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1.
Artigo em Chinês | MEDLINE | ID: mdl-33540994

RESUMO

Objective:The aim of this study is to analyze the mutation characteristics of GJB2 and SLC26A4 gene in patients with delayed non-syndromic hearing loss, which is beneficial to the early detection and intervention of delayed deafness. Methods:Sanger sequencing technology was used to detect two common genes in 139 patients with non-syndromic deafness, six hot spot mutations in GJB2 gene and SLC26A4 gene, and single heterozygous mutations found in GJB2 gene and SLC26A4 gene were detected by whole exome sequencing. Results:Among the 25 patients with deafness caused by GJB2 gene mutation, 12 of them passed universal newborn hearing screening and then developed delayed extremely severe hearing loss. The onset time of hearing loss was 6-48 months. All the genotypes were homozygous or compound heterozygous mutation of c. 235delC, especially genotype of GJB2 c. 235delC homozygous and c. 235delC/c. 299-300 delAT compound heterozygous mutations, and the CT manifestations were normal. Among the 42 patients with deafness caused by SLC26A4 gene mutation, 30 of them passed universal newborn hearing screening and developed delayed deafness. The onset time of hearing loss was three months to ten years old. Among them, the genotypes of 21 patients were compound heterozygous mutation, and 9 patients were homozygous mutation of c. 919-2A>G, especially genotypes were SLC26A4 c. 919-2A>G/c. 665G>T and c. 919-2A>G /c. 2027T>A compound heterozygous mutation. The CT findings of 19 cases showed single enlarged vestibular aqueduct, and 11 cases showed enlarged vestibular aqueduct with Mondini malformation. Conclusion:For the children who have passed universal newborn hearing screening, the genotypes detected are GJB2 c. 235delC homozygous, SLC26A4 c. 919-2A>G homozygous or compound heterozygous mutations, especially genotypes GJB2 c. 235delC homozygous, c. 235delC/c. 299-300delAT compound heterozygous mutations and SLC26A4 c. 919-2A>G/c. 665G>T and c. 919-2A>G/c. 2027T>A compound heterozygous mutation. Attention should be paid to the hearing problems of children all the time, and the possibility of delayed deafness in the future should be considered.


Assuntos
Conexinas , Surdez , Criança , China , Conexinas/genética , Análise Mutacional de DNA , Surdez/genética , Genótipo , Humanos , Lactente , Recém-Nascido , Mutação , Fenótipo , Transportadores de Sulfato/genética
2.
Zhonghua Yi Xue Za Zhi ; 101(2): 103-107, 2021 Jan 12.
Artigo em Chinês | MEDLINE | ID: mdl-33455124

RESUMO

Objective: To explore the application value of high-throughput gene detection method of copy number variations (CNV) in the diagnosis of enlarged vestibular aqueduct (EVA). Methods: A total of 46 nonsyndromic hearing loss patients with EVA were recruited between May 2014 and December 2016 from Department of Otolaryngology of Xiangya Hospital, Central South University. A high-throughput multiplex analysis method based on double ligation and multiple fluorescent PCR was designed and performed to detect CNV in the three EVA-related genes (SLC26A4, FOXI1 and KCNJ10). The data were analyzed by GeneMapper v4.1. Healthy volunteers (n=100) were selected as normal controls. Results: A total of 46 EVA patients were detected (32 males, 14 females, aged 1 to 26 years). In 4 EVA patients, deletions of exons 1-3 of SLC26A4 gene (4/46, 8.7%) were detected, which were not reported in the database of genomic variants (DGV), and were absent in 100 normal controls. There was no CNV detected in FOXI1 and KCNJ10 in the study. Conclusions: In the current study, three known EVA-related genes were designed as the target area for CNV detection by high-throughput ligation-dependent probe amplification (HLPA) analysis. This method can be used as a supplementary analysis of point mutation detection of hearing loss, which helps achieve the accurate genetic diagnosis of EVA.


