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PURPOSE: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11% to 28% of NL and/or NC, suggesting that additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC. METHODS: Exome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious, rare OXGR1 variants were functionally characterized. RESULTS: Exome sequencing revealed a heterozygous OXGR1 missense variant (c.371T>G, p.L124R) cosegregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multigenerational family with 5 affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate [AKG]) receptor 1 in the distal nephron. In response to its ligand AKG, OXGR1 stimulates the chloride-bicarbonate exchanger, pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologs and paralogs, severe in silico prediction scores, and extreme rarity in exome population databases suggested that the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified 5 additional deleterious dominant variants in 5 families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in patients with NL/NC compared with Exome Aggregation Consortium controls (χ2 = 7.117, P = .0076). Wild-type OXGR1-expressing Xenopus oocytes exhibited AKG-responsive Ca2+ uptake. Of 5 NL/NC-associated missense variants, 5 revealed impaired AKG-dependent Ca2+ uptake, demonstrating loss of function. CONCLUSION: Rare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.
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Nefrolitíase , Receptores Purinérgicos P2 , Humanos , Oxalato de Cálcio , Nefrolitíase/genética , Mutação de Sentido Incorreto/genética , Transportadores de Sulfato/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismoRESUMO
Purpose: Modeling disease variants in animals is useful for drug discovery, understanding disease pathology, and classifying variants of uncertain significance (VUS) as pathogenic or benign. Methods: Using Clustered Regularly Interspaced Short Palindromic Repeats, we performed a Whole-gene Humanized Animal Model procedure to replace the coding sequence of the animal model's unc-18 ortholog with the coding sequence for the human STXBP1 gene. Next, we used Clustered Regularly Interspaced Short Palindromic Repeats to introduce precise point variants in the Whole-gene Humanized Animal Model-humanized STXBP1 locus from 3 clinical categories (benign, pathogenic, and VUS). Twenty-six phenotypic features extracted from video recordings were used to train machine learning classifiers on 25 pathogenic and 32 benign variants. Results: Using multiple models, we were able to obtain a diagnostic sensitivity near 0.9. Twenty-three VUS were also interrogated and 8 of 23 (34.8%) were observed to be functionally abnormal. Interestingly, unsupervised clustering identified 2 distinct subsets of known pathogenic variants with distinct phenotypic features; both p.Tyr75Cys and p.Arg406Cys cluster away from other variants and show an increase in swim speed compared with hSTXBP1 worms. This leads to the hypothesis that the mechanism of disease for these 2 variants may differ from most STXBP1-mutated patients and may account for some of the clinical heterogeneity observed in the patient population. Conclusion: We have demonstrated that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in STXBP1 and identify variant-specific intensities of aberrant activity suggesting a genotype-to-phenotype correlation is likely to occur in human clinical variations of STXBP1.
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BACKGROUND: Children with molar-incisor hypomineralisation (MIH) frequently seek aesthetic treatment for incisor opacities. Surprisingly, few studies have evaluated the clinical success of such interventions. AIM: To quantify the effectiveness of minimally invasive treatments in reducing enamel opacity visibility in children with MIH. DESIGN: This in vitro study used digital clinical images of 23 children aged 8-16 years with MIH who underwent microabrasion and/or resin infiltration for the management of incisor opacities. Standard images were taken pre-treatment and 6 months post-treatment. Image software (Image-Pro Plus® V7) was employed to convert 24-bit RGB images to 16-bit greyscale and 145× magnification. Measurement repeatability was assessed using intra-class correlation coefficients (ICCs). Post-treatment changes in visible opacity area (mm2 ) and brightness (greyscale value) were tested using the Wilcoxon signed-rank test for related samples. RESULTS: The mean total opacity surface area significantly reduced from 14.3 mm2 (SD = 7.5) to 9.4 mm2 (SD = 9.0) post-treatment. The proportion of tooth surface affected by the opacity also significantly reduced from 22.5% (SD = 10.5) to 14.7% (SD = 12.7). The mean maximum opacity brightness significantly reduced from 53 066 greyscale value (SD = 4740) to 49 040 (SD = 3796). ICC was good/excellent (0.75-1.0). CONCLUSION: Minimally invasive treatment is effective in reducing the size and brightness of discrete incisor opacities. Future research should compare objective findings with patient-reported outcomes.
