Mutation analysis and phenotypic investigation in patients with primary distal renal tubular acidosis / 中华肾脏病杂志

Objective:To analyze the gene variants in patients with primary distal renal tubular acidosis (dRTA), and explore the correlation between the genotype and phenotype.Methods:The Sanger direct sequencing or whole-exome sequencing was used to identify causal variants and the variation pathogenicity was evaluated according to 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines in 44 dRTA patients (37 families) diagnosed in the Affiliated Qingdao Municipal Hospital of Qingdao University and the Affiliated Hospital of Qingdao University from April 2010 to September 2020. The clinical features of the patients were summarized, and the correlation between the genotype and phenotype was investigated.Results:Seven variants of SLC4A1 gene, 17 variants of ATP6V0A4 gene, and 15 variants of ATP6V1B1 gene were identified in 44 patients with dRTA, and of which 11 variants were new ones. According to ACMG guidelines, the pathogenic, likely pathogenic, benign variants among the 39 variants were 22, 16 and 1, respectively. Nine patients were autosomal dominant hereditary dRTA caused by SLC4A1 gene mutation, 4 patients with autosomal recessive hereditary dRTA complicated with Southeast Asian ovalocytosis and anemia were caused by SLC4A1 gene mutation, and 14 patients caused by ATP6V0A4 gene mutation and 8 patients caused by ATP6V1B1 gene mutation were autosomal recessive hereditary dRTA; Two children with dRTA were found to carry one monoallelic defect in ATP6V1B1, and no causal gene mutation was identified in 7 patients. One patient showed incomplete dRTA, and the other 43 patients showed complete dRTA. The prevalence of sensory neural hearing loss caused by ATP6V0A4 and ATP6V1B1 mutation were 2/14 and 6/10 respectively. The frequency of chronic kidney disease in adults, children and infants were 4/4, 2/4, and 1/36, separately. After the drug treatment based on potassium citrate and sodium citrate, the growth and development (28/40) and electrolyte disturbance (41/44) of most patients were significantly improved. Conclusions:The present study has identified 39 variants of SLC4A1, ATP6V0A4 and ATP6V1B1 genes in 44 patients with dRTA, including 11 novel ones. There is a close relationship between genotype and phenotype in dRTA patients and most patients' conditions were improved after proper treatment. This study enriches the human gene mutation database and provides valuable references for diagnosis, treatment and genetic counseling in patients with dRTA.

Mutation analysis of KCNJ1 gene and investigation of phenotype in 5 Chinese patients with Bartter syndrome type 2 / 中华肾脏病杂志

Objective@#To identify and analyze the variants of the KCNJ1 gene in five Chinese patients with Bartter syndrome type 2 (BS2), and to describe their clinical features as well as treatment results.@*Methods@#Data and blood samples of five BS2 patients and their relatives confirmed by Qingdao Municipal Hospital from June 2012 to January 2019 were collected. Whole-exome-sequencing (WES) based on the second generation high throughput sequencing was performed to detect variants. The 2015 American College of Medical Genetics and Genomics Standards and Guidelines were applied to analyze the pathogenicity of the variants. The clinical features and laboratory results were retrospectively studied. The response to treatment and follow-up data were reviewed.@*Results@#Ten variants including six novel ones of KCNJ1 gene were identified through WES and verified by Sanger dideoxy sequencing. Missense variants accounted for the highest proportion. The common symptoms and signs of five BS2 patients from high to low incidence were polydipsia and polyuria (5/5), one of them (1/5) presented with diabetes insipidus; maternal polyhydramnios and premature delivery (4/5); growth retardation (3/5). Initially, two patients presented with hypochloremic metabolic alkalosis and hypokalemia, whereas the acid-base disturbance was absent in the others. One patient experienced hyperkalemia. In terms of calcium-phosphorus metabolism, one patient had evident parathyroid hormone (PTH) resistance (hypocalcemia, hyperphosphatemia and markedly elevated serum intact PTH levels), three presented with PTH overacting (hypercalcemia, hypophosphatemia and mild elevated serum intact PTH levels), and one showed normal blood calcium and phosphorus concentrations with high-normal serum intact PTH levels. All patients had nephrocalcinosis or hypercalciuria, and one of them complicated with nephrolithiasis. Indomethacin helped to correct the growth retardation, halt polydipsia polyuria, decrease the elevated urinary calcium excretion, and normalize electrolyte disturbance as well as PTH parameters in some patients.@*Conclusions@#This investigation identifies ten variants of KCNJ1 gene, including six ones that have not been previously reported, which will enrich the human gene mutation database (HGMD). These patients in our study have atypical BS phenotype, so that careful differentiation from other parathyroid diseases will be required for clinicians.

