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.
Caco-2 Cells ; Chloride-Bicarbonate Antiporters ; genetics ; Diarrhea ; congenital ; genetics ; Humans ; Intestinal Mucosa ; Metabolism, Inborn Errors ; genetics ; Polymorphism, Single Nucleotide ; Sulfate Transporters ; genetics ; Tight Junctions ; Tumor Necrosis Factor-alpha

Hereditary spherocytosis (HS) is caused by mutations in the SPTA1, SPTB, ANK1, SLC4A1, and EPB42 genes, all of which encode erythrocyte membrane proteins. Mutations in SLC4A1, which encodes band 3 protein, have rarely been reported as the causative factor among Korean patients with HS. Here, we report two Korean patients with HS carrying mutations in SLC4A1. Patient 1 was a 3-year-old girl with unremarkable past and family histories and was evaluated for anemia that was detected after a complete blood count. She was suspected of having HS considering the spherocytosis of her peripheral blood smear, increased osmotic fragility, hemolytic features in blood chemistry tests, and splenomegaly. Sequence analysis revealed that the patient harbored a single heterozygous missense mutation, c.2278C>T (p.Arg760Trp) in exon 17 of SLC4A1. Patient 2 was a 23-year-old man who had a prior history of intermittent jaundice. Although the patient did not have anemia, a genetic test for HS was performed due to evidence of hemolytic features in the blood chemistry test, splenomegaly, and a family history of HS. The test confirmed a single heterozygous missense mutation, c.2423G>T (p.Arg808Leu) in exon 18 of SLC4A1.
Anemia ; Anion Exchange Protein 1, Erythrocyte ; Blood Cell Count ; Chemistry ; Child, Preschool ; Erythrocyte Membrane ; Exons ; Female ; Humans ; Jaundice ; Mutation, Missense ; Osmotic Fragility ; Sequence Analysis ; Splenomegaly ; Young Adult

OBJECTIVE: To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). METHODS: Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. RESULTS: Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. CONCLUSION The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.
Anion Exchange Protein 1, Erythrocyte ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA Primers ; Heterozygote ; Humans ; Mutation ; Spherocytosis, Hereditary ; genetics

OBJECTIVETo assess the association of single nucleotide polymorphisms (SNPs) and haplotypes of solute-linked carrier family 26 member A3 (SLC26A3) gene with ulcerative colitis (UC) among Chinese patients.

METHODSFor 416 UC patients and 584 controls, 5 SNPs of the SLC26A3 gene (rs17154444, rs7810937, rs7785539, rs2108225 and rs6951457) were determined with a SNaPshot method. Linkage disequilibrium (LD) and haplotype were analyzed for all subjects.

RESULTSThe G allele and AG+GG genotype of rs2108225 were more prevalent in UC patients compared with the controls (65.14% vs. 58.65%, P=0.030; 87.02% vs. 81.85%, P=0.012, respectively). The C allele and TC+CC genotype of rs17154444 were more prevalent in patients with severe UC than in other patients (14.00% vs. 6.01%, P<0.01; 28.00% vs. 11.48%, all P<0.01). Similar conclusion may also be drawn for C allele and GC+CC genotype of rs7785539 (8.00% vs. 7.38%, P=0.011; 16.00% vs. 13.93%, P=0.017, respectively). The SNPs rs17154444, rs7810937, rs7785539 and rs2108225 were found to be in strong LD. Compared with the controls, the T-A-G-G haplotype was more prevalent in UC patients (62.60% vs. 58.20%, P=0.017), whereas the T-G-G-A haplotype was less common in UC patients (27.40% vs. 31.60%, P=0.041).

CONCLUSIONVariations of the SLC26A3 rs2108225 may enhance the risk of UC. The rs17154444 and rs7785539 polymorphisms of the SLC26A3 gene are correlated with the severity of UC. The T-A-G-G haplotype formed by rs17154444, rs781093, rs7785539 and rs2108225 of the SLC26A3 gene may increase the risk for UC, whereas the T-G-G-A haplotype may decrease this risk.

Adult ; Asian Continental Ancestry Group ; genetics ; China ; Chloride-Bicarbonate Antiporters ; genetics ; Colitis, Ulcerative ; genetics ; Female ; Genotype ; Haplotypes ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

OBJECTIVETo study the clinical features of two families with distal renal tubular acidosis (dRTA) and mutations in the pathogenic gene SLC4A1.