Assuntos
Perda Auditiva Neurossensorial , Proteínas de Membrana Transportadoras , Adolescente , Adulto , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Feminino , Fatores de Transcrição Forkhead/genética , Dosagem de Genes , Perda Auditiva Neurossensorial/genética , Humanos , Lactente , Masculino , Proteínas de Membrana Transportadoras/genética , Mutação , Transportadores de Sulfato/genética , Aqueduto Vestibular/anormalidades , Adulto Jovem
3.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(11): 1213-1216, 2020 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-33179223

RESUMO

OBJECTIVE: To detect additional variants for newborn carriers of single heterozygous variants of the GJB2 or SLC26A4 gene by genechip analysis in Changsha area, and explore the variation spectrum of deafness-related genes in this region. METHODS: For 462 newborns carrying single heterozygous variants of the GJB2 or SLC26A4 gene, all exons of the genes were subjected to Sanger sequencing. The pathogenicity of the variants was analyzed by database and literature search. RESULTS: For 305 newborns carrying a heterozygous GJB2 variant, 143 (46.49%) were found to carry additional variants, including 29 (9.51%) with c.109G>A likely pathogenic variant, and 1 (6.48%) with c.551G>A pathogenic variant. Among 153 newborns carrying single heterozygous variant of the SLC26A4 gene, 2 (1.31%) were found with a c.281C>T variant, and 1 (0.65%) with a c.1547_1548ins pathogenic variant. Among 4 newborns simultaneously carrying GJB2 and SLC26A4 variants, two were found to carry c.109G>A and c.844T>C variants (clinical significance unknown), respectively. CONCLUSION: For newborns carrying single heterozygous variants of the GJB2 or SLC26A4 gene by genechip analysis, the detection rate for other variants is quite high. Sanger sequencing can significantly improve the detection rate of high-risk newborns and enrich the variant spectrum of deafness genes.


Assuntos
Conexinas/genética , Triagem de Portadores Genéticos , Transportadores de Sulfato/genética , Análise Mutacional de DNA , Surdez/diagnóstico , Surdez/genética , Heterozigoto , Humanos , Recém-Nascido , Mutação , Análise de Sequência com Séries de Oligonucleotídeos
4.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(9): 958-961, 2020 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-32820507

RESUMO

OBJECTIVE: To determine the carrier rate of deafness-related genetic variants among 53 873 newborns from Zhengzhou. METHODS: Heel blood samples of the newborns were collected with informed consent from the parents, and 15 loci of 4 genes related to congenital deafness were detected by microarray. RESULTS: In total 2770 newborns were found to carry deafness-related variants, with a carrier rate of 5.142%. 1325 newborns (2.459%) were found to carry heterozygous variants of the GJB2 gene, 1071 (1.988%) were found with SLC26A4 gene variants, 205 were found with GJB3 gene variants (0.381%), and 120 were found with 12S rRNA variants (0.223%). Five newborns have carried homozygous GJB2 variants, two have carried homozygous SLC26A4 variants, five have carried compound heterozygous GJB2 variants, and four have carried compound heterozygous SLC26A4 variants. 33 neonates have carried heterozygous variants of two genes at the same time. CONCLUSION: The carrier rate of deafness-related variants in Zhengzhou, in a declining order, is for GJB2, SLC26A4, GJB3 and 12S rRNA. The common variants included GJB2 235delC and SLC26A4 IVS7-2A>G, which are similar to other regions in China. To carry out genetic screening of neonatal deafness can help to identify congenital, delayed and drug-induced deafness, and initiate treatment and follow-up as early as possible.


Assuntos
Coloboma/genética , Conexinas , Heterozigoto , Diagnóstico Pré-Natal , Insuficiência Renal/genética , Refluxo Vesicoureteral/genética , China , Coloboma/diagnóstico , Conexinas/genética , Análise Mutacional de DNA , Surdez/genética , Feminino , Feto , Homozigoto , Humanos , Recém-Nascido , Mutação , Fenótipo , Gravidez , Insuficiência Renal/diagnóstico , Transportadores de Sulfato/genética , Refluxo Vesicoureteral/diagnóstico
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(9): 962-967, 2020 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-32820508