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Cárie Dentária , Hipoplasia do Esmalte Dentário , Incisivo , Criança , Hipoplasia do Esmalte Dentário/terapia , Humanos , Incisivo/cirurgia , Dente Molar/cirurgia , PrevalênciaRESUMO
OBJECTIVES: To identify clinical and psychosocial predictors of oral health-related quality of life (OHRQoL) in children with molar incisor hypomineralisation (MIH) following aesthetic treatment of incisor opacities. METHODS: Participants were 7- to 16-year-old children referred to a UK Dental Hospital for management of incisor opacities. Prior to treatment (To), participants completed validated questionnaires to assess OHRQoL and overall health status (C-OHIP-SF19), and self-concept (Harter's Self-Perception Profile for Children [SPPC]). Interventions for MIH included microabrasion, resin infiltration, tooth whitening or composite resin restoration. Children were reviewed after six months (T1) when they re-completed the C-OHIP-SF19 and SPPC questionnaires. The relationships of predictors with improvement of children's OHRQoL (T1-To) and children's overall health status at T1 were assessed using linear and ordinal logistic regression respectively, guided by the Wilson and Cleary's theoretical model. RESULTS: Of 103 participants, 86 were reviewed at T1 (83.5 % completion rate). Their mean age was 11-years (rangeâ¯=â¯7-16) and 60 % were female. Total and domain OHRQoL scores significantly increased (improved OHRQoL) following MIH treatment. There was a significant positive change in SPPC physical appearance subscale score between To and T1. A higher number of anterior teeth requiring aesthetic treatment were associated with poor improvement of socio-emotional wellbeing at T1 (Coef =-0.43). Higher self-concept at To was associated with greater improvement of socio-emotional wellbeing at T1 (ßâ¯=â¯3.44). Greater orthodontic treatment need (i.e. higher IOTN-AC score) at T0 was linked to worse overall oral health at T1 (ORâ¯=â¯0.43). CONCLUSIONS: Psychosocial factors and dental clinical characteristics were associated with change in children's OHRQoL following minimal interventions for incisor opacities. CLINICAL SIGNIFICANCE: MIH is a common condition and clinicians should be aware of the negative impacts some children experience, particularly those with multiple anterior opacities, poor tooth alignment and low self-concept. However, simple, minimally invasive treatments can provide good clinical and psychosocial outcomes and should be offered to children reporting negative effects.
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Hipoplasia do Esmalte Dentário , Qualidade de Vida , Adolescente , Criança , Esmalte Dentário , Hipoplasia do Esmalte Dentário/terapia , Estética Dentária , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Saúde BucalRESUMO
BACKGROUND: Alport syndrome (AS) and atypical hemolytic-uremic syndrome (aHUS) are rare forms of chronic kidney disease (CKD) that can lead to a severe decline of renal function. Steroid-resistant nephrotic syndrome (SRNS) is more common than AS and aHUS and causes 10% of childhood-onset CKD. In recent years, multiple monogenic causes of AS, aHUS and SRNS have been identified, but their relative prevalence has yet to be studied together in a typical pediatric cohort of children with proteinuria and hematuria. We hypothesized that identification of causative mutations by whole exome sequencing (WES) in known monogenic nephritis and nephrosis genes would allow distinguishing nephritis from nephrosis in a typical pediatric group of patients with both proteinuria and hematuria at any level. METHODS: We therefore conducted an exon sequencing (WES) analysis for 11 AS, aHUS and thrombotic thrombocytopenic purpura-causing genes in an international cohort of 371 patients from 362 families presenting with both proteinuria and hematuria before age 25 years. In parallel, we conducted either WES or high-throughput exon sequencing for 23 SRNS-causing genes in all patients. RESULTS: We detected pathogenic mutations in 18 of the 34 genes analyzed, leading to a molecular diagnosis in 14.1% of families (51 of 362). Disease-causing mutations were detected in 3 AS-causing genes (4.7%), 3 aHUS-causing genes (1.4%) and 12 NS-causing genes (8.0%). We observed a much higher mutation detection rate for monogenic forms of CKD in consanguineous families (35.7% versus 10.1%). CONCLUSIONS: We present the first estimate of relative frequency of inherited AS, aHUS and NS in a typical pediatric cohort with proteinuria and hematuria. Important therapeutic and preventative measures may result from mutational analysis in individuals with proteinuria and hematuria.
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Sequenciamento do Exoma/métodos , Marcadores Genéticos , Mutação , Nefrite/diagnóstico , Nefrite/genética , Nefrose/diagnóstico , Nefrose/genética , Adolescente , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Diagnóstico Diferencial , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/genética , PrognósticoRESUMO
Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.
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Síndrome Nefrótica/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Xenopus/genética , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.