Mutation analysis of KCNJ1 gene and investigation of phenotype in 5 Chinese patients with Bartter syndrome type 2 / 中华肾脏病杂志

Objective:To identify and analyze the variants of the KCNJ1 gene in five Chinese patients with Bartter syndrome type 2 (BS2), and to describe their clinical features as well as treatment results. Methods:Data and blood samples of five BS2 patients and their relatives confirmed by Qingdao Municipal Hospital from June 2012 to January 2019 were collected. Whole-exome-sequencing (WES) based on the second generation high throughput sequencing was performed to detect variants. The 2015 American College of Medical Genetics and Genomics Standards and Guidelines were applied to analyze the pathogenicity of the variants. The clinical features and laboratory results were retrospectively studied. The response to treatment and follow-up data were reviewed.Results:Ten variants including six novel ones of KCNJ1 gene were identified through WES and verified by Sanger dideoxy sequencing. Missense variants accounted for the highest proportion. The common symptoms and signs of five BS2 patients from high to low incidence were polydipsia and polyuria (5/5), one of them (1/5) presented with diabetes insipidus; maternal polyhydramnios and premature delivery (4/5); growth retardation (3/5). Initially, two patients presented with hypochloremic metabolic alkalosis and hypokalemia, whereas the acid-base disturbance was absent in the others. One patient experienced hyperkalemia. In terms of calcium-phosphorus metabolism, one patient had evident parathyroid hormone (PTH) resistance (hypocalcemia, hyperphosphatemia and markedly elevated serum intact PTH levels), three presented with PTH overacting (hypercalcemia, hypophosphatemia and mild elevated serum intact PTH levels), and one showed normal blood calcium and phosphorus concentrations with high-normal serum intact PTH levels. All patients had nephrocalcinosis or hypercalciuria, and one of them complicated with nephrolithiasis. Indomethacin helped to correct the growth retardation, halt polydipsia polyuria, decrease the elevated urinary calcium excretion, and normalize electrolyte disturbance as well as PTH parameters in some patients. Conclusions:This investigation identifies ten variants of KCNJ1 gene, including six ones that have not been previously reported, which will enrich the human gene mutation database (HGMD). These patients in our study have atypical BS phenotype, so that careful differentiation from other parathyroid diseases will be required for clinicians.

Mutation analysis of SLC5A2 gene and determination of the renal threshold for glucose excretion in Chinese patients with familial renal glucosuria / 中华肾脏病杂志

Objective To analyze and identify the mutations in SGLT2 gene of nine Chinese families with FRG, and determine the renal threshold for glucose excretion (RTG), so as to explore the association of genotype and RTG. Methods All coding regions of SGLT2 gene, including intron exon boundaries, were analyzed using PCR followed by direct sequence analysis. Quantitative test for 24?hour urine glucose and RTG were measured among 9 probands (21 patients) and their family members from 9 pedigrees (total 25 subjects). The differences in renal glucose thresholds between patients with different genotypes (heterozygotes and compound heterozygotes; c.886(-10_-31) del heterozygotes and other heterozygotes) were compared. Results Twelve mutations were identified by SGLT2 gene analysis, including 10 novel ones that were not included in HGMD:c.331T>C, p.W111R;c.374T>C, p.M125T; c.394C>T, p.R132C; c.612G>C, p.Q204H; c.829C>T, p.P277S; c.880G>A, p.D294N;c.1129G>A, p.G377S; c.1194C>A, p.F398L; c.1540C>T, p.P514S; c.1573C>T, p.H525Y. In thisstudy, the mutation c.886(-10_-31)del that is specific to Chinese population accounted for about 28％of the total alleles (5/18). The RTG values of patients with compound heterozygous mutations were much lower than those with simple heterozygous mutations [(1.28 ±0.10) vs (5.14±0.77) mmol/L; P<0.001];and c.886(-10_-31)del heterozygotes had significant lower RTG values than others [(4.43 ± 0.37) vs (5.70 ± 0.51) mmol/L, P<0.001]. Conclusions Ten novel mutations which may be related to FRG are found in this study, and c.886(-10-31)del may be a hot?spot mutation in Chinese patients. Compound heterozygotes had much lower RTG values than simple heterozygotes.