RESUMO

OBJECTIVE: To determine the types and frequency of deafness-related variants among 7875 newborns from Dongying area of Shandong Province. METHODS: One hundred loci of 18 common deafness genes were subjected to semiconductor sequencing. Variant site, frequency and distribution of the variants were analyzed. RESULTS: In total 552 deafness gene variants were detected among the 7875 newborns, which yielded a detection rate of 7.01%. Among these, common variant sites for GJB2, SLC26A4 and GJB3 genes were c.235delC, IVS7-2A>G and c.538C>T, respectively. The variant frequencies of matrilinear inheritance deafness genes MT-CO1, MT-RNR1, MT-TL1 and MT-TS1 were 0.38%, 0.25%, 0.1% and 0.01%, respectively. Four newborns were diagnosed with deafness, among which one had unilateral hearing loss. Analysis of the proportions of neonatal deafness-related variants in five counties of Dongying showed that the highest variant rate for the SLC26A4 gene compared with GJB2 was in Lijin county (51.76% vs. 40%), while the lowest was in Hekou county (30.77% vs. 56.41%). CONCLUSION: The carrier rate of deafness-related variants in Dongying area is higher than other regions of China, which may be attributed to the increased types and variant sites covered by the semiconductor sequencing method compared with the chip method and time-of-flight mass spectrometry. Due to geographical and population aggregation factors, the proportion of deafness variants in the five counties of Dongying differed significantly. Above results may provide a guide for the prevention of congenital deafness in Dongying area.


Assuntos
Conexinas , Surdez , Triagem Neonatal , China , Conexinas/genética , Análise Mutacional de DNA , Surdez/diagnóstico , Surdez/genética , Humanos , Recém-Nascido , Mutação , RNA Ribossômico , Transportadores de Sulfato/genética
6.
Medicine (Baltimore) ; 99(19): e19763, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32384426

RESUMO

INTRODUCTION: Pendred syndrome (PDS)/DFNB 4 is a disorder with fluctuating and progressive hearing loss, vertigo, and thyroid goiter. We identified pathophysiology of a neurodegenerative disorder in PDS patient derived cochlear cells that were induced via induced pluripotent stem cells and found sirolimus, an mTOR inhibitor, as an inhibitor of cell death with the minimum effective concentration less than 1/10 of the approved dose for other diseases. Given that there is no rational standard therapy for PDS, we planned a study to examine effects of low dose oral administration of sirolimus for the fluctuating and progressive hearing loss, and the balance disorder of PDS by daily monitor of their audio-vestibular symptoms. METHODS AND ANALYSIS: This is a phase I/IIa double blind parallel-group single institute trial in patient with PDS/DFNB4. Sixteen of outpatients with fluctuating hearing diagnosed as PDS in SLC26A4 genetic testing aged in between 7 and 50 years old at the time of consent are given either placebo or sirolimus tablet (NPC-12T). In NPC-12T placebo arm, placebo will be given for 36 weeks; in active substance arm, placebo will be given for 12 weeks and the NPC-12T for 24 weeks. Primary endpoints are safety and tolerability. The number of occurrences and types of adverse events and of side effects will be sorted by clinical symptoms and by abnormal change of clinical test results. A 2-sided 95% confidence interval of the incidence rate by respective dosing arms will be calculated using the Clopper-Pearson method. Clinical effects on audio-vestibular tests performed daily and precise physiological test at each visit will also be examined as secondary and expiratory endpoints. TRIAL REGISTRATION NUMBER: JMA-IIA00361; Pre-results.


Assuntos
Bócio Nodular/tratamento farmacológico , Perda Auditiva Neurossensorial/tratamento farmacológico , Sirolimo/administração & dosagem , Aqueduto Vestibular/anormalidades , Adolescente , Adulto , Audiometria , Criança , Método Duplo-Cego , Feminino , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Pessoa de Meia-Idade , Transportadores de Sulfato/genética , Resultado do Tratamento , Testes de Função Vestibular , Adulto Jovem
7.
Proc Natl Acad Sci U S A ; 117(20): 10865-10875, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366666

RESUMO

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.


Assuntos
Endocitose/fisiologia , Miosinas/química , Miosinas/metabolismo , Polieletrólitos/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular , Clatrina/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Modelos Moleculares , Doença de Parkinson/metabolismo , Conformação Proteica , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
8.
Am J Physiol Cell Physiol ; 319(2): C331-C344, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432926

RESUMO

The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl-/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Interleucina-17/genética , Mucosa Respiratória/metabolismo , Transportadores de Sulfato/genética , Fator de Necrose Tumoral alfa/genética , Álcalis/metabolismo , Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Interleucina-17/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
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
10.
Int J Pediatr Otorhinolaryngol ; 134: 110018, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32251972

RESUMO

Sensorineural hearing loss is a heterogeneous disease caused by mutations in many genes. However, in the presence of enlarged vestibular aqueduct, it is frequently associated with mutations in the solute carrier family 26 member 4 (SLC26A4), a gene causative of a syndromic form (Pendred) as well as a non-syndromic form of hearing loss (DFNB4). We describe a clinical case presenting bilateral sensorineural hearing loss and enlarged vestibular aqueduct in which a novel homozygous SLC26A4 mutation was identified. Despite a late diagnosis of hearing loss, a peculiar rehabilitation therapy strategy was identified that provided excellent results.