METHODSFamily investigation, medical history collection, and measurement of biochemical parameters were performed to analyze the clinical phenotype and genetic characteristics of dRTA. Direct sequencing was used to detect SLC4A1 gene mutations.

RESULTSThree patients in these two families (two of them were mother and son) were diagnosed with dRTA with typical clinical features, including short stature, metabolic acidosis, alkaline urine, hypokalemia, and nephrocalcinosis. SLC4A1 gene analysis showed that all the three patients had a pathogenic missense mutation R589H (c.1766G>A). The child in family 1 had a de novo mutation of SLC4A1, and the child in family 2 had an SLC4A1 gene mutation inherited from the mother, which met the characteristic of autosomal dominant inheritance.

CONCLUSIONSThis study reports the R589H mutation in SLC4A1 gene in families with hereditary dRTA for the first time in China. Clinical physicians should perform gene detection for patients suspected of hereditary dRTA to improve the diagnosis and treatment of this disease.

Acidosis, Renal Tubular ; genetics ; Anion Exchange Protein 1, Erythrocyte ; genetics ; Child ; Humans ; Male ; Mutation

To demonstrate the effect of AB serum on terminal erythroid differentiation ex vivo.
 Methods: After separation of CD34+ cells from cord blood, the cells were cultured and divided into a control group and an experimental group. The effects of AB serum were examined by the expressions of different markers (GPA, Band3 and α4-integrin) for erythroblast differentiation and enucleation by flow cytometry. 
 Results: The CD34+ cells were successfully differentiated to enucleated red blood cells. There were evident differences among the expressions of GPA, Band3 and α4-integrin between the 2 groups. The percentage of GPA positive cells in the experimental group was bigger than that in the control group in every time point. The expression of Band3 in the experimental group was higher than that in the control group. The expression of α4-integrin in the experimental group was lower than that in the control group. In addition, the enucleation rate in the experimental group was higher than that in the control group.
 Conclusion: AB serum can promote the cell differentiation and enucleation during terminal erythroid differentiation in vitro.
ABO Blood-Group System ; blood ; physiology ; Anion Exchange Protein 1, Erythrocyte ; metabolism ; Antigens, CD34 ; blood ; Cell Differentiation ; genetics ; physiology ; Cell Nucleus ; Cells, Cultured ; Erythrocytes ; physiology ; ultrastructure ; Erythropoiesis ; genetics ; physiology ; Fetal Blood ; cytology ; physiology ; Flow Cytometry ; Glycophorins ; metabolism ; Humans ; Integrin alpha4beta1 ; metabolism

BACKGROUND: The eosin-5'-maleimide (EMA) binding test using flow cytometry is a common method to measure reduced mean channel fluorescence (MCF) of EMA-labeled red blood cells (RBCs) from patients with red cell membrane disorders. The basic principle of the EMA-RBC binding test involves the covalent binding of EMA to lysine-430 on the first extracellular loop of band 3 protein. METHODS: In the present study, the MCF of EMA was analyzed for samples derived from 12 healthy volunteers (controls) to determine the stability (i.e., the percentage decrease in fluorescence) of EMA over a period of 1 year. RESULTS: Comparison of periodical MCF readings over time, that is, at 2-month intervals, showed that there were no significant changes in mean channel fluorescence for up to 6 months; however, there was a significant decrease in MCF at 8 months. CONCLUSION: For optimal dye utilization, EMA remained stable only for up to 6 months. Therefore, we recommend reconstitution of the dye every 6 months when implementing this test and storage at -80degrees C in dark conditions.
Anion Exchange Protein 1, Erythrocyte ; Cell Membrane* ; Erythrocytes ; Flow Cytometry ; Fluorescence ; Healthy Volunteers ; Humans ; Reading