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Resistência a Medicamentos/genética , Glucocorticoides/farmacologia , Síndrome Nefrótica/tratamento farmacológico , Mapas de Interação de Proteínas/genética , Proteína rhoA de Ligação ao GTP/genética , Adulto , Animais , Criança , Pré-Escolar , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Glucocorticoides/uso terapêutico , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Síndrome Nefrótica/genética , Linhagem , Podócitos , RNA Interferente Pequeno/metabolismo , Resultado do Tratamento , Sequenciamento do Exoma , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Midaortic syndrome (MAS) is a rare cause of severe childhood hypertension characterized by narrowing of the abdominal aorta in children and is associated with extensive vascular disease. It may occur as part of a genetic syndrome, such as neurofibromatosis, or as consequence of a pathological inflammatory disease. However, most cases are considered idiopathic. We hypothesized that in a high percentage of these patients, a monogenic cause of disease may be detected by evaluating whole exome sequencing data for mutations in 1 of 38 candidate genes previously described to cause vasculopathy. We studied a cohort of 36 individuals from 35 different families with MAS by exome sequencing. In 15 of 35 families (42.9%), we detected likely causal dominant mutations. In 15 of 35 (42.9%) families with MAS, whole exome sequencing revealed a mutation in one of the genes previously associated with vascular disease (NF1, JAG1, ELN, GATA6, and RNF213). Ten of the 15 mutations have not previously been reported. This is the first report of ELN, RNF213, or GATA6 mutations in individuals with MAS. Mutations were detected in NF1 (6/15 families), JAG1 (4/15 families), ELN (3/15 families), and one family each for GATA6 and RNF213 Eight individuals had syndromic disease and 7 individuals had isolated MAS. Whole exome sequencing can provide conclusive molecular genetic diagnosis in a high fraction of individuals with syndromic or isolated MAS. Establishing an etiologic diagnosis may reveal genotype/phenotype correlations for MAS in the future and should, therefore, be performed routinely in MAS.
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Estenose da Valva Aórtica , Hipertensão , Proteína Jagged-1/genética , Neurofibromatoses , Neurofibromina 1/genética , Adolescente , Aorta Abdominal/patologia , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Estudos de Associação Genética , Humanos , Hipertensão/diagnóstico , Hipertensão/genética , Masculino , Mutação , Neurofibromatoses/diagnóstico , Neurofibromatoses/genética , Linhagem , Síndrome , Estados Unidos , Sequenciamento do Exoma/métodosRESUMO
The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.
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Sequenciamento do Exoma , Mutação , Nefrocalcinose/genética , Nefrolitíase/genética , Adolescente , Idade de Início , Criança , Pré-Escolar , Progressão da Doença , Feminino , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Hereditariedade , Humanos , Lactente , Masculino , Nefrocalcinose/diagnóstico por imagem , Nefrocalcinose/epidemiologia , Nefrolitíase/diagnóstico por imagem , Nefrolitíase/epidemiologia , Linhagem , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Tomografia Computadorizada por Raios X , Ultrassonografia , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVES: Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. RESULTS: In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. CONCLUSIONS: Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome.
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Análise Mutacional de DNA/métodos , Sequenciamento do Exoma , Marcadores Genéticos , Mutação , Síndrome Nefrótica/congênito , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Hereditariedade , Humanos , Lactente , Masculino , Taxa de Mutação , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/epidemiologia , Síndrome Nefrótica/genética , Síndrome Nefrótica/terapia , Linhagem , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Adulto JovemRESUMO
Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.
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Hérnia Hiatal/genética , Microcefalia/genética , Complexos Multiproteicos/genética , Mutação , Nefrose/genética , Animais , Apoptose/genética , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Movimento Celular , Citoesqueleto/ultraestrutura , Reparo do DNA/genética , Estresse do Retículo Endoplasmático/genética , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Modelos Moleculares , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Conformação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/metabolismo , Homeostase do Telômero/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
Nephrolithiasis, a condition in which urinary supersaturation leads to stone formation in the urinary system, affects about 5%-10% of individuals worldwide at some point in their lifetime and results in significant medical costs and morbidity. To date, mutations in more than 30 genes have been described as being associated with nephrolithiasis, and these mutations explain about 15% of kidney stone cases, suggesting that additional nephrolithiasis-associated genes remain to be discovered. To identify additional genes whose mutations are linked to nephrolithiasis, we performed targeted next-generation sequencing of 18 hypothesized candidate genes in 348 unrelated individuals with kidney stones. We detected biallelic mutations in SLC26A1 (solute carrier family 26 member 1) in two unrelated individuals with calcium oxalate kidney stones. We show by immunofluorescence, immunoblotting, and glycosylation analysis that the variant protein mimicking p.Thr185Met has defects in protein folding or trafficking. In addition, by measuring anion exchange activity of SLC26A1, we demonstrate that all the identified mutations in SLC26A1 result in decreased transporter activity. Our data identify SLC26A1 mutations as causing a recessive Mendelian form of nephrolithiasis.