Assuntos
Códon sem Sentido/genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/reabilitação , Transportadores de Sulfato/genética , Aqueduto Vestibular/anormalidades , Criança , Feminino , Perda Auditiva Neurossensorial/genética , Homozigoto , Humanos
11.
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
12.
Nat Commun ; 11(1): 1343, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165640

RESUMO

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.


Assuntos
Efrina-A2/genética , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Transportadores de Sulfato/genética , Sequência de Aminoácidos , Animais , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A2/química , Efrina-A2/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Bócio Nodular/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação Puntual , Ligação Proteica , Transportadores de Sulfato/química , Transportadores de Sulfato/metabolismo
13.
Artigo em Chinês | MEDLINE | ID: mdl-32086922

RESUMO

Objective:To detect 20 common deafness gene mutations in non- syndromic deafness patients in China using PCR- RDB, and analyze and summarize the mutation data to explore the clinical value of this method. Method:The PCR- RDB and Sanger sequencing were used to detect 20 common mutations of four deafness genes(GJB2, GJB3, SLC26A4 and mtDNA) in 500 patients with non- syndromic hearing loss . The Sanger sequencing was used to compare the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the deafness mutation detected by PCR- RDB. Result:A total of 500 samples were detected. 147 wild- type samples, 81 homozygous mutant samples, 240 heterozygous mutant samples, 32 composite heterozygous mutant samples were detected using the PCR- RDB within the range of 20 gene mutations, which were identical to the Sanger sequencing results. GJB2 c.235delC and SLC26A4 c.919- 2 A>G are the most common hotspot mutations in this study, followed by mtDNA m. 1555 A>G. Compared with the Sanger sequencing method, the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the real- time fluorescence PCR melting curve method were 100%, and the Kappa value was one. Conclusion:PCR reverse dot-blot hybridization is a simple, rapid, sensitive and specific method for detecting 20 mutations of 4 common deafness genes in Chinese population, it is expected to be used in clinical detection of deafness genes in the future.


Assuntos
Análise Mutacional de DNA , Surdez/genética , China , Conexinas/genética , DNA Mitocondrial/genética , Humanos , Mutação , Reação em Cadeia da Polimerase , Transportadores de Sulfato/genética
14.
J Am Soc Nephrol ; 31(3): 483-499, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32054691

RESUMO

BACKGROUND: Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium's role in this response and the receptor's effect on intercalated and principal cell function in the cortical collecting duct (CCD). METHODS: We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell-specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. RESULTS: Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin's relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone's effect on pendrin total and apical abundance and subcellular distribution. CONCLUSIONS: With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin's total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.


Assuntos
Aldosterona/metabolismo , Proteínas de Transporte de Ânions/fisiologia , Túbulos Renais Coletores/metabolismo , Potássio/sangue , Receptores de Mineralocorticoides/metabolismo , Transportadores de Sulfato/genética , Angiotensina II/farmacologia , Animais , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Técnicas In Vitro , Transporte de Íons/fisiologia , Túbulos Renais Coletores/citologia , Camundongos , Camundongos Knockout , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Canais de Sódio/genética
15.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R320-R328, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913688

RESUMO

The modifications of the hemodynamic system and hydromineral metabolism are physiological features characterizing a normal gestation. Thus, the ability to expand plasma volume without increasing the level of blood pressure is necessary for the correct perfusion of the placenta. The kidney is essential in this adaptation by reabsorbing avidly sodium and fluid. In this study, we observed that the H,K-ATPase type 2 (HKA2), an ion pump expressed in kidney and colon and already involved in the control of the K+ balance during gestation, is also required for the correct plasma volume expansion and to maintain normal blood pressure. Indeed, compared with WT pregnant mice that exhibit a 1.6-fold increase of their plasma volume, pregnant HKA2-null mice (HKA2KO) only modestly expand their extracellular volume (×1.2). The renal expression of the epithelial Na channel (ENaC) α- and γ-subunits and that of the pendrin are stimulated in gravid WT mice, whereas the Na/Cl- cotransporter (NCC) expression is downregulated. These modifications are all blunted in HKA2KO mice. This impeded renal adaptation to gestation is accompanied by the development of hypotension in the pregnant HKA2KO mice. Altogether, our results showed that the absence of the HKA2 during gestation leads to an "underfilled" situation and has established this transporter as a key player of the renal control of salt and potassium metabolism during gestation.


