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.

Role of ATP6V1H gene