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BACKGROUND: Nephronophthisis (NPH) comprises a heterogeneous group of inherited renal ciliopathies clinically characterized by progressive kidney failure. So far, definite diagnosis is based on molecular testing only. Here, we studied the feasibility of NPHP1 and NPHP4 immunostaining of nasal epithelial cells to secure and accelerate the diagnosis of NPH. METHODS: Samples of 86 individuals with genetically determined renal ciliopathies were analyzed for NPHP1 localization using immunofluorescence microscopy (IF). A sub-cohort of 35 individuals was also analyzed for NPHP4 localization. Western blotting was performed to confirm IF results. RESULTS: NPHP1 and NPHP4 were both absent in all individuals with disease-causing NPHP1 variants including one with a homozygous missense variant (c.1027G > A; p.Gly343Arg) formerly classified as a "variant of unknown significance." In individuals with an NPHP4 genotype, we observed a complete absence of NPHP4 while NPHP1 was severely reduced. IF results were confirmed by immunoblotting. Variants in other genes related to renal ciliopathies did not show any impact on NPHP1/NPHP4 expression. Aberrant immunostaining in two genetically unsolved individuals gave rise for a further genetic workup resulting in a genetic diagnosis for both with disease-causing variants in NPHP1 and NPHP4, respectively. CONCLUSIONS: IF of patient-derived respiratory epithelial cells may help to secure and accelerate the diagnosis of nephronophthisis-both by verifying inconclusive genetic results and by stratifying genetic diagnostic approaches. Furthermore, we provide in vivo evidence for the interaction of NPHP1 and NPHP4 in a functional module.
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Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.
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Background: Genomic disorders caused by copy number variations (CNVs) are prevalent in patients with kidney disease; however, their contribution to chronic kidney failure (KF) of undetermined aetiology (uKF) is unclear. We screened patients with uKF aged 50 years or younger to establish the prevalence of causative CNVs. Methods: We enrolled patients with an onset of KF ≤50 years from suspected undetermined aetiology for initial review of medical records to exclude patients with clear-cut clinical or histopathological kidney diagnoses or patients with already established genetic kidney diseases. Next, we performed single nucleotide polymorphism (SNP) array-based CNV screening. All the detected CNVs were systematically classified and evaluated as possible causes of the patient's kidney disease. Patients with CNVs not explaining the kidney phenotype were additionally screened for causal variants in 540 genes using whole-genome sequencing. Results: We enrolled 172 patients, of whom 123 underwent SNP-array. Pathogenic CNVs corresponding to known genomic disorders were identified in 12 patients (9.8%). The identified genomic disorders provided a causative kidney diagnosis in three patients, all of whom had reached KF by age 18 years. The remaining nine patients had CNVs with unclear kidney disease causality. Subsequently, whole-genome sequencing provided a causative genetic diagnosis in an additional four patients, including two diagnostic sequence variants unrelated to the detected CNVs. Conclusions: Genomic disorders were prevalent in this cohort with uKF, and causative CNVs were identified in 5 of 123 patients. Further studies combining the analysis of CNVs and sequence variants are needed to clarify the causal role of genomic disorders in kidney disease.
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We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or â¼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.
Subject(s)
Genome , Primates , Animals , Humans , Base Sequence , Primates/classification , Primates/genetics , Biological Evolution , Sequence Analysis, DNA , Genomic Structural VariationABSTRACT
A precise diagnosis in medicine allows appropriate disease-specific management. Kidney failure of unknown aetiology remains a frequent diagnostic label within the haemodialysis unit and kidney transplant clinic, accounting for 15-20% of these patients. Approximately 10% of such cases may have an underlying monogenic cause of kidney failure. Modern genetic approaches can provide a precise diagnosis for patients and their families. A search for extra-renal disease manifestations is also important as this may point to a specific genetic diagnosis. Here, we present two patients where molecular genetic testing was performed because of kidney failure of unknown aetiology and associated retinal phenotypes. The first patient reached kidney failure at 16 years of age but only presented with a retinal phenotype at 59 years of age and was found to have evidence of rod-cone dystrophy. The second patient presented with childhood kidney failure at the age of 15 years and developed visual difficulties and photophobia at the age of 32 years and was diagnosed with cone dystrophy. In both cases, genetic tests were performed which revealed a homozygous whole-gene deletion of NPHP1-encoding nephrocystin-1, providing the unifying diagnosis of Senior-Løken syndrome type 1. We conclude that reviewing kidney and extra-renal phenotypes together with targeted genetic testing was informative in these cases of kidney failure of unknown aetiology and associated retinal phenotypes. The involvement of an interdisciplinary team is advisable when managing such patients and allows referral to other relevant specialities. The long time lag and lack of diagnostic clarity and clinical evaluation in our cases should encourage genetic investigations for every young patient with unexplained kidney failure. For these and similar patients, a more timely genetic diagnosis would allow for improved management, a risk assessment of kidney disease in relatives, and the earlier identification of extra-renal disease manifestations. Supplementary Information: The online version contains supplementary material available at 10.1007/s44162-024-00031-4.