Mutation analysis of SLC12A1 gene in nine Chinese patients with Bartter syndrome type Ⅰ / 中华肾脏病杂志

Objective To analyze the mutations of SLC12A1 gene in nine Chinese families with Bartter syndrome type I (BS1),and analyze the relationship between genotype and phenotype.Methods The next generation sequencing was used to detect mutations in nine BS1 patients including eight with antenatal BS (aBS) and one with classical BS (cBS).Clinical characteristics and biochemical findings at the first admission as well as follow-up were reviewed.Results 15 different mutations of SLC12A1 gene were identified,including 11 novel ones.Among nine probands,seven were compound heterozygotes,two were homozygotes.All patients presented with polydipsia and polyuria,and eight with growth retardation.All patients had lower than-normal serum chloride concentration,metabolic alkalosis,and elevated basal renin activity and aldosterone,and seven had hypokalemia.Through treatment of indomethacin and/or potassium chloride,biochemical indicators could roughly restored normal.Conclusion These findings will enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis for Chinese population.

Analysis on the mutation of SLC5A2 gene in Chinese patients with familial renal glucosuria and investigation on the association of genotype and phenotype / 中华肾脏病杂志

Objective To describe and analyze the clinical characters of patients with FRG from 7 Chinese families.Then analyze and identify their mutations in SGLT2 gene,and explore the association of genotype and phenotype.Methods Quantitative test for 24-hour urine glucose and other laboratory tests were carried out among 7 probands (14 patients in all) and their family members from 7 pedigrees (totaling 23 subjects).All coding regions,including intron-exon boundaries,were analyzed using PCR followed by direct sequence analysis.Results Five novel mutations in SLC5A2 gene were identified in this investigation,including four missense mutations (A Serine to Glycine at position 335 (c.1003A＞G,p.S335G),a Glutamine to Arginine at position 448 (c.1343A ＞ G,p.Q448R),an alanine to proline at position 474 (p.A474P,c.1420G ＞ C) and a glycine to aspartic acid at position 580 (c.1739G ＞ A,p.G580D) and a deletion in intron 7 (c.886(-10_-31)del).By the minigene studies using the pSPL3 plasmids,we confirmed the deletion c.886(-10_-31)del as a splicing mutation.In this study,the mutation c.886(-10_-31)del accounted for about 43％ of the total alleles (12/28).These patients with compound heterozygous or homozygous mutations manifested middle degree or severe glycosuria (Quantitative test for 24-hour urine glucose:10.56-50.68 g/1.73 m2),however those with heterozygous variants presented with mild to moderate glycosuria (Quantitative test for 24-hour urine glucose ≤ 2.45 g/1.73 m2).This fits co-dominant inheritance pattern.Conclusions Five novel mutations which may be related to FRG are found in this study,and c.886(-10-31) del may be a high frequency mutation in Chinese patients.

Lidamycin inhibits angiogenesis of zebrafish embryo via down-regulation of VEGF / 药学学报

Lidamycin (LDM) is a potent antitumor antibiotic. Previous studies have shown that LDM could inhibit proliferation and migration in endothelial cells. In the present report, the effect of LDM on angiogenesis of zebrafish embryo was studied. The results showed that treatment of zebrafish embryos with LDM resulted in significant inhibition of angiogenesis. Morphological observation, quantitative endogenous alkaline phosphatase (EAP) assay, alkaline phosphatase staining, and transgenic zebrafish assay were performed to evaluate vascular development defects in zebrafish. The results indicated that after the zebrafish embryos were exposed to LDM, angiogenesis defects of zebrafish embryos were observed, including pericardial edema, reduced numbers of circulating red blood cells, suppression of zebrafish vessel growth, and absences of SIV (subintestinal vein). The expression of VEGF was detected by RT-PCR assay, quantitative reverse transcriptase real-time PCR (qRT-PCR) assay and Western blotting analysis. The results revealed that LDM could inhibit the expression of VEGF protein, while the expression of mRNA was not significantly affected. The study suggests that LDM could inhibit the zebrafish embryo angiogenesis by down-regulation ofVEGF expression.