Assuntos
Pressão Sanguínea , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Rim/enzimologia , Volume Plasmático , Potássio/metabolismo , Sódio/metabolismo , Animais , Aquaporina 2/metabolismo , Colo/enzimologia , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , ATPase Trocadora de Hidrogênio-Potássio/deficiência , ATPase Trocadora de Hidrogênio-Potássio/genética , Homeostase , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
16.
FASEB J ; 34(1): 720-734, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914611

RESUMO

Confusion persists over pathogenesis of spondylolysis. To confirm pathogenicity of the previously identified causative mutation of spondylolysis and investigate the genetic etiology, we generate a new mouse line harboring D673V mutation in the Slc26a2 gene. D673V mutation induces delayed endochondral ossification characterized by transiently reduced chondrocyte proliferation in mice at the early postnatal stage. Adult D673V homozygotes exhibit dysplastic isthmus and reduced bone volume of the dorsal vertebra resembling the detached vertebral bony structure when spondylolysis occurs, including the postzygopophysis, vertebral arch, and spinous process, which causes biomechanical alterations around the isthmic region of L4-5 vertebrae indicated by finite element analysis. Consistently, partial ablation of Slc26a2 in vertebral skeletal cells using Col1a1-Cre; Slc26a2 fl/fl mouse line recapitulates a similar but worsened vertebral phenotype featured by lamellar isthmus. In addition, when reaching late adulthood, D673V homozygotes develop an evident bone-loss phenotype and show impaired osteogenesis. These findings support a multifactorial etiology, involving congenitally predisposed isthmic conditions, altered biomechanics, and age-dependent bone loss, which leads to SLC26A2-related spondylolysis.


Assuntos
Vértebras Lombares/cirurgia , Espondilólise/patologia , Transportadores de Sulfato/efeitos dos fármacos , Envelhecimento , Animais , Vértebras Lombares/patologia , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Fenótipo , Espondilólise/etiologia , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
17.
PLoS One ; 15(1): e0225368, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31971949

RESUMO

Single Nucleotide Polymorphisms (SNPs) are the most common candidate mutations in human beings that play a vital role in the genetic basis of certain diseases. Previous studies revealed that Solute Carrier Family 26 Member 4 (SLC26A4) being an essential gene of the multi-faceted transporter family SLC26 facilitates reflexive movement of Iodide into follicular lumen through apical membrane of thyrocyte. SLC26A4 gene encodes Pendred protein, a membrane glycoprotein, highly hydrophobic in nature, present at the apical membrane of thyrocyte functioning as transporter of iodide for thyroid cells. A minor genetic variation in SLC26A4 can cause Pendred syndrome, a syndrome associated with thyroid glands and deafness. In this study, we performed in-silico analysis of 674 missense SNPs of SLC26A4 using different computational platforms. The bunch of tools including SNPNEXUS, SNAP-2, PhD-SNP, SNPs&GO, I-Mutant, ConSurf, and ModPred were used to predict 23 highly confident damaging and disease causing nsSNPs (G209V, G197R, L458P, S427P, Q101P, W472R, N392Y, V359E, R409C, Q235R, R409P, G139V, G497S, H723R, D87G, Y127H, F667C, G334A, G95R, S427C, R291W, Q383H and E384G) that could potentially alter the SLC26A4 gene. Moreover, protein structure prediction, protein-ligand docking and Molecular Dynamics simulation were performed to confirm the impact of two evident alterations (Y127H and G334A) on the protein structure and function.


