , Animais , Simulação por Computador , Síndrome de Fanconi/complicações , Síndrome de Fanconi/metabolismo , Síndrome de Fanconi/patologia , Feminino , Heterozigoto , Humanos , Lactente , Inflamassomos/metabolismo , Falência Renal Crônica/etiologia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Masculino , Camundongos , Camundongos Knockout , Conformação Molecular , Mutação , Mutação de Sentido Incorreto , Linhagem , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de DNA , Adulto JovemRESUMO
Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic ß cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2 We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy.
Assuntos
Hiperinsulinismo Congênito/complicações , Hiperinsulinismo Congênito/genética , Mutação , Fosfotransferases (Fosfomutases)/genética , Doenças Renais Policísticas/complicações , Doenças Renais Policísticas/genética , Regiões Promotoras Genéticas/genética , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing ß-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity, mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.
Assuntos
Síndrome de Fanconi/metabolismo , Rim/metabolismo , Rim/patologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Animais , Transporte Biológico , Extratos Celulares , Metabolismo Energético , Síndrome de Fanconi/complicações , Síndrome de Fanconi/patologia , Ácidos Graxos/metabolismo , Células LLC-PK1 , Microscopia Confocal , Doenças Mitocondriais/complicações , Doenças Mitocondriais/patologia , Mutação/genética , Oxirredução , Enzima Bifuncional do Peroxissomo/metabolismo , Proteômica , ATPase Trocadora de Sódio-Potássio/metabolismo , Frações Subcelulares/metabolismo , SuínosRESUMO
Renal Fanconi syndrome (RFS) refers to the generalized dysfunction of the proximal tubule (PT) (Kleta R. Fanconi or not Fanconi? Lowe syndrome revisited. Clin J Am Soc Nephrol 2008; 3: 1244-1245). In its isolated form, RFS only affects the PT, but not the other nephron segments. The study of isolated RFS can thus provide specific insights into the function of the PT. In a recent paper, Klootwijk et al. investigated one such form of isolated RFS and revealed the underlying molecular basis (Klootwijk ED, Reichold M, Helip-Wooley A et al. Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome. N Engl J Med 2014; 370: 129-138). The affected family had been described previously, demonstrating the typical features of RFS, such as low-molecular weight proteinuria, aminoaciduria, glycosuria and phosphaturia with consequent rickets; yet, importantly, patients had no evidence of impaired glomerular filtration (Tolaymat A, Sakarcan A, Neiberger R. Idiopathic Fanconi syndrome in a family. Part I. Clinical aspects. J Am Soc Nephrol 1992; 2: 1310-1317). Inheritance was consistent with an autosomal dominant mode. Klootwijk et al. discovered a surprising explanation: a heterozygous missense mutation causing partial mistargeting of the peroxisomal enzyme EHHADH to the mitochondria. Notably, disease causing was not the absence of the enzyme in the peroxisome, but its interference with mitochondrial function. The discovery of this novel disease mechanism not only confirmed the importance of mitochondrial function for PT transport, but also demonstrated the critical dependence of PT on fatty acid metabolism for energy generation.
Assuntos
Síndrome de Fanconi/patologia , Túbulos Renais Proximais/patologia , DNA Mitocondrial/genética , Síndrome de Fanconi/genética , Heterozigoto , Humanos , Mutação de Sentido Incorreto/genética , Enzima Bifuncional do Peroxissomo/genéticaAssuntos
Desmineralização Patológica Óssea/genética , Claudinas/genética , Hipercalciúria/genética , Mutação , Nefrocalcinose/genética , Erros Inatos do Transporte Tubular Renal/genética , Adulto , Humanos , Hipocalcemia/genética , Hipoparatireoidismo/congênito , Hipoparatireoidismo/genética , Falência Renal Crônica/genética , MasculinoRESUMO
BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).
Assuntos
Síndrome de Fanconi/genética , Túbulos Renais Proximais/metabolismo , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Enzima Bifuncional do Peroxissomo/genética , Sequência de Aminoácidos , Animais , População Negra , Cromossomos Humanos Par 3 , Modelos Animais de Doenças , Síndrome de Fanconi/etnologia , Feminino , Ligação Genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Linhagem , Enzima Bifuncional do Peroxissomo/química , Enzima Bifuncional do Peroxissomo/metabolismo , Fenótipo , Análise de Sequência de DNARESUMO
BACKGROUND/AIMS: Mutations in the inwardly-rectifying K(+)-channel KCNJ10/Kir4.1 cause autosomal recessive EAST syndrome (epilepsy, ataxia, sensorineural deafness and tubulopathy). KCNJ10 is expressed in the distal convoluted tubule of the kidney, stria vascularis of the inner ear and brain glial cells. Patients diagnosed clinically with EAST syndrome were genotyped and mutations in KCNJ10 were studied functionally. METHODS: Patient DNA was amplified and sequenced, and new mutations were identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K(+) currents were measured by 2-electrode voltage clamping and channel expression was analysed by Western blotting. RESULTS: We identified 3 homozygous mutations in KCNJ10 (p.F75C, p.A167V and p.V91fs197X), with mutation p.A167V previously reported in a compound heterozygous state. Oocytes expressing wild-type human KCNJ10 showed inwardly rectified currents, which were significantly reduced in all of the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba(2+) demonstrated a large residual function in p.A167V only, which was not compatible with causing disease. However, co-expression with KCNJ16 abolished function in these heteromeric channels almost completely. CONCLUSION: This study provides an explanation for the pathophysiology of the p.A167V KCNJ10 mutation, which had previously not been considered pathogenic on its own. These findings provide evidence for the functional cooperation of KCNJ10 and KCNJ16. Thus, in vitro ascertainment of KCNJ10 function may necessitate co-expression with KCNJ16.