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OBJECTIVE: To explore the activation of tumor necrosis factor-α (TNF-α) signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells. METHODS: A human proximal renal tubular cell (HK2) model of lentivirus-mediated NPHP1 knockdown (NPHP1KD) was constructed, and the expressions of TNF-α, p38, and C/EBPß and the inflammatory factors CXCL5, CCL20, IL-1ß, IL-6 and MCP-1 were detected using RT-qPCR, Western blotting or enzyme-linked immunosorbent assay. A small interfering RNA (siRNA) was transfected in wild-type and NPHP1KDHK2 cells, and the changes in the expressions of TNF-α, p38, and C/EBPß and the inflammatory factors were examined. RESULTS: NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α, C/EBPß, CXCL5, IL-1ß, and IL-6 (P < 0.05), protein expressions of phospho-p38 and C/EBPß (P < 0.05), and IL-6 level in the culture supernatant (P < 0.05), and these changes were significantly blocked by transfection of cells with siRNA-C/EBPß (P < 0.05). CONCLUSION: TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells, and C/EBPß may serve as a key transcription factor to mediate these changes.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta , Tumor Necrosis Factor-alpha , Humans , Adaptor Proteins, Signal Transducing/metabolism , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cytoskeletal Proteins/metabolism , Epithelial Cells/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Renal epithelial cells are subjected to fluid shear stress of urine flow. Several cellular structures act as mechanosensors-the primary cilium, microvilli and cell adhesion complexes-that directly relay signals to the cytoskeleton to regulate various processes including cell differentiation and renal cell functions. Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy leading to end-stage kidney failure before adulthood. NPHP1 and NPHP4 are the major genes which code for proteins that form a complex at the transition zone of the primary cilium, a crucial region required for the maintenance of the ciliary composition integrity. These two proteins also interact with signaling components and proteins associated with the actin cytoskeleton at cell junctions. Due to their specific subcellular localization, we wondered whether NPHP1 and NPHP4 could ensure mechanosensory functions. Using a microfluidic set up, we showed that murine inner medullary collecting ductal cells invalidated for Nphp1 or Nphp4 are more responsive to immediate shear exposure with a fast calcium influx, and upon a prolonged shear condition, an inability to properly regulate cilium length and actin cytoskeleton remodeling. Following a transcriptomic study highlighting shear stress-induced gene expression changes, we showed that prolonged shear triggers both cholesterol biosynthesis pathway and uptake, processes that do not seem to involve neither NPHP1 nor NPHP4. To conclude, our study allowed us to determine a moderate role of NPHP1 and NPHP4 in flow sensation, and to highlight a new signaling pathway induced by shear stress, the cholesterol biosynthesis and uptake pathways, which would allow cells to cope with mechanical stress by strengthening their plasma membrane through the supply of cholesterol.
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Our case report and review contribute to the understanding of ocular manifestations in NPHP1 ciliopathies by reinforcing the relationship between pathogenic genetic variants and a wide array of ophthalmic abnormalities.
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BACKGROUND: Joubert syndrome (JS) is a group of rare ciliopathies, mainly characterized by cerebellar dysplasia representing the "molar tooth sign (MTS)" on neuroimaging, hypotonia, and developmental delay. Having a complicated genotype-phenotype correlation due to its rich genetic heterogeneity, JS is usually combined with other organic defects affecting the retina, kidney, and liver. This report aimed to present new cases and novel variants of JS. CASE PRESENTATION: Five unrelated patients who were diagnosed with JS, with or without typical clinical characteristics, received integrated examinations, including whole-exome sequencing (WES) and Sanger sequencing. We identified nine pathogenic variants in the TCTN2, CPLANE1, INPP5E, NPHP1, and CC2D2A genes. CONCLUSION: Four novel pathogenic mutations in the TCTN2, CPLANE1, and INPP5E genes were reported. The findings broadened the genotypic spectrum of JS and contributed to a better understanding of genotype-phenotype correlation.
Subject(s)
Abnormalities, Multiple , Eye Abnormalities , Kidney Diseases, Cystic , Humans , Abnormalities, Multiple/genetics , Cerebellum/diagnostic imaging , East Asian People , Eye Abnormalities/genetics , Kidney Diseases, Cystic/genetics , Membrane Proteins , Phosphoric Monoester Hydrolases/genetics , Retina , MutationABSTRACT
Background: Nephronophthisis (NPH) is the most common genetic cause of end-stage renal disease (ESRD) in childhood, and NPHP1 is the major pathogenic gene. Cyst formation at the corticomedullary junction is a pathological feature of NPH, but the mechanism underlying cystogenesis is not well understood. The isolation and identification of cystic cell subpopulation could help to identify their origins and provide vital clues to the mechanisms underlying cystogenesis in NPH. Methods: Single-nucleus RNA sequencing (snRNA-seq) was performed to produce an atlas of NPHP1 renal cells. Kidney samples were collected from WT (Nphp1 +/+) mice and NPHP1 (Nphp1 del2-20/del2-20) model mice. Results: A comprehensive atlas of the renal cellular landscape in NPHP1 was generated, consisting of 14 basic renal cell types as well as a subpopulation of DCT cells that was overrepresented in NPHP1 kidneys compared to WT kidneys. GO analysis revealed significant downregulation of genes associated with tubular development and kidney morphogenesis in this subpopulation. Furthermore, the reconstruction of differentiation trajectories of individual cells within this subpopulation confirmed that a specific group of cells in NPHP1 mice become arrested at an early stage of differentiation and proliferate to form cysts. We demonstrate that Niban1 is a specific molecular marker of cystic cells in both mice and human NPHP1. Conclusion: In summary, we report a novel subpopulation of DCT cells, marked by Niban1, that are classified as cystic cells in the NPHP1 mice kidney. These results offer fresh insights into the cellular and molecular basis of cystogenesis in NPH.
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Nephronophthisis (NPHP) is a common pediatric cystic kidney disease, accounting for approximately 10% of end-stage renal failure cases in children. NPHP is primarily diagnosed through the identification of indel mutations and copy number variants (CNVs), and patients carrying NPHP1 mutations usually progress to renal failure at a mean age of 13 years old. However, the association between CNVs containing NPHP1 variations and the progression of NPHP-induced disease remains unclear. Here, we report three NPHP patients in a family. The proband had developed stage 4 chronic kidney disease (CKD) at 9 years old, and her younger brother and older sister had developed renal failure at 8 and 10 years old, respectively. A genetic diagnosis showed that they carried two rare CNVs, including homozygous loss of NPHP1, MALL, ACTR1AP1, MTLN, and LOC100507334. Heterozygous deletions mainly consisted of non-coding RNA genes on both sides of the CNVs. The proband was in stage 4 of CKD while her brother had progressed to renal failure, probably due to more extensive heterozygous deletion of a 67.115 kbp fragment, which included LIMS3-LOC440895, LOC440895, GPAA1P1, ZBTB45P1, and LINC0112 genes. This report demonstrates that larger CNV deletions, including homozygous NPHP1, MALL, and MTLN mutations and heterozygous deletions, presumably accelerate disease progression. Therefore, early genetic diagnosis plays a crucial role in the intervention and prognosis of these patients.
Subject(s)
Kidney Failure, Chronic , Renal Insufficiency, Chronic , Humans , Male , Child , Female , Adolescent , DNA Copy Number Variations , Sequence Deletion , Cytoskeletal Proteins/genetics , Membrane Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Kidney Failure, Chronic/genetics , Renal Insufficiency, Chronic/geneticsABSTRACT
BACKGROUND: Hypoxia-inducible factors (HIFs) are the most essential endogenous transcription factors in the hypoxic microenvironment and regulate multiple genes involved in the proliferation, migration, invasion, and EMT of hepatocellular carcinoma (HCC) cells. However, the regulatory mechanism of HIFs in driving HCC progression remains poorly understood. METHODS: Gain- and loss-of-function experiments were carried out to investigate the role of TMEM237 in vitro and in vivo. The molecular mechanisms involved in HIF-1α-induced TMEM237 expression and TMEM237-mediated enhancement of HCC progression were confirmed by luciferase reporter, ChIP, IP-MS and Co-IP assays. RESULTS: TMEM237 was identified as a novel hypoxia-responsive gene in HCC. HIF-1α directly bound to the promoter of TMEM237 to transactivate its expression. The overexpression of TMEM237 was frequently detected in HCC and associated with poor clinical outcomes in patients. TMEM237 facilitated the proliferation, migration, invasion, and EMT of HCC cells and promoted tumor growth and metastasis in mice. TMEM237 interacted with NPHP1 and strengthened the interaction between NPHP1 and Pyk2 to trigger the phosphorylation of Pyk2 and ERK1/2, thereby contributing to HCC progression. The TMEM237/NPHP1 axis mediates hypoxia-induced activation of the Pyk2/ERK1/2 pathway in HCC cells. CONCLUSIONS: Our study demonstrated that HIF-1α-activated TMEM237 interacted with NPHP1 to activate the Pyk2/ERK pathway, thereby promoting HCC progression.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Mice , Adaptor Proteins, Signal Transducing/metabolism , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cytoskeletal Proteins/metabolism , Focal Adhesion Kinase 2/metabolism , Hypoxia/genetics , Liver Neoplasms/metabolism , MAP Kinase Signaling System , Tumor Microenvironment , Hypoxia-Inducible Factor 1, alpha Subunit/metabolismABSTRACT
Homozygous deletion of NPHP1 can lead to isolated nephronophthisis (NPHP) and syndromic disorders. However, the phenotype of scalp tumor and hydroureteronephrosis in NPHP patients with homozygous deletion of NPHP1 has not been reported. Clinical data, laboratory results, and genetic testing of 4 NPHP patients were collected. Examination of their eyes, heart, and urinary tract and of their hepatobiliary, skeletal, and central nervous systems was evaluated. Isolated NPHP was observed in 1 case, and syndromic disorders were observed in the other 3 patients. Their syndromic disorders showed NPHP combined with central nervous system defects, eye involvement, scalp tumor, arachnoid cyst, or hydroureteronephrosis. Large homozygous deletions covering the whole NPHP1 gene locus were identified in all 4 patients. We report a novel phenotype of scalp tumor and hydroureteronephrosis in NPHP patients with homozygous deletion of NPHP1, paving an avenue for further research on NPHP1-associated deformity in the skin and the urinary system.
Subject(s)
Kidney Diseases, Cystic , Kidney Failure, Chronic , Neoplasms , Humans , Homozygote , Kidney Failure, Chronic/genetics , Membrane Proteins/genetics , Cytoskeletal Proteins/genetics , Scalp/pathology , Sequence Deletion , Adaptor Proteins, Signal Transducing/genetics , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/pathologyABSTRACT
BACKGROUND: Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease and the most common hereditary disease leading to end-stage renal disease in children and adolescents. The NPHP1 gene was the first NPHP gene to be discovered. Pathogenic variation of the NPHP1 gene can cause juvenile renal wasting disease type 1. CASE PRESENTATION: Here, we report the first case of living related kidney transplantation of monozygotic twins with NPHP1 nephronophthisis in China; one of these cases involved cross-blood type kidney transplantation. Our experience shows that patients with NPHP1 nephronophthisis have almost no risk recurrent kidney disease following living related kidney transplantation and genetic testing. The two twins recovered well without any complications. CONCLUSIONS: This is the first report of living related kidney transplantation of monozygotic twins with heterozygous deletion of the NPHP1 gene in a Chinese family with NPHP. In addition, genetic testing provides an efficient means of evaluating the safety of living related kidney transplantation in patients with NPHP1 nephronophthisis.
Subject(s)
Kidney Transplantation , Humans , Adaptor Proteins, Signal Transducing/genetics , Cytoskeletal Proteins/genetics , East Asian People , Homozygote , Living Donors , Membrane Proteins/genetics , Sequence Deletion , Twins, MonozygoticABSTRACT
Siblings with nephronophthisis occasionally show different clinical courses; however, the reasons for this remain unclear. We herein report cases of nephronophthisis in a pair of dizygotic twins with different clinical courses. The brother developed end-stage kidney disease at 17 years old; however, his sister did not show kidney insufficiency. Kidney biopsies revealed severe tubulointerstitial damage at 14 and 22 years old in the brother and sister, respectively. Both had a homozygous NPHP1 deletion with different heterozygous mutations related to hereditary cystic kidney disease. Since the dizygotic twins were exposed to similar environmental factors, genetic factors may have influenced their clinical course more strongly than environmental factors.
Subject(s)
Kidney Diseases, Cystic , Polycystic Kidney Diseases , Male , Female , Humans , Adolescent , Young Adult , Adult , Twins, Dizygotic , Membrane Proteins/genetics , Cytoskeletal Proteins , Adaptor Proteins, Signal Transducing/genetics , Kidney Diseases, Cystic/genetics , Disease ProgressionABSTRACT
Introduction: Nephronophthisis (NPHP) is a tubulointerstitial kidney disorder with an autosomal recessive inheritance pattern. Its genetic heterogeneity contributes to phenotype variability. The most frequent etiology of juvenile nephronophthisis is a mutation in the nephronophthisis type 1 (NPHP1) gene. This study aimed to evaluate the genotype-phenotype correlation in NPHP1 gene mutation. Methods: A multicenter retrospective study was performed over 20 years from 1998 to 2018 to describe the clinical, biological, and radiological features associated with the large deletion NPHP1 gene in 32 patients. Results: The incidence of NPHP1 was 1.6/204041. Eighty-one percent of our patients were born out of consanguineous marriages. The mean age at diagnosis was 14 ± 7 years. The patients were divided into three groups: isolated nephronophthisis (72%), syndromic nephronophthisis (19%), and patients without recognizable syndrome (9%). Intrafamilial and geographical variability was observed in syndrome diagnoses and in age at the onset of CKD stage 5. Genotype frequency varied between 50% and 100% in genealogical data. Juvenile (47%), adolescent (37%), and adult (13%) clinical forms have been distinguished by the onset of CKD stage 5. The five-year survival rate of renal transplantation was 80%. Conclusion: Given the broad clinical spectrum of NPHP1 associated with the large deletion of the NPHP1 gene, no genotype-phenotype correlation could be established.
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OBJECTIVE: We present prenatal diagnosis and molecular cytogenetic characterization of a de novo duplication of 2q12.2âq13 encompassing MALL, NPHP1, RGPD6 and BUB1. CASE REPORT: A 36-year-old, primigravid woman underwent amniocentesis at 16 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 46,XX,dup(2) (q12.2q13). Simultaneous array comparative genomic hybridization (aCGH) analysis revealed a 6.1-Mb 2q12.2q13 duplication. aCGH analysis of the parental bloods did not find such a duplication. Prenatal ultrasound was unremarkable. After genetic counseling, the parents decided to terminate the pregnancy at 21 weeks of gestation, and a 420-g female fetus was delivered with no gross abnormalities. Postnatal cytogenetic analysis of the umbilical cord confirmed the prenatal diagnosis. The parental karyotypes were normal. aCGH analysis of the umbilical cord revealed the result of arr [GRCh37 (hg19)] 2q12.2q13 (107, 132, 950-113,065,779) × 3.0 with a 2q12.2âq13 duplication encompassing 20 OMIM genes including MALL, NPHP1, RGPD6 and BUB1. Polymorphic DNA marker analysis of quantitative fluorescence polymerase chain reaction (QF-PCR) on the DNAs extracted from the umbilical cord and parental bloods confirmed a maternal origin of the duplication of 2q12.2âq13. CONCLUSION: Amniocentesis may incidentally detect a de novo chromosomal segmental duplication of maternal origin in the fetus. The genetic information acquired by molecular analyses such as aCGH and QF-PCR are useful for genetic counseling under such a circumstance.
Subject(s)
Amniocentesis , Prenatal Diagnosis , Pregnancy , Female , Humans , Adult , Comparative Genomic Hybridization , Karyotyping , Cytogenetic Analysis , Chromosome Duplication/genetics , DNA , Cytoskeletal Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Protein Serine-Threonine Kinases/geneticsABSTRACT
OBJECTIVES: Joubert syndrome is a spectrum of rare genetic disorders, mainly characterized by a distinctive cerebellar and brain stem malformation called the "molar tooth sign" (MTS), hypotonia, and intellectual disability/developmental delay. METHODS: In this study, 4 pediatric cases with developmental delay and oculomotor abnormities were recruited, and submitted to a clinical evaluation and magnetic resonance imaging (MRI) examination. Afterwards, genetic detection with whole exome sequencing (WES) was conducted on the 4 patients. RESULTS: Imaging results demonstrated cerebellar dysplasia in all probands, yet the MTS findings varied in severity. WES detected diagnostic variations in all four probands, which were distributed in four genes, namely CC2D2A, NPHP1, AHI1, and C5orf42. Two variants were novelly identified, which were the CC2D2A: c.2444delC (p.P815fs*2) and the AIH1: exon (15-17) del. In silico analysis supported the pathogenicity of the variations in this study. CONCLUSIONS: Our findings expanded the mutation spectrum of Joubert syndrome related disorders, and provided solid evidence to the affected families for further genetic counseling and pregnancy guidance.
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Objective:To investigate and analyze the clinical phenotypes and genotypes in children diagnosed with nephronophthisis (NPHP), and to provide references for clinical diagnosis.Methods:Clinical data of 9 children with NPHP diagnosed by genetic testing in the Department of Nephrology, Wuhan Children′s Hospital from April 2017 to January 2022 were retrospectively collected. The clinical characteristics and genetic test results were analyzed.Results:The median onset age was 11.2(3.4, 14.2) years old in 9 patients, including 5 females and 4 males. There were 8 cases of glomerular proteinuria, 8 cases of renal tubular proteinuria, and 7 cases of reduced urinary gravity in 9 patients. All the children had varying degrees of impaired renal function at the time of diagnosis. Seven cases entered chronic kidney disease (CKD) stage 5, 1 case entered CKD stage 3, and 1 case entered CKD stage 4 at the time of diagnosis. All the children had renal ultrasound abnormalities of varying degrees: size change (3/9), echo enhancement (8/9) and cysts (3/9). Extrarenal phenotypes were present in 3 children. Genetic test showed that 6 patients had mutation of NPHP1 gene, 1 patient had mutation of WDR19 gene, 1 patient had mutation of NPHP3 gene and 1 patient had mutation of NPHP5 gene. Conclusions:Deletion mutation of NPHP1 gene is the most common, while NPHP3, NPHP5 and extremely rare WDR19 mutations have also been found in NPHP patients. The clinical manifestations of NPHP are not typical, so it is necessary to find a specific diagnosis method in the early.
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BACKGROUND: Nephronophthisis (NPHP) is a kind of ciliopathy. Interstitial fibrosis occurs at the early stage of the disease. TGF-ß/Smad is a key signaling pathway in regulating interstitial fibrosis and epithelial-mesenchymal transition (EMT). In this study, we explored the activation of the TGF-ß/Smad signaling pathway and EMT in NPHP1-defective MDCK cells to further understand the pathogenesis of NPHP. METHODS: NPHP1-knockdown (NPHP1KD) MDCK cells were constructed by recombinant lentiviral short hairpin RNA, and NPHP1-knockout (NPHP1KO) MDCK cells were constructed by using the CRISPR/Cas9 technique. The morphology and migration ability were observed under a microscope. Western blotting was used to detect the expression of E-cadherin, ß-catenin, α-smooth muscle actin (α-SMA), fibroblast-specific protein-1(FSP1), TGF-ß1, Smad2, Smad3, p-Smad3, Smad4 and Smad7. The localization of Smad3 was determined by immunofluorescence assay. RESULTS: NPHP1KD and NPHP1KO MDCK cells were spindle-shaped and presented EMT-like changes. E-cadherin and ß-catenin expression decreased, while α-SMA and FSP1 expression increased; the TGF-ß/Smad signaling pathway was activated, Smad2, Smad3, p-Smad3 and Smad4 expression increased, Smad3 translocated to nuclear and Smad7 expression decreased compared with those in wild type MDCK cells. Overexpression of Smad7 reversed these changes to different degrees. CONCLUSIONS: Our results indicate that NPHP1 defects induce the activation of the TGF-ß/Smad signaling pathway and EMT in MDCK cells. These factors may be implicated in the pathogenesis of interstitial fibrosis in NPHP.