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1.
BMC Med Genet ; 21(1): 79, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32295532

RESUMO

BACKGROUND: Congenital chloride diarrhea (CLD; OMIM 214700) is a rare autosomal recessive disorder caused by pathogenic variations in the solute carrier family 26 member A3 (SLC26A3) gene. Without salt substitution, this chronic diarrheal disorder causes severe dehydration and electrolyte disturbances. Homozygous variants in the nearby gene SLC26A4 disrupt anion exchange in the inner ear and the thyroid, causing Pendred syndrome (PDS; OMIM 274600), which is the most frequent form of syndromic deafness. CASE PRESENTATION: We report an unusual co-occurrence of two rare homozygous mutations in both the SLC26A3 and SLC26A4 genes, causing a rare combination of both CLD and PDS in two siblings. Although the clinical pictures were typical, the combined loss of these anion transporters might modulate the risk of renal injury associated with CLD. CONCLUSIONS: Familial presentation of two rare autosomal recessive disorders with loss of function of different SLC26 anion transporters is described. Independent homozygous variants in the SLC26A3 and SLC26A4 genes cause CLD and PDS in siblings, shedding light on co-occurrence of rare recessive traits in the progeny of consanguineous couples.


Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , Diarreia/congênito , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Erros Inatos do Metabolismo/genética , Transportadores de Sulfato/genética , Diarreia/diagnóstico , Diarreia/genética , Diarreia/patologia , Feminino , Genes Recessivos/genética , Testes Genéticos , Bócio Nodular/diagnóstico , Bócio Nodular/patologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/patologia , Humanos , Lactente , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/patologia , Mutação , Linhagem , Gravidez , Doenças Raras/diagnóstico , Doenças Raras/genética , Doenças Raras/patologia , Irmãos
2.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G854-G869, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32116023

RESUMO

DRA (downregulated in adenoma, SLC26A3) and NHE3 (Na+/H+ exchanger 3, SLC9A3) together mediate intestinal electroneutral NaCl absorption. Both transporters contain PDZ (postsynaptic density 95, disc large, zonula occludens 1) binding motifs and interact with PDZ adaptor proteins regulating their activity and recycling. SNX27 (sorting nexin 27) contains a PDZ domain and is involved in the recycling of cargo proteins including NHE3. The interaction of SNX27 with DRA and its potential role for the activity and recycling of DRA have been evaluated in this study. SNX27 specifically interacts with DRA via its PDZ domain. The knockdown (KD) of SNX27 reduced DRA activity by 50% but was not accompanied by a decrease of DRA surface expression. This indicates that DRA is trafficked to specific functional domains in the plasma membrane in which DRA is particularly active. Consistently, the disruption of lipid raft integrity by methyl-ß-cyclodextrin has an inhibitory effect on DRA activity that was strongly reduced after SNX27 KD. In differentiated intestinal Caco2 cells, superresolution microscopy and a novel quantitative axial approach revealed that DRA and SNX27 colocalize in rab5-positive early endosomes at the apical pole. SNX27 regulates the activity of DRA in the apical plasma membrane through binding with its PDZ domain. This interaction occurs in rab5-positive early endosomes at the apical pole of differentiated intestinal Caco2 cells. SNX27 is involved in the direct recycling of DRA to the plasma membrane where it is inserted into lipid rafts facilitating increased activity.NEW & NOTEWORTHY SNX27 has a PDZ domain and is involved in the regulation and recycling of transmembrane proteins. The role of SNX27 on the activity and recycling of the intestinal Cl-/HCO3- exchanger DRA has not yet been studied. This study shows that SNX27 directly interacts with DRA in early endosomes at the apical pole of intestinal Caco2 cells and mediates its direct recycling to facilitate high activity in lipid rafts in the apical plasma membrane.