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Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Mutação/genética , Nefrolitíase/etiologia , Sequência de Aminoácidos , Proteínas de Transporte de Ânions/química , Bicarbonatos/metabolismo , Imunofluorescência , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Immunoblotting , Nefrolitíase/patologia , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , Transportadores de Sulfato , Sulfatos/metabolismoRESUMO
Nucleoporins are essential components of the nuclear pore complex (NPC). Only a few diseases have been attributed to NPC dysfunction. Steroid-resistant nephrotic syndrome (SRNS), a frequent cause of chronic kidney disease, is caused by dysfunction of glomerular podocytes. Here we identify in eight families with SRNS mutations in NUP93, its interaction partner NUP205 or XPO5 (encoding exportin 5) as hitherto unrecognized monogenic causes of SRNS. NUP93 mutations caused disrupted NPC assembly. NUP93 knockdown reduced the presence of NUP205 in the NPC, and, reciprocally, a NUP205 alteration abrogated NUP93 interaction. We demonstrate that NUP93 and exportin 5 interact with the signaling protein SMAD4 and that NUP93 mutations abrogated interaction with SMAD4. Notably, NUP93 mutations interfered with BMP7-induced SMAD transcriptional reporter activity. We hereby demonstrate that mutations of NUP genes cause a distinct renal disease and identify aberrant SMAD signaling as a new disease mechanism of SRNS, opening a potential new avenue for treatment.
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Carioferinas/genética , Síndrome Nefrótica/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Idade de Início , Sequência de Aminoácidos , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Resistência a Medicamentos/genética , Feminino , Genes Recessivos , Estudos de Associação Genética , Ligação Genética , Células HEK293 , Humanos , Lactente , Carioferinas/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Síndrome Nefrótica/tratamento farmacológico , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Estresse Oxidativo , Podócitos/fisiologia , Análise de Sequência de DNA , Esteroides/farmacologia , Esteroides/uso terapêutico , Xenopus laevisRESUMO
BACKGROUND: The term nephronophthisis-related ciliopathies (NPHP-RC) describes a group of rare autosomal-recessive cystic kidney diseases, characterised by broad genetic and clinical heterogeneity. NPHP-RC is frequently associated with extrarenal manifestations and accounts for the majority of genetically caused chronic kidney disease (CKD) during childhood and adolescence. Generation of a molecular diagnosis has been impaired by this broad genetic heterogeneity. However, recently developed high-throughput exon sequencing techniques represent powerful and efficient tools to screen large cohorts for dozens of causative genes. METHODS: Therefore, we performed massively multiplexed targeted sequencing using the modified molecular inversion probe strategy (MIPs) in an international cohort of 384 patients diagnosed with NPHP-RC. RESULTS: As a result, we established the molecular diagnoses in 81/384 unrelated individuals (21.1%). We detected 127 likely disease-causing mutations in 18 of 34 evaluated NPHP-RC genes, 22 of which were novel. We further compared a subgroup of current findings to the results of a previous study in which we used an array-based microfluidic PCR technology in the same cohort. While 78 likely disease-causing mutations were previously detected by the array-based microfluidic PCR, the MIPs approach identified 94 likely pathogenic mutations. Compared with the previous approach, MIPs redetected 66 out of 78 variants and 28 previously unidentified variants, for a total of 94 variants. CONCLUSIONS: In summary, we demonstrate that the modified MIPs technology is a useful approach to screen large cohorts for a multitude of established NPHP genes in order to identify the underlying molecular cause. Combined application of two independent library preparation and sequencing techniques, however, may still be indicated for Mendelian diseases with extensive genetic heterogeneity in order to further increase diagnostic sensitivity.