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder with the hallmark of persistent watery Cl(-)-rich diarrhea from birth. Mutations in the solute carrier family 26, member 3 (SLC26A3) gene, which encodes a coupled Cl-/HCO3- exchanger in the ileum and colon, are known to cause CLD. Although there are a few reports of CLD patients in Korea, none of these had been confirmed by genetic analysis. Here, we describe the case of a Korean infant with clinical features of CLD. Using direct sequencing analysis, we identified 2 sequence variants: a missense variant of unknown significance (c.525G>C; p.Arg175 Ser) and a splicing mutation (c.2063-1G>T) in the SLC26A3 gene; these had been inherited from the father and mother, respectively. Whilst CLD is rare, its main symptom, diarrhea, is very common in infants. Hence, the diagnosis of CLD can prove difficult. Mutational analysis of the SLC26A3 gene should be considered as a viable method to confirm a diagnosis of CLD in Korean infants with persistent diarrhea.
Asian Continental Ancestry Group/*genetics ; Chloride-Bicarbonate Antiporters/*genetics ; DNA Mutational Analysis ; Diarrhea/*congenital/diagnosis/genetics/radiography ; Heterozygote ; Humans ; Infant ; Male ; Metabolism, Inborn Errors/*diagnosis/genetics/radiography ; Mutation ; Mutation, Missense ; RNA Splicing ; Republic of Korea ; Ultrasonography, Prenatal

OBJECTIVETo evaluate the safety and efficacy of retroperitoneal laparoscopic radical nephrectomy in the treatment of renal cancer.

METHODSThe clinical data of 53 cases who underwent retroperitoneal laparoscopic radical nephrectomy were analyzed retrospectively.

RESULTSFifty-two cases achieved successful retroperitoneal laparoscopic radical nephrectomy, a conversion to open surgery was required in one case because of severe adhesion. The operation time was 75 min to 220 min (mean, 125 min), the blood loss was 50 ml to 420 ml (mean, 120 ml), and the postoperative hospital stay was 6 d to 12 d. Complications occurred in 4 cases. Pathological examination showed that 47 cases were of renal clear cell carcinoma, 5 of chromophobe carcinoma, and 1 of cystic renal cell carcinoma. Follow-up for 1 month to 5 years showed no tumor recurrence and metastasis.

CONCLUSIONRetroperitoneal laparoscopic radical nephrectomy is a safe and effective treatment for patients with stage T1 - 2N0M0 renal cell carcinoma.

Adult ; Aged ; Anion Exchange Protein 1, Erythrocyte ; metabolism ; Antiporters ; metabolism ; Carcinoma, Renal Cell ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Humans ; Keratin-7 ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; surgery ; Laparoscopy ; Male ; Middle Aged ; Neoplasm Staging ; Nephrectomy ; methods ; Neprilysin ; metabolism ; Retroperitoneal Space ; Retrospective Studies

The molecular approaches to distal renal tubular acidosis (dRTA) associated AE1 mutations lead us to understand the genetic and pathophysiological aspects of the acidification defects. An unanticipated high value of the urine-blood (U-B) PCO2 after NaHCO3 loading observed in a case of dRTA and southeast Asian ovalocytosis (SAO) might be from a mistarget of the AE1 to the luminal membrane of type A intercalated cells. The mutations of the AE1 gene resulted in SAO and also affected renal acidification function. Notwithstanding, after the NH4Cl loading in 20 individuals with SAO, the acidification in the distal nephron was normal. The presence of both SAO and G701D mutations of AE1 gene would explain the abnormal urinary acidification in the patients with the compound heterozogosity. In terms of the effect of the mutations on trafficking of AE1, truncated kidney isoform (kAE1) of wild-type showed a 'dominant-positive effect' in rescuing the recessive mutant kAE1 (S773P or G701D) trafficking to the plasma membrane, in contrast with the dominant mutant kAE1 (R589H) resulting in a 'dominant-negative effect' when heterodimerized with the wild-type kAE1. It is notable that the dominant mutants kAE1 (R901X or G609R) expression in MDCK cells clearly results in aberrant surface expression with some mutant protein appearing at the apical membrane. These might result in net bicarbonate secretion and increasing U-B PCO2 in the distal nephron. The molecular physiological and genetic approaches have permitted identification of the molecular defects, predominantly in transporter proteins, and should in turn prompt development of novel therapeutic strategies.
Acidosis, Renal Tubular ; Anion Exchange Protein 1, Erythrocyte ; Asian Continental Ancestry Group ; Cell Membrane ; Humans ; Kidney ; Madin Darby Canine Kidney Cells ; Membranes ; Mutant Proteins ; Nephrons ; Organometallic Compounds ; Phenobarbital ; Proteins