Assuntos
Polaridade Celular , Antiportadores de Cloreto-Bicarbonato/metabolismo , Endossomos/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Nexinas de Classificação/metabolismo , Transportadores de Sulfato/metabolismo , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/genética , Humanos , Microdomínios da Membrana/metabolismo , Domínios PDZ , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Nexinas de Classificação/genética , Transportadores de Sulfato/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
3.
Fluids Barriers CNS ; 17(1): 2, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31906971

RESUMO

BACKGROUND: Genetic disruption of slc4a10, which encodes the sodium-dependent chloride/bicarbonate exchanger Ncbe, leads to a major decrease in Na+-dependent HCO3- import into choroid plexus epithelial cells in mice and to a marked reduction in brain intraventricular fluid volume. This suggests that Ncbe functionally is a key element in vectorial Na+ transport and thereby for cerebrospinal fluid secretion in the choroid plexus. However, slc4a10 disruption results in severe changes in expression of Na+,K+-ATPase complexes and other major transport proteins, indicating that profound cellular changes accompany the genetic manipulation. METHODS: A tandem mass tag labeling strategy was chosen for quantitative mass spectrometry. Alterations in the broader patterns of protein expression in the choroid plexus in response to genetic disruption of Ncbe was validated by semi-quantitative immunoblotting, immunohistochemistry and morphometry. RESULTS: The abundance of 601 proteins were found significantly altered in the choroid plexus from Ncbe ko mice relative to Ncbe wt. In addition to a variety of transport proteins, particularly large changes in the abundance of proteins involved in cellular energy metabolism were detected in the Ncbe ko mice. In general, the abundance of rate limiting glycolytic enzymes and several mitochondrial enzymes were reduced following slc4a10 disruption. Surprisingly, this was accompanied by increased ATP levels in choroid plexus cells, indicating that the reduction in capacity for energy metabolism was adaptive to high ATP rather than causal for a decreased capacity for ion and water transport. Ncbe-deficient cells also had a reduced cell area and decreased K+ content. CONCLUSION: Our findings suggest that the lack of effective Na+-entry into the epithelial cells of the choroid plexus leads to a profound change in the cellular phenotype, shifting from a high-rate secretory function towards a more dormant state; similar to what is observed during ageing or Alzheimer's disease.


Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , Plexo Corióideo/metabolismo , Epitélio/metabolismo , Transporte de Íons/fisiologia , Simportadores de Sódio-Bicarbonato/genética , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Antiportadores de Cloreto-Bicarbonato/fisiologia , Células Epiteliais/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Camundongos Knockout
4.
Nat Rev Gastroenterol Hepatol ; 17(2): 93-110, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31819247

RESUMO

Primary biliary cholangitis is a chronic, seropositive and female-predominant inflammatory and cholestatic liver disease, which has a variable rate of progression towards biliary cirrhosis. Substantial progress has been made in patient risk stratification with the goal of personalized care, including early adoption of next-generation therapy with licensed use of obeticholic acid or off-label fibrate derivatives for those with insufficient benefit from ursodeoxycholic acid, the current first-line drug. The disease biology spans genetic risk, epigenetic changes, dysregulated mucosal immunity and altered biliary epithelial cell function, all of which interact and arise in the context of ill-defined environmental triggers. A current focus of research on nuclear receptor pathway modulation that specifically and potently improves biliary excretion, reduces inflammation and attenuates fibrosis is redefining therapy. Patients are benefiting from pharmacological agonists of farnesoid X receptor and peroxisome proliferator-activated receptors. Immunotherapy remains a challenge, with a lack of target definition, pleiotropic immune pathways and an interplay between hepatic immune responses and cholestasis, wherein bile acid-induced inflammation and fibrosis are dominant clinically. The management of patient symptoms, particularly pruritus, is a notable goal reflected in the development of rational therapy with apical sodium-dependent bile acid transporter inhibitors.