Assuntos
Perda Auditiva Neurossensorial/genética , Deficiência Intelectual/genética , Mutação Puntual , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Convulsões/genética , Alanina/genética , Animais , Feminino , Genótipo , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/química , Multimerização Proteica , Convulsões/metabolismo , Convulsões/patologia , Análise de Sequência de DNA , Valina/genética , XenopusRESUMO
BACKGROUND: Arterial calcification (AC) is a major health problem associated with extreme morbidity and a shortened survival. It is currently without any effective treatment. ATP and the purinergic system in general are now emerging as being important in the pathogenesis of AC and potentially provide a new focus for novel therapies. METHODS: This review systematically analyses and discusses the current literature examining the relevance of the purinergic system to AC. Particular emphasis is given to the enzymes associated with ATP metabolism and their role in maintaining a balance between promotion and inhibition of arterial mineralization. Points of controversy are highlighted, and areas for future research are suggested. CONCLUSION: The potential roles of ATP and the purinergic system in AC are beginning to be elucidated. While further work is necessary, current knowledge suggests that several components of the purinergic system could be targeted to develop new treatments for AC.
Assuntos
Trifosfato de Adenosina/fisiologia , Receptores Purinérgicos/metabolismo , Calcificação Vascular/metabolismo , Artérias , HumanosRESUMO
Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.
Assuntos
Modelos Animais de Doenças , Nefropatias/genética , Animais , Biomarcadores/metabolismo , Carboidratos Epimerases/genética , Proteínas de Transporte/genética , Suplementos Nutricionais , Avaliação Pré-Clínica de Medicamentos/métodos , Hexosaminas/uso terapêutico , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/embriologia , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Mutação , Ácido N-Acetilneuramínico/fisiologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sialoglicoproteínas/metabolismoRESUMO
BACKGROUND/AIMS: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood. METHODS: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing. RESULTS: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified. CONCLUSIONS: This autosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.
Assuntos
Amelogênese Imperfeita/genética , Proteínas do Esmalte Dentário/genética , Predisposição Genética para Doença/genética , Mutação , Nefrocalcinose/genética , Adolescente , Adulto , Amelogênese Imperfeita/complicações , Amelogênese Imperfeita/patologia , Criança , Consanguinidade , Exoma/genética , Saúde da Família , Feminino , Genes Recessivos/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Nefrocalcinose/complicações , Nefrocalcinose/patologia , Linhagem , Análise de Sequência de DNA/métodos , Síndrome , Adulto JovemRESUMO
Renal fibromuscular dysplasia (FMD) can cause hypertension, and previous reports suggest that FMD is familial. We hypothesized that, in families containing an individual with proven FMD, relatives of index cases would have an increased risk of hypertension. ACTA2 mutations cause a spectrum of extra-renal arteriopathy, leading to our second hypothesis that mutations are implicated in FMD. The blood pressure of first-degree relatives was measured using standard devices and, when indicated, with 24-h ambulatory monitoring. Leucocyte DNA was obtained from FMD index cases and ACTA2 sequenced. Thirteen unrelated index cases, aged 2-32 (median 15) years, were recruited. Blood pressure was assessed in 40 first-degree relatives, comprising 22 parents aged 28-58 (median 44) years and 18 siblings aged 3-30 (median 13) years. Hypertension was evident in six (27%) parents but in none of the eight adult siblings. Of the ten screened siblings aged less than 18 years, one teenager was pre-hypertensive (90th-95th centile), the remainder being normotensive. No ACTA2 mutations were found in 13 index cases. Hypertension was evident in 20% of all assessed adult first-degree relatives and is therefore not increased relative to 25% of the adult population. Although hypertensive parents did not undergo angiography to assign FMD status, this observation, together with the lack of hypertension in 18 siblings, indicates that FMD is unlikely to confer an excess hypertension risk in first-degree relatives up to middle-age. Furthermore, in our cohort, FMD was not caused by ACTA2 mutations.
Assuntos
Actinas/genética , Displasia Fibromuscular/complicações , Displasia Fibromuscular/genética , Hipertensão/epidemiologia , Hipertensão/etiologia , Adolescente , Adulto , Idade de Início , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Criança , Pré-Escolar , Estudos de Coortes , DNA/genética , Família , Feminino , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Linhagem , Circulação Renal/fisiologia , Irmãos , Adulto JovemRESUMO
3-Methylglutaconic aciduria type III (3-MGCA type III), caused by recessive mutations in the 2-exon gene OPA3, is characterized by early-onset bilateral optic atrophy, later-onset extrapyramidal dysfunction, and increased urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. Here we report the identification of a novel third OPA3 coding exon, the apparent product of a segmental duplication event, resulting in two gene transcripts, OPA3A and OPA3B. OPA3A deficiency (as in optic atrophy type 3) causes up-regulation of OPA3B. OPA3 protein function remains unknown, but it contains a putative mitochondrial leader sequence, mitochondrial sorting signal and a peroxisomal sorting signal. Our green fluorescent protein tagged OPA3 expression studies found its localization to be predominantly mitochondrial. These findings thus place the cellular metabolic defect of 3-MGCA type III in the mitochondrion rather than the peroxisome and implicate loss of OPA3A rather than gain of OPA3B in disease etiology.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Glutaratos/urina , Mitocôndrias/genética , Atrofias Ópticas Hereditárias/genética , Proteínas/genética , Erros Inatos do Metabolismo dos Aminoácidos/urina , Sequência de Aminoácidos , Éxons , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
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.