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Sequenciamento de Nucleotídeos em Larga Escala , Doenças Renais Císticas/genética , Técnicas de Diagnóstico Molecular , Heterogeneidade Genética , Humanos , Sensibilidade e EspecificidadeRESUMO
Chronically increased echogenicity on renal ultrasound is a sensitive early finding of chronic kidney disease that can be detected before manifestation of other symptoms. Increased echogenicity, however, is not specific for a certain etiology of chronic kidney disease. Here, we performed whole exome sequencing in 79 consanguineous or familial cases of suspected nephronophthisis in order to determine the underlying molecular disease cause. In 50 cases, there was a causative mutation in a known monogenic disease gene. In 32 of these cases whole exome sequencing confirmed the diagnosis of a nephronophthisis-related ciliopathy. In 8 cases it revealed the diagnosis of a renal tubulopathy. The remaining 10 cases were identified as Alport syndrome (4), autosomal-recessive polycystic kidney disease (2), congenital anomalies of the kidney and urinary tract (3), and APECED syndrome (1). In 5 families, in whom mutations in known monogenic genes were excluded, we applied homozygosity mapping for variant filtering and identified 5 novel candidate genes (RBM48, FAM186B, PIAS1, INCENP, and RCOR1) for renal ciliopathies. Thus, whole exome sequencing allows the detection of the causative mutation in 2/3 of affected individuals, thereby presenting the etiologic diagnosis, and allows identification of novel candidate genes.
Assuntos
Insuficiência Renal Crônica/genética , Idade de Início , Estudos de Coortes , Análise Mutacional de DNA , Exoma , Humanos , Doenças Renais Císticas/congênito , Doenças Renais Císticas/genética , Insuficiência Renal Crônica/diagnóstico por imagemRESUMO
BACKGROUND: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. METHODS: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. RESULTS: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. CONCLUSIONS: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.
Assuntos
Cílios/genética , Cílios/patologia , Olho/patologia , Rim/patologia , Proteínas Musculares/genética , Humanos , Mutação , Análise de Sequência de DNARESUMO
Skeletal muscle is essential for mobility, stability and whole body metabolism, and muscle loss, for instance, during sarcopenia, has profound consequences. Satellite cells (muscle stem cells) have been hypothesized, but not yet demonstrated, to contribute to muscle homeostasis and a decline in their contribution to myofibre homeostasis to play a part in sarcopenia. To test their role in muscle maintenance, we genetically labelled and ablated satellite cells in adult sedentary mice. We demonstrate via genetic lineage experiments that, even in the absence of injury, satellite cells contribute to myofibres in all adult muscles, although the extent and timing differs. However, genetic ablation experiments showed that satellite cells are not globally required to maintain myofibre cross-sectional area of uninjured adult muscle.
Assuntos
Fibras Musculares Esqueléticas/patologia , Alelos , Animais , Cruzamentos Genéticos , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX7/metabolismo , Regeneração , Sarcopenia/genética , Células Satélites de Músculo Esquelético/citologia , Fatores de TempoRESUMO
The diaphragm is an essential mammalian skeletal muscle, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common and often lethal birth defect. The diaphragm is derived from multiple embryonic sources, but how these give rise to the diaphragm is unknown, and, despite the identification of many CDH-associated genes, the etiology of CDH is incompletely understood. Using mouse genetics, we show that the pleuroperitoneal folds (PPFs), which are transient embryonic structures, are the source of the diaphragm's muscle connective tissue and regulate muscle development, and we show that the striking migration of PPF cells controls diaphragm morphogenesis. Furthermore, Gata4 mosaic mutations in PPF-derived muscle connective tissue fibroblasts result in the development of localized amuscular regions that are biomechanically weaker and more compliant, leading to CDH. Thus, the PPFs and muscle connective tissue are critical for diaphragm development, and mutations in PPF-derived fibroblasts are a source of CDH.
Assuntos
Diafragma/embriologia , Hérnias Diafragmáticas Congênitas/patologia , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Tecido Conjuntivo/patologia , Diafragma/patologia , Modelos Animais de Doenças , Fibroblastos/fisiologia , Fator de Transcrição GATA4/genética , Deleção de Genes , Fator de Crescimento de Hepatócito/metabolismo , Hérnias Diafragmáticas Congênitas/genética , Pulmão/diagnóstico por imagem , Pulmão/embriologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Desenvolvimento Muscular , Mioblastos/fisiologia , Microtomografia por Raio-XRESUMO
Adult muscle's exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/ß-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/ß-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated ß-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/ß-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which ß-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/ß-catenin signaling that is important for muscle regeneration.
Assuntos
Inativação Gênica , Músculos/fisiologia , Regeneração , Células-Tronco/citologia , Via de Sinalização Wnt , beta Catenina/genética , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Músculos/lesões , Mioblastos/citologia , Mioblastos/metabolismo , Células-Tronco/metabolismoRESUMO
The vertebrate head-trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head-trunk interface.