Assuntos
Autoimunidade/imunologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/imunologia , Cirrose Hepática Biliar/imunologia , Imunidade Adaptativa/imunologia , Animais , Ácidos e Sais Biliares/metabolismo , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/uso terapêutico , Antiportadores de Cloreto-Bicarbonato/genética , Colagogos e Coleréticos/uso terapêutico , Progressão da Doença , Exposição Ambiental , Epigênese Genética , Humanos , Imunidade Inata/imunologia , Cirrose Hepática , Cirrose Hepática Biliar/tratamento farmacológico , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/metabolismo , MicroRNAs/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Simportadores/antagonistas & inibidores , Ácido Ursodesoxicólico/uso terapêutico
5.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(11): 1131-1137, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31753097

RESUMO

OBJECTIVE: To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function. METHODS: The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group. RESULTS: The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (P<0.05), and at the same time, the P131R group had a significantly greater increase in cell membrane permeability after the induction with 100 ng/mL TNF-α (P<0.05). Western blot showed that compared with the normal control group, the P131R group had a significant reduction in the expression of SLC26A3 protein (P=0.001). CONCLUSIONS: SLC26A3 c.392C>G (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.


Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , Diarreia/congênito , Erros Inatos do Metabolismo , Transportadores de Sulfato/genética , Células CACO-2 , Diarreia/genética , Humanos , Mucosa Intestinal , Erros Inatos do Metabolismo/genética , Polimorfismo de Nucleotídeo Único , Junções Íntimas , Fator de Necrose Tumoral alfa
6.
Rev Esp Enferm Dig ; 111(10): 775-788, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31538484

RESUMO

BACKGROUND AND AIMS: heterozygous ABCB4, ABCB11 and ATP8B1 sequence variants were previously reported to be associated with low phospholipid-associated cholelithiasis, intrahepatic cholestasis of pregnancy, benign recurrent intrahepatic cholestasis and biliary lithiasis. The present study aimed to identify the presence of sequence variations in genes responsible for Mendelian liver disorders in patients with cholestatic liver disease. METHODS: targeted massive parallel sequencing of a panel of genes involved in bile acid homeostasis was performed in 105 young and adult patients with cholestatic liver disease in our laboratory for molecular diagnosis. The effects of novel variants were evaluated using bioinformatics prediction tools and the Protter and Phyre2 software programs were used to create 2D, 3D topology protein modeling. Genotype-phenotype correlation was established according to molecular analysis and clinical records. RESULTS: twenty novel heterozygous ABCB4 sequence variations, one heterozygous ABCB4 large intragenic deletion and only one novel missense variant in ABCB11 and ATP8B1 were identified. Interestingly, heterozygous and homozygous SLC4A2 missense variants were detected in patients with low phospholipid-associated cholelithiasis. Two patients harbored heterozygous GPBAR1 variants. Common variants such as homozygous ABCB11 p.Val444Ala and heterozygous ABCG8 p.Asp19His were also identified in 12 cases. CONCLUSIONS: forty-eight variants were identified in five genes including ABCB4, ABCB11, ATP8B1, SLC4A2 and GPBAR1, twenty-five of which were novel. This study expands the phenotypic and mutational spectrum in genes involved in bile acid homeostasis and highlights the genetic and phenotypic heterogeneity in patients with inherited liver disorders.


Assuntos
Colestase Intra-Hepática/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Adolescente , Adulto , Ácidos e Sais Biliares/genética , Ácidos e Sais Biliares/metabolismo , Criança , Antiportadores de Cloreto-Bicarbonato/genética , Feminino , Predisposição Genética para Doença , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Receptores Acoplados a Proteínas-G/genética , Adulto Jovem
7.
J Pediatr ; 214: 151-157.e6, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31477378

RESUMO

OBJECTIVE: To clarify clinical and genetic features of Japanese children with congenital chloride diarrhea (CCD). STUDY DESIGN: This was a multi-institutional, retrospective survey of 616 pediatric centers in Japan with identified patients with CCD between 2014 and 2018. Mutations involving SLC26A3 were detected by Sanger sequencing. RESULTS: Thirteen patients met all entry criteria including mutations in SLC26A3, and 14 patients satisfied clinical diagnostic criteria. Homozygous or compound heterozygous mutations in SLC26A3, including 6 novel mutations, were identified in 13 of these 14 patients (93%). The most common (detected in 7 of 13) was c.2063-1g>t. Median age at diagnosis was 1 day. Nine of the patients meeting all criteria were diagnosed as neonates (69%). Median follow-up duration was 10 years. When studied, 8 patients had <5 stools daily (62%), and all had fewer than in infancy. Only 1 patient had nephrocalcinosis, and 3 (23%) had mild chronic kidney disease. Neurodevelopment was generally good; only 1 patient required special education. Five patients (38%) received long-term sodium, potassium, and chloride supplementation. CONCLUSIONS: Early fetal ultrasound diagnosis and prompt long-term sodium, potassium, and chloride supplementation were common management features. Genetic analysis of SLC26A3 provided definitive diagnosis of CCD. In contrast with previously reported localities, c.2063-1g>t might be a founder mutation in East Asia.


Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , DNA/genética , Diarreia/congênito , Previsões , Erros Inatos do Metabolismo/genética , Mutação , Vigilância da População , Transportadores de Sulfato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Análise Mutacional de DNA , Diarreia/epidemiologia , Diarreia/genética , Diarreia/metabolismo , Feminino , Seguimentos , Testes Genéticos , Humanos , Incidência , Recém-Nascido , Japão/epidemiologia , Masculino , Erros Inatos do Metabolismo/epidemiologia , Erros Inatos do Metabolismo/metabolismo , Estudos Retrospectivos , Transportadores de Sulfato/metabolismo , Taxa de Sobrevida/tendências , Fatores de Transcrição
8.
J Obstet Gynaecol Res ; 45(11): 2280-2283, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499577

RESUMO

Congenital chloride diarrhea (CCD) is an autosomal recessive hereditary disease manifested by persistent, watery, profuse diarrhea with high chloride concentration (>90 mmol/L). Postnatally, neonates suffer from hypochloremia, hyponatremia, hypokalemia, metabolic alkalosis, dehydration, developmental retardation, or even death. Prenatal diagnosis is of great importance for the prognosis of CCD. We report a prenatal recurrent case of CCD. Prenatal ultrasound revealed fetal diffuse intestinal dilation with the typical honeycomb sign and polyhydramnios with high amniotic fluid index. The whole exome capture and massively-parallel DNA sequencing showed an abnormal mutation of Solute Carrier Family 26, Member 3 (SLC26A3), c.1039G>A (p.Ala347Thr), and the mutation sites were verified by sanger sequencing. When prenatal ultrasound shows polyhydramnios and diffuse intestinal dilation, CCD should be suspected. Molecular genetic testing can be helpful for the diagnosis.


Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , Diarreia/congênito , Erros Inatos do Metabolismo/genética , Diagnóstico Pré-Natal/métodos , Transportadores de Sulfato/genética , Adulto , Diarreia/diagnóstico , Diarreia/embriologia , Diarreia/genética , Feminino , Humanos , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/embriologia , Mutação , Poli-Hidrâmnios/diagnóstico , Poli-Hidrâmnios/genética , Gravidez , Recidiva
9.
Am J Physiol Cell Physiol ; 317(6): C1205-C1212, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31483700

RESUMO

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.


Assuntos
Antiporters/genética , Antiportadores de Cloreto-Bicarbonato/genética , Criptosporidiose/genética , Cryptosporidium parvum/patogenicidade , Regulação da Expressão Gênica/genética , Mucosa Intestinal/metabolismo , Transportadores de Sulfato/genética , Animais , Anticorpos Neutralizantes/farmacologia , Antiporters/antagonistas & inibidores , Antiporters/metabolismo , Células CACO-2 , Antiportadores de Cloreto-Bicarbonato/antagonistas & inibidores , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Criptosporidiose/metabolismo , Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Interações Hospedeiro-Parasita/genética , Humanos , Íleo/metabolismo , Íleo/parasitologia , Mucosa Intestinal/parasitologia , Transporte de Íons , Janus Quinases/genética , Janus Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Organoides/parasitologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transportadores de Sulfato/antagonistas & inibidores , Transportadores de Sulfato/metabolismo
10.
J Gene Med ; 21(10): e3118, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31408246

RESUMO

BACKGROUND: The present study aimed to investigate the functions and regulation mechanism of the transmembrane protease, serine 3 (TMPRSS3), which plays an important role in sensorineural hearing loss. METHODS: House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, comprising auditory-related cells, were used in the present study. An overexpression vector and small hairpin RNA target on TMPRSS3 were designed and transfected into HEI-OC1 cells. Circular RNA (circRNA) sequencing was conducted and expression profiles were obtained. The circular structure of circRNAs was validated with a polymerase chain reaction and Sanger sequencing using convergent and divergent primers. RESULTS: Overexpression of TMPRSS3 increased cell viability, whereas suppression of TMPRSS3 increased the percentage of apoptotic cells and decreased cell viability, compared to the control group. circRNA sequencing provided expression profiles indicating that the overexpression of TMPRSS3 increased the expression level of 195 circRNAs. Results of GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) studies indicated that the circRNAs are focused on the RAS signaling pathway. The pathway, circ-Slc41a2 (chr10: 82744115|82767120), miR-182 and Akt, might comprise one of the key cascades of TMPRSS3. CONCLUSIONS: TMPRSS3 is an important molecule in the regulation of cell viability and cell apoptosis of HEI-OC1 cells. Its functions are dependent on the circ-Slc41a2, miR-182 and Akt cascade.


Assuntos
Apoptose/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina Endopeptidases/genética , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Antiportadores de Cloreto-Bicarbonato/genética , Perfilação da Expressão Gênica , Humanos , Órgão Espiral/citologia , RNA Circular , Transdução de Sinais
12.
Orphanet J Rare Dis ; 14(1): 41, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760291

RESUMO

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.


Assuntos
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 Completo do Exoma/métodos
13.
Biochem Cell Biol ; 97(3): 290-306, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30462520

RESUMO

The human solute carrier 26 (SLC26) gene family of anion transporters consists of 10 members (SLC26A1-A11, A10 being a pseudogene) that encode membrane glycoproteins with 14 transmembrane segments and a C-terminal cytoplasmic sulfate transporter anti-sigma antagonist domain. Thus far, mutations in eight members of the SLC26 family (A1-A6, A8, and A9) have been linked to diseases in humans. Our goal is to characterize the role of N-glycosylation and the effect of mutations in SLC26A2 and A3 proteins on their functional expression in transfected HEK-293 cells. We found that certain mutants were retained in the endoplamic reticulum via an interaction with the lectin chaperone calnexin. Some could escape protein quality control and traffic to the cell surface upon removal of the N-glycosylation sites. Furthermore, we found that loss of N-glycosylation reduced expression of SLC26A2 at the cell surface. Loss of N-glycosylation had no effect on the stability of SLC26A3, yet resulted in a profound decrease in transport activity. Thus, N-glycosylation plays three roles in the functional expression of SLC26 proteins: (1) to retain misfolded proteins in the endoplamic reticulum, (2) to stabilize the protein at the cell surface, and (3) to maintain the transport protein in a functional state.


Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Transportadores de Sulfato/metabolismo , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Transportadores de Sulfato/química , Transportadores de Sulfato/genética
14.
Int J Colorectal Dis ; 33(9): 1169-1172, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29855681

RESUMO

PURPOSE: Abnormalities of the solute-linked carrier family 26 member A3 (SLC26A3) are implicated in the pathogenesis of several diseases including ulcerative colitis (UC). The short communication aimed at investigating the associations of UC with SLC26A3 (rs17154444, rs7810937, rs7785539, rs2108225 and rs6951457) polymorphisms and its expression in colonic tissues. METHODS: The techniques of SNaPshot method, quantitative real-time PCR and immunohistochemical analysis were conducted. RESULTS AND CONCLUSION: We found that the rs2108225 variation in SLC26A3 might increase the risk of UC and affect its expression at both the mRNA and protein levels in colonic tissues of patients with UC. Moreover, the rs17154444 variation might influence the severity of UC.


Assuntos
Antiportadores de Cloreto-Bicarbonato/genética , Colite Ulcerativa/genética , Transportadores de Sulfato/genética , Grupo com Ancestrais do Continente Asiático , China , Genótipo , Haplótipos , Humanos , Polimorfismo de Nucleotídeo Único
15.
BMC Cancer ; 18(1): 356, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29609569

RESUMO

BACKGROUND: The accumulated evidence has indicated the diagnostic role of cytokeratin (CK) and vimentin protein immunoassay in primary esophageal spindle cell carcinoma (PESC), which is a rare malignant tumor with epithelial and spindle components. However, it is largely unknown for the expression of CK and vimentin in pathological changes and prognosis of PESC. METHODS: Eighty-two PESC patients were identified from the esophageal and gastric cardia cancer database established by Henan Key Laboratory for Esophageal Cancer Research of Zhengzhou University. We retrospectively evaluated CK and vimentin protein expressions in PESC. Clinicopathological features were examined by means of univariate and multivariate survival analyses. Furthermore, the co-expression value of cytokeratin and vimentin was analyzed by receiver operating characteristic (ROC) curve. RESULTS: The positive pan-cytokeratins AE1/AE3 (AE1/AE3 for short) staining was chiefly observed in cytoplasm of epithelial component tumor cells, with a positive detection rate of 85.4% (70/82). Interestingly, 19 cases showed AE1/AE3 positive staining both in epithelial and spindle components (23.2%). However, AE1/AE3 expression was not observed with any significant association with age, gender, tumor location, gross appearance, lymph node metastasis and TNM stage. Furthermore, AE1/AE3 protein expression does not show any effect on survival. Similar results were observed for vimentin immunoassay. However, in comparison with a single protein, the predictive power of AE1/AE3 and vimentin proteins signature was increased apparently than with single signature [0.75 (95% CI = 0.68-0.82) with single protein v.s. 0.89 (95% CI = 0.85-0.94) with AE1/AE3 and vimentin proteins]. The 1-, 3-, 5- and 7-year survival rates for PESC patients in this study were 79.3%, 46.3%, 28.0% and 15.9%, respectively. Multivariate analysis demonstrated age and TNM stage were independent prognostic factors for overall survival (P = 0.036 and 0.003, respectively). It is noteworthy that only 17.1% patients had a PESC accurate diagnosis by biopsy pathology before surgery (14/82). 72.4% PESC patients with biopsy pathology before surgery had been diagnosed as squamous cell carcinoma. CONCLUSION: The present study demonstrates that cytokeratin and vimentin protein immunoassay is a useful biomarker for PESC accurate diagnosis, but not prognosis. The co-expression of cytokeratin and vimentin in both epithelial and spindle components suggest the possibility of single clone origination for PESC.


Assuntos
Neoplasias Esofágicas/metabolismo , Queratinas/metabolismo , Sarcoma/metabolismo , Vimentina/metabolismo , Adulto , Idoso , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Biomarcadores Tumorais , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Queratinas/genética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Curva ROC , Sarcoma/genética , Vimentina/genética
16.
Am J Physiol Renal Physiol ; 315(2): F313-F322, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29667915

RESUMO

Prolonged lithium treatment is associated with various renal side effects and is known to induce inner medullary collecting duct (IMCD) remodeling. In animals treated with lithium, the fraction of intercalated cells (ICs), which are responsible for acid-base homeostasis, increases compared with renal principal cells (PCs). To investigate the intricacies of lithium-induced IMCD remodeling, male Sprague-Dawley rats were fed a lithium-enriched diet for 0,1, 2, 3, 6, 9, or 12 wk. Urine osmolality was decreased at 1 wk, and from 2 to 12 wk, animals were severely polyuric. After 6 wk of lithium treatment, approximately one-quarter of the cells in the initial IMCD expressed vacuolar H+-ATPase, an IC marker. These cells were localized in portions of the inner medulla, where ICs are not normally found. Pendrin, a Cl-/[Formula: see text] exchanger, is normally expressed only in two IC subtypes found in the convoluted tubule, the cortical collecting duct, and the connecting tubule. At 6 wk of lithium treatment, we observed various patterns of pendrin localization and expression in the rat IMCD, including a novel phenotype wherein pendrin was coexpressed with aquaporin-4. These observations collectively suggest that renal IMCD cell plasticity may play an important role in lithium-induced IMCD remodeling.


Assuntos
Plasticidade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antiportadores de Cloreto-Bicarbonato/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Carbonato de Lítio/toxicidade , Transportadores de Sulfato/metabolismo , Compostos de Amônio/urina , Animais , Aquaporina 4/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Esquema de Medicação , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Masculino , Concentração Osmolar , Fenótipo , Poliúria/induzido quimicamente , Poliúria/patologia , Poliúria/urina , Ratos Sprague-Dawley , Transdução de Sinais , Transportadores de Sulfato/genética , Fatores de Tempo , ATPases Vacuolares Próton-Translocadoras/metabolismo
17.
J Biol Chem ; 293(17): 6259-6268, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29530983

RESUMO

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.


Assuntos
Células Acinares/metabolismo , Antiporters/metabolismo , Membrana Celular/metabolismo , Ácido Oxálico/metabolismo , Glândula Submandibular/metabolismo , Transportadores de Sulfato/metabolismo , Células Acinares/citologia , Animais , Antiporters/genética , Bicarbonatos/metabolismo , Células CHO , Membrana Celular/genética , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Cricetulus , Camundongos , Camundongos Knockout , Saliva/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Glândula Submandibular/citologia , Transportadores de Sulfato/genética
18.
Lab Invest ; 98(6): 745-754, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29540861

RESUMO

Dysregulated autophagy may be a central player in trehe pathogenesis of primary biliary cholangitis (PBC) by inducing autoimmune processes via abnormal expression of mitochondrial antigens such as pyruvate dehydrogenase complex, E2 component (PDC-E2) and also by inducing cellular senescence in biliary epithelial cells (BECs) in bile duct lesions in PBC. We examined the association of an impaired "biliary bicarbonate umbrella" due to dysfunction of anion exchanger 2 (AE2) with dysregulated autophagy and cellular senescence in PBC. The expression of AE2 was examined in cultured BECs treated with bile acids such as glycochenodeoxycholic acid (GCDC) and tauro-ursodeoxycholic acid (TUDCA), various cytokines (IL-4, IL-13, IFNγ, TNFα, TGFß), and serum deprivation. The effect of AE2 knockdown using siRNA on autophagy, cell surface expression of PDC-E2, and cellular senescence was also examined. The expression of AE2 and its association with autophagy-related markers and senescent markers p16INK4a and p21WAF1/Cip1 were immunohistochemically determined in livers taken from the patients with PBC (n = 50) and 69 control diseased and normal livers. The expression of AE2 was significantly induced in the cultured BECs shortly treated with GCDC and other stresses, whereas it was significantly decreased in senescent BECs induced by GCDC and other stresses (p < 0.05). Dysregulated autophagy, cell surface expression of PDC-E2, and cellular senescence were significantly increased by knockdown of AE2 (p < 0.05). The expression of AE2 was significantly decreased in cholangitis in PBC, compared to control livers (p < 0.05). The decreased expression of AE2 was correlated with dysregulated autophagy, abnormal expression of PDC-E2, and cellular senescence in bile duct lesions in PBC. In conclusion, an impaired biliary bicarbonate umbrella may be involved in the pathogenesis of PBC by inducing dysregulated autophagy.


Assuntos
Autofagia , Bicarbonatos/metabolismo , Ductos Biliares/metabolismo , Antiportadores de Cloreto-Bicarbonato/fisiologia , Colangite/etiologia , Animais , Células Cultivadas , Senescência Celular , Antiportadores de Cloreto-Bicarbonato/genética , Colangite/metabolismo , Colangite/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C
19.
Am J Physiol Regul Integr Comp Physiol ; 314(4): R552-R562, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29351430

RESUMO

Recent research has shown that nitric oxide (NO) produced by nitric oxide synthases (NOS) is an inhibitor of ion transporter activity and a modulator of epithelial ion transport in fish, but little is known on changes in the NOS/NO system during osmotic stress. We hypothesized that the NOS/NO system responds to salinity changes as an integrated part of the acclimation process. Expression and localization of nos1/Nos1 and nos2/Nos2 were investigated in gill, kidney, and intestine of freshwater (FW)- and seawater (SW)-transferred trout using quantitative PCR, Western blotting, and immunohistochemistry, along with expressional changes of major ion transporters in the gill. The classical branchial ion transporters showed expected expressional changes upon SW transfer, there among a rapid decrease in Slc26a6 mRNA, coding a branchial Cl-/[Formula: see text] exchanger. There was a major downregulation of nos1/ nos2/Nos2 expression in the gill during SW acclimation. A significant decrease in plasma nitrite supported an overall decreased Nos activity and NO production. In the middle intestine, Nos1 was upregulated during SW acclimation, whereas no changes in nos/Nos expression were observed in the posterior intestine and the kidney. Nos1 was localized along the longitudinal axis of the gill filament, beneath smooth muscle fibers of the intestine wall and in blood vessel walls of the kidney. Nos2 was localized within the epithelium adjacent to the gill filament axis and in hematopoietic tissues of the kidney. We conclude that downregulation of branchial NOS is integrated to the SW acclimation process likely to avoid the inhibitory effects of NO on active ion extrusion.


Assuntos
Aclimatação , Proteínas de Peixes/metabolismo , Óxido Nítrico Sintase/metabolismo , Oncorhynchus mykiss/metabolismo , Água do Mar , Animais , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Proteínas de Peixes/genética , Regulação Enzimológica da Expressão Gênica , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Oncorhynchus mykiss/genética , Osmorregulação , Pressão Osmótica , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Fatores de Tempo , Distribuição Tecidual
20.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt B): 1232-1239, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28962898

RESUMO

Primary biliary cholangitis (PBC) is a chronic fibrosing cholangiopathy characterized by an autoimmune stereotype and defective biliary bicarbonate secretion due to down-regulation of anion exchanger 2 (AE2). Despite the autoimmune features, immunosuppressants are ineffective while two bile acid-based therapies (ursodeoxycholic acid and obeticholic acid) have been shown to improve biochemical and histological features of cholestasis and long-term prognosis. However, the etiology and pathogenesis of PBC is largely unknown. Recently, it has been shown that microRNA-506 (miR-506) on chromosome X is up-regulated in PBC cholangiocytes and suppresses AE2 expression, which sensitizes cholangiocytes to bile salt-induced apoptosis by activating soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. In this review, we discuss the experimental evidence for the emerging role of the miR-506-AE2-sAC axis in PBC pathogenesis. We further hypothesize that the initial disease trigger induces an X-linked epigenetic change, leading to a female-biased activation of the miR-506-AE2-sAC axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.


Assuntos
Adenilil Ciclases/metabolismo , Apoptose , Doenças Autoimunes/etiologia , Colangite/etiologia , Células Epiteliais/patologia , Doenças Autoimunes/patologia , Bicarbonatos/metabolismo , Ácidos e Sais Biliares/metabolismo , Ductos Biliares/citologia , Ductos Biliares/imunologia , Ductos Biliares/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Colangite/patologia , Epigênese Genética/genética , Epigênese Genética/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Genes Ligados ao Cromossomo X/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima
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