Assuntos
Biologia Computacional , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Transportadores de Sulfato/genética , Surdez/genética , Surdez/patologia , Bócio Nodular/genética , Bócio Nodular/patologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Humanos , Ligantes , Simulação de Dinâmica Molecular , Mutação/genética , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transportadores de Sulfato/química
18.
J Clin Invest ; 130(1): 272-286, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31581148

RESUMO

Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40%-50% of adults with CF. The age at onset of CF-related diabetes (CFRD) (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age at onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that 2 common DNA haplotypes formed by the risk variants account for the association with diabetes. Single-cell RNA sequencing (scRNA-Seq) indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells and frequently coexpressed with CF transmembrane conductance regulator (CFTR) along with transcription factors that have binding sites 5' of SLC26A9. These findings were replicated upon reanalysis of scRNA-Seq data from 4 independent studies. DNA fragments derived from the 5' region of SLC26A9-bearing variants from the low-risk haplotype generated 12%-20% higher levels of expression in PANC-1 and CFPAC-1 cells compared with the high- risk haplotype. Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age at onset of diabetes, suggesting a CFTR-agnostic treatment for a major complication of CF.


Assuntos
Antiporters/biossíntese , Fibrose Cística/metabolismo , Diabetes Mellitus/metabolismo , Haplótipos , Transportadores de Sulfato/biossíntese , Antiporters/genética , Linhagem Celular , Fibrose Cística/complicações , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diabetes Mellitus/etiologia , Diabetes Mellitus/genética , Feminino , Humanos , Masculino , RNA-Seq , Transportadores de Sulfato/genética
19.
Mol Plant ; 13(1): 99-111, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31610248

RESUMO

During plant growth and development mineral elements are preferentially delivered to different organs and tissues to meet the differential demand. It has been shown that the preferential distribution of mineral nutrients in gramineous plants is mediated by node-based transporters, but the mechanisms of preferential distribution in dicots are poorly understood. Here, we report a distinct mechanism for the preferential distribution of phosphorus (P) in Arabidopsis plants, revealed by detailed functional analysis of AtSPDT/AtSULTR3;4 (SULTR-like P Distribution Transporter), a homolog of rice OsSPDT. Like OsSPDT, AtSPDT is localized at the plasma membrane and showed proton-dependent transport activity for P. Interestingly, we found that AtSPDT is mainly expressed in the rosette basal region and leaf petiole, and its expression is up-regulated by P deficiency. Tissue-specific analysis showed that AtSPDT is mainly located in the vascular cambium of different organs, as well as in the parenchyma tissues of both xylem and phloem regions. Knockout of AtSPDT inhibited the growth of new leaves under low P due to decreased P distribution to those organs. The seed yields of the wild-type and atspdt mutant plants are similar, but the seeds of mutant plants contain - less P. These results indicate that AtSPDT localized in the vascular cambium is involved in preferential distribution of P to the developing tissues, through xylem-to-phloem transfer mainly at the rosette basal region and leaf petiole.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Fósforo/metabolismo , Feixe Vascular de Plantas/fisiologia , Transportadores de Sulfato/genética , Simportadores/genética , Arabidopsis/genética , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo
20.
Urolithiasis ; 48(1): 1-8, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31201468

RESUMO

Oxalobacter sp. promotion of enteric oxalate excretion, correlating with reductions in urinary oxalate excretion, was previously reported in rats and mice, but the mechanistic basis for this affect has not been described. The main objective of the present study was to determine whether the apical oxalate transport proteins, PAT1 (slc26a6) and DRA (slc26a3), are involved in mediating the Oxalobacter-induced net secretory flux across colonized mouse cecum and distal colon. We measured unidirectional and net fluxes of oxalate across tissues removed from colonized PAT1 and DRA knockout (KO) mice and also across two double knockout (dKO) mouse models with primary hyperoxaluria, type 1 (i.e., deficient in alanine-glyoxylate aminotransferase; AGT KO), including PAT1/AGT dKO and DRA/AGT dKO mice compared to non-colonized mice. In addition, urinary oxalate excretion was measured before and after the colonization procedure. The results demonstrate that Oxalobacter can induce enteric oxalate excretion in the absence of either apical oxalate transporter and urinary oxalate excretion was reduced in all colonized genotypes fed a 1.5% oxalate-supplemented diet. We conclude that there are other, as yet unidentified, oxalate transporters involved in mediating the directional changes in oxalate transport across the Oxalobacter-colonized mouse large intestine.


Assuntos
Antiporters/metabolismo , Mucosa Intestinal/metabolismo , Oxalatos/metabolismo , Oxalobacter formigenes/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Ceco/metabolismo , Ceco/microbiologia , Colo/metabolismo , Colo/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Knockout , Oxalobacter formigenes/isolamento & purificação , Eliminação Renal , Transportadores de Sulfato/genética , Simbiose
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