RESUMEN
Background and Aims: Survival rates for esophageal squamous cell carcinoma (ESCC) are extremely low due to the late diagnosis of most cases. An understanding of the early molecular processes that lead to ESCC may facilitate opportunities for early diagnosis; however, these remain poorly defined. Tylosis with esophageal cancer (TOC) is a rare syndrome associated with a high lifetime risk of ESCC and germline mutations in RHBDF2, encoding iRhom2. Using TOC as a model of ESCC predisposition, this study aimed to identify early-stage transcriptional changes in ESCC development. Methods: Esophageal biopsies were obtained from control and TOC individuals, the latter undergoing surveillance endoscopy, and adjacent diagnostic biopsies were graded as having no dysplasia or malignancy. Bulk RNA-Seq was performed, and findings were compared with sporadic ESCC vs normal RNA-Seq datasets. Results: Multiple transcriptional changes were identified in TOC samples, relative to controls, and many were detected in ESCC. Accordingly, pathway analyses predicted an enrichment of cancer-associated processes linked to cellular proliferation and metastasis, and several transcription factors were predicted to be associated with TOC and ESCC, including negative enrichment of GRHL2. Subsequently, a filtering strategy revealed 22 genes that were significantly dysregulated in both TOC and ESCC. Moreover, Keratin 17, which was upregulated in TOC and ESCC, was also found to be overexpressed at the protein level in 'normal' TOC esophagus tissue. Conclusion: Transcriptional changes occur in TOC esophagus prior to the onset of dysplasia, many of which are associated with ESCC. These findings support the utility of TOC to help reveal the early molecular processes that lead to sporadic ESCC.
RESUMEN
Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
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Acidosis/metabolismo , Túbulos Renales Distales/metabolismo , Mutación , Seudohipoaldosteronismo/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Acidosis/genética , Acidosis/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Células HEK293 , Humanos , Túbulos Renales Distales/patología , Ratones , Ratones Mutantes , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/patología , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Xenopus laevisRESUMEN
Dehydrated hereditary stomatocytosis is a genetic condition with defective red blood cell membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations in the mechanically activated PIEZO1 (FAM38A) ion channel were associated with dehydrated hereditary stomatocytosis. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated dehydrated hereditary stomatocytosis cases, we identify three novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for dehydrated hereditary stomatocytosis. All the dehydrated hereditary stomatocytosis-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in red blood cells of dehydrated hereditary stomatocytosis patients. Our findings also suggest a new role for mechanotransduction in red blood cell biology and pathophysiology.
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Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Activación del Canal Iónico/genética , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mutación/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Fenómenos Biomecánicos , Niño , Análisis Mutacional de ADN , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Canales Iónicos/química , Cinética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Proteínas Recombinantes/metabolismo , Adulto JovenAsunto(s)
Proteínas Portadoras/genética , Proteínas Cullin/genética , Seudohipoaldosteronismo/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/fisiología , Cloruros/metabolismo , Proteínas Cullin/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Transporte Iónico , Túbulos Renales Distales/metabolismo , Proteínas de Microfilamentos , Antígenos de Histocompatibilidad Menor , Modelos Biológicos , Potasio/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/fisiología , Estructura Terciaria de Proteína , Sodio/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na(+)-Cl(-) cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.
Asunto(s)
Proteínas Portadoras/genética , Transporte Iónico/genética , Nefronas/metabolismo , Seudohipoaldosteronismo/genética , Simportadores del Cloruro de Sodio/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Presión Sanguínea/genética , Niño , Femenino , Humanos , Riñón/metabolismo , Masculino , Proteínas de Microfilamentos , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Seudohipoaldosteronismo/metabolismo , Seudohipoaldosteronismo/fisiopatología , Análisis de Secuencia de ADN , Transducción de Señal , Simportadores del Cloruro de Sodio/genética , Adulto JovenRESUMEN
By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.