RESUMO
Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.
Assuntos
Cílios , Ciliopatias , Humanos , Animais , Camundongos , Cílios/genética , Cílios/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas/genética , Aminoácidos/metabolismo , Mamíferos/metabolismo , Proteínas do Citoesqueleto/genéticaRESUMO
Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.
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Estruturas Embrionárias , Fatores de Transcrição Forkhead , Nefropatias , Rim , Néfrons , Sistema Urinário , Anormalidades Urogenitais , Refluxo Vesicoureteral , Adulto , Animais , Humanos , Camundongos , Estudo de Associação Genômica Ampla , Rim/anormalidades , Rim/embriologia , Nefropatias/genética , Camundongos Knockout , Néfrons/embriologia , Fatores de Transcrição/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/metabolismoRESUMO
Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.
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Padronização Corporal , Dineínas/genética , Síndrome de Kartagener/genética , Mutação/genética , Proteínas Nucleares/genética , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/ultraestrutura , Feminino , Genes Recessivos , Humanos , Mutação com Perda de Função/genética , Masculino , Cauda do Espermatozoide/metabolismoRESUMO
Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located ß-HC DNAH11 (defining ODA type 1), and the distally localized ß-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the ß-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient ß-HCs such as that of the unicellular Chlamydomonas reinhardtii.
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Dineínas do Axonema/genética , Cílios/genética , Dineínas/genética , Mutação/genética , Axonema/genética , Transtornos da Motilidade Ciliar/genética , Humanos , Síndrome de Kartagener/genética , FenótipoRESUMO
Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance. Here we report maternally inherited and de novo mutations in PIH1D3 in four men affected with PCD. PIH1D3 is located on the X chromosome and is involved in the preassembly of both outer (ODA) and inner (IDA) dynein arms of cilia and sperm flagella. Loss-of-function mutations in PIH1D3 lead to absent ODAs and reduced to absent IDAs, causing ciliary and flagellar immotility. Further, PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4. This result has clinical and genetic counseling implications for genetically unsolved male case subjects with a classic PCD phenotype that lack additional phenotypes such as intellectual disability or retinitis pigmentosa.
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Cílios/patologia , Transtornos da Motilidade Ciliar/genética , Dineínas/metabolismo , Genes Ligados ao Cromossomo X , Mutação/genética , Cauda do Espermatozoide/patologia , Cílios/metabolismo , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Citoplasma/metabolismo , Feminino , Humanos , Masculino , Linhagem , Fenótipo , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/metabolismoRESUMO
BACKGROUND: Putative nucleotidyltransferase MAB21L1 is a member of an evolutionarily well-conserved family of the male abnormal 21 (MAB21)-like proteins. Little is known about the biochemical function of the protein; however, prior studies have shown essential roles for several aspects of embryonic development including the eye, midbrain, neural tube and reproductive organs. OBJECTIVE: A homozygous truncating variant in MAB21L1 has recently been described in a male affected by intellectual disability, scrotal agenesis, ophthalmological anomalies, cerebellar hypoplasia and facial dysmorphism. We employed a combination of exome sequencing and homozygosity mapping to identify the underlying genetic cause in subjects with similar phenotypic features descending from five unrelated consanguineous families. RESULTS: We identified four homozygous MAB21L1 loss of function variants (p.Glu281fs*20, p.Arg287Glufs*14 p.Tyr280* and p.Ser93Serfs*48) and one missense variant (p.Gln233Pro) in 10 affected individuals from 5 consanguineous families with a distinctive autosomal recessive neurodevelopmental syndrome. Cardinal features of this syndrome include a characteristic facial gestalt, corneal dystrophy, hairy nipples, underdeveloped labioscrotal folds and scrotum/scrotal agenesis as well as cerebellar hypoplasia with ataxia and variable microcephaly. CONCLUSION: This report defines an ultrarare but clinically recognisable Cerebello-Oculo-Facio-Genital syndrome associated with recessive MAB21L1 variants. Additionally, our findings further support the critical role of MAB21L1 in cerebellum, lens, genitalia and as craniofacial morphogenesis.
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Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Proteínas de Homeodomínio/genética , Mutação com Perda de Função , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Consanguinidade , Fácies , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas de Homeodomínio/química , Homozigoto , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Linhagem , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Síndrome , Sequenciamento do ExomaRESUMO
Next-generation sequencing (NGS) has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome (WES) and whole genome sequencing (WGS) in four independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter, and thinning of the corpus callosum, we identified four previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co-immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin-488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by biallelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway.
Assuntos
Alelos , Endocitose , Mutação com Perda de Função/genética , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Fosfoproteínas Fosfatases/genética , Adulto , Criança , Pré-Escolar , Endossomos/metabolismo , Endossomos/ultraestrutura , Feminino , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Linhagem , Fosfoproteínas Fosfatases/química , Síndrome , Transferrina/metabolismoRESUMO
Renal tubular cells respond to mechanical stimuli generated by urinary flow to regulate the activity and transcript abundance of important genes for ion handling, cellular homeostasis, and proper renal development. The primary cilium, a mechanosensory organelle, is postulated to regulate this mRNA response. The aim of this study is to reveal the transcriptome changes of tubular epithelia in response to fluid flow and determine the role of primary cilia in this process. Inner-medullary collecting duct (CD) cells were subjected to either static or physiologically relevant fluid flow (â¼0.6 dyn/cm2). RNA-sequencing analysis of ciliated cells subjected to fluid flow showed up-regulation of 1379 genes and down-regulation of 1294 genes compared with static control cells. Strikingly, only 54 of these genes were identified as gene candidates sensitive to primary cilia sensing of fluid flow, of which 16 were linked to ion or water transport pathways in the CD. Validation by quantitative real-time PCR revealed that only the expression of transferrin receptor, which is involved in iron transport; and tribbles pseudokinase 3, which is involved in insulin signaling, were unequivocally regulated by primary cilia sensing of fluid flow. This study shows that the involvement of primary cilia in ion transport in the collecting duct is exceptionally specific.-Mohammed, S. G., Arjona, F. J., Verschuren, E. H. J., Bakey, Z., Alkema, W., van Hijum, S., Schmidts, M., Bindels, R. J. M., Hoenderop, J. G. J. Primary cilia-regulated transcriptome in the renal collecting duct.
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Cílios/metabolismo , Túbulos Renais Coletores/metabolismo , Transcriptoma , Animais , Linhagem Celular , Túbulos Renais Coletores/citologia , Camundongos , MicrofluídicaRESUMO
The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.
Assuntos
Proteínas de Transporte/genética , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Rim/metabolismo , Rim/patologia , Proteínas Nucleares/genética , Animais , Repetição de Anquirina , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Feminino , Homozigoto , Humanos , Rim/embriologia , Doenças Renais Císticas/fisiopatologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Alça do Néfron/metabolismo , Alça do Néfron/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Fenótipo , Podócitos/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Proteínas de Ligação a RNA/metabolismo , RatosRESUMO
The matrix metalloproteinase MMP9 influences cellular morphology and function, and plays important roles in organogenesis and disease. It exerts both protective and deleterious effects in renal pathology, depending upon its specific substrates. To explore new functions for MMP9 in kidney cysts formation and disease progression, we generated a mouse model by breeding juvenile cystic kidney (jck) mice with MMP9 deficient mice. Specifically, we provide evidence that MMP9 is overexpressed in cystic tissue where its enzymatic activity is increased 7-fold. MMP9 deficiency in cystic kidney worsen cystic kidney diseases by decreasing renal function, favoring cyst expansion and fibrosis. In addition, we find that periostin is a new critical substrate for MMP9 and in its absence periostin accumulates in cystic lining cells. As periostin promotes renal cyst growth and interstitial fibrosis in polycystic kidney diseases, we propose that the control of periostin by MMP9 and its associated intracellular signaling pathways including integrins, integrin-linked kinase and focal adhesion kinase confers to MMP9 a protective effect on the severity of the disease.
Assuntos
Metaloproteinase 9 da Matriz , Doenças Renais Policísticas , Animais , Camundongos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Doenças Renais Policísticas/patologia , Rim/patologia , Transdução de Sinais , FibroseRESUMO
Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.
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Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in two multiplex families with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in family 1 and a missense variant in family 2 with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.
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Laterality defects are defined by the perturbed left-right arrangement of organs in the body, occurring in a syndromal or isolated fashion. In humans, primary ciliary dyskinesia (PCD) is a frequent underlying condition of defective left-right patterning, where ciliary motility defects also result in reduced airway clearance, frequent respiratory infections, and infertility. Non-motile cilia dysfunction and dysfunction of non-ciliary genes can also result in disturbances of the left-right body axis. Despite long-lasting genetic research, identification of gene mutations responsible for left-right patterning has remained surprisingly low. Here, we used whole-exome sequencing with Copy Number Variation (CNV) analysis to delineate the underlying molecular cause in 35 mainly consanguineous families with laterality defects. We identified causative gene variants in 14 families with a majority of mutations detected in genes previously associated with PCD, including two small homozygous CNVs. None of the patients were previously clinically diagnosed with PCD, underlining the importance of genetic diagnostics for PCD diagnosis and adequate clinical management. Identified variants in non-PCD-associated genes included variants in PKD1L1 and PIFO, suggesting that dysfunction of these genes results in laterality defects in humans. Furthermore, we detected candidate variants in GJA1 and ACVR2B possibly associated with situs inversus. The low mutation detection rate of this study, in line with other previously published studies, points toward the possibility of non-coding genetic variants, putative genetic mosaicism, epigenetic, or environmental effects promoting laterality defects.
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Background: Nephropathic Cystinosis, the most common cause of renal Fanconi syndrome, is a lysosomal transport disorder with an autosomal recessive inheritance pattern. A large number of mutations in CTNS have been identified as causative to date. A 57 kb deletion encompassing parts of CTNS is most commonly identified in Caucasians but this allele has not been identified in individuals of Eastern Mediterranean, Middle Eastern, Persian, or Arab origin to date. Methods and Results: Implementing whole exome sequencing (WES) in a consanguineous Iranian family, we identified this large deletion affecting CTNS in a patient initially presenting with hypokalemic metabolic alkalosis symptoms and considerable proteinuria. Conclusion: We show WES is a cost and time efficient genetic diagnostics modality to identify the underlying molecular pathology in Cystinosis individuals and provide a summary of all previously reported CTNS alleles in the Middle east population. Our work also highlights the importance to consider the 57-kb deletion as underlying genetic cause in non-European populations, including the Middle East. Limited diagnostic modalities for Cystinosis in developing countries could account for the lack of previously reported cases in these populations carrying this allele. Further, our findings emphasize the utility of WES to define genetic causes in clinically poorly defined phenotypes and demonstrate the requirement of Copy number variation (CNV) analysis of WES data.
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BACKGROUND: Bartter Syndrome is a rare, genetically heterogeneous, mainly autosomal recessively inherited condition characterized by hypochloremic hypokalemic metabolic alkalosis. Mutations in several genes encoding for ion channels localizing to the renal tubules including SLC12A1, KCNJ1, BSND, CLCNKA, CLCNKB, MAGED2 and CASR have been identified as underlying molecular cause. No genetically defined cases have been described in the Iranian population to date. Like for other rare genetic disorders, implementation of Next Generation Sequencing (NGS) technologies has greatly facilitated genetic diagnostics and counseling over the last years. In this study, we describe the clinical, biochemical and genetic characteristics of patients from 15 Iranian families with a clinical diagnosis of Bartter Syndrome. RESULTS: Age range of patients included in this study was 3 months to 6 years and all patients showed hypokalemic metabolic alkalosis. 3 patients additionally displayed hypercalciuria, with evidence of nephrocalcinosis in one case. Screening by Whole Exome Sequencing (WES) and long range PCR revealed that 12/17 patients (70%) had a deletion of the entire CLCNKB gene that was previously identified as the most common cause of Bartter Syndrome in other populations. 4/17 individuals (approximately 25% of cases) were found to suffer in fact from pseudo-Bartter syndrome resulting from congenital chloride diarrhea due to a novel homozygous mutation in the SLC26A3 gene, Pendred syndrome due to a known homozygous mutation in SLC26A4, Cystic Fibrosis (CF) due to a novel mutation in CFTR and apparent mineralocorticoid excess syndrome due to a novel homozygous loss of function mutation in HSD11B2 gene. 1 case (5%) remained unsolved. CONCLUSIONS: Our findings demonstrate deletion of CLCNKB is the most common cause of Bartter syndrome in Iranian patients and we show that age of onset of clinical symptoms as well as clinical features amongst those patients are variable. Further, using WES we were able to prove that nearly 1/4 patients in fact suffered from Pseudo-Bartter Syndrome, reversing the initial clinical diagnosis with important impact on the subsequent treatment and clinical follow up pathway. Finally, we propose an algorithm for clinical differential diagnosis of Bartter Syndrome.
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Síndrome de Bartter/diagnóstico , Síndrome de Bartter/genética , Diagnóstico Diferencial , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Algoritmos , Síndrome de Bartter/epidemiologia , Criança , Pré-Escolar , Canais de Cloreto/genética , Antiportadores de Cloreto-Bicarbonato/genética , Feminino , Humanos , Lactente , Irã (Geográfico)/epidemiologia , Masculino , Transportadores de Sulfato/genética , Sequenciamento do Exoma/métodosRESUMO
INTRODUCTION: Monogenetic renal diseases, including recessively inherited nephrotic syndromes, represent a significant health burden despite being rare conditions. Precise diagnosis, including identification of the underlying molecular cause, is especially difficult in low-income countries and/or if affected individuals are unavailable for biochemical testing. Whole-exome sequencing (WES) has opened up novel diagnostic perspectives for these settings. However, sometimes the DNA of affected individuals is not suitable for WES due to low amounts or degradation. METHODS: We report on the use of parental WES with implementation of specific stepwise variant filtering to identify the underlying molecular cause of the childhood-onset nephrotic syndrome as nephrosialidosis resulting from a mutation in NEU1. RESULTS: Sequencing both parents enabled a nephrosialidosis diagnosis in the deceased child. To date, only 16 other cases of nephrosialidosis have been reported in the literature, with only 1 genetically confirmed case. After we reviewed the clinical information of all reported cases, we found that most patients presented with proteinuria, which started at between 2 and 3 years of age. Renal pathology showed mainly focal segmental glomerulosclerosis (FSGS)with vacuolated cells, and steroid treatment was always unsuccessful. Hepatomegaly was present in nearly all cases, whereas corneal clouding and a cherry red spot on the macula was observed in only approximately 50% of cases. Fourteen of 16 previously reported cases were no longer alive at the time of reporting. CONCLUSIONS: Our findings demonstrate the power of parental WES to diagnose rare genetic diseases, such as childhood-onset nephrotic syndrome. We further provide a comprehensive overview of the clinical course of nephrosialidosis and raise awareness of this ultra-rare condition as an underlying cause of FSGS.
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BACKGROUND: Mainzer-Saldino syndrome (MZSDS) is a skeletal ciliopathy and part of the short-rib thoracic dysplasia (SRTD) group of ciliary disorders. The main characteristics of MZSDS are short limbs, mild narrow thorax, blindness, and renal failure. Thus far, variants in two genes are associated with MZSDS: IFT140, and IFT172. In this study, we describe a 1-year-old girl presenting with mild skeletal abnormalities, Leber congenital amaurosis, and bilateral hearing difficulties. For establishing an accurate diagnosis, we combined clinical, molecular, and functional analyses. METHODS: We performed diagnostic whole-exome sequencing (WES) analysis to determine the genetic cause of the disease and analyzed two gene panels, containing all currently known genes in vision disorders, and in hearing impairment. Upon detection of the likely causative variants, ciliary phenotyping was performed in patient urine-derived renal epithelial cells (URECs) and rescue experiments were performed in CRISPR/Cas9-derived Ift140 knock out cells to determine the pathogenicity of the detected variants in vitro. Cilium morphology, cilium length, and intraflagellar transport (IFT) were evaluated by immunocytochemistry. RESULTS: Diagnostic WES revealed two novel compound heterozygous variants in IFT140, encoding IFT140. Thorough investigation of WES data did not reveal any variants in candidate genes associated with hearing impairment. Patient-derived URECs revealed an accumulation of IFT-B protein IFT88 at the ciliary tip in 41% of the cells indicative of impaired retrograde IFT, while this was absent in cilia from control URECs. Furthermore, transfection of CRISPR/Cas9-derived Ift140 knock out cells with an IFT140 construct containing the patient mutation p.Tyr923Asp resulted in a significantly higher percentage of IFT88 tip accumulation than transfection with the wild-type IFT140 construct. CONCLUSIONS: By combining the clinical, genetic, and functional data from this study, we could conclude that the patient has SRTD9, also called Mainzer-Saldino syndrome, caused by variants in IFT140. We suggest the possibility that variants in IFT140 may underlie hearing impairment. Moreover, we show that urine provides an excellent source to obtain patient-derived cells in a non-invasive manner to study the pathogenicity of variants detected by genetic testing.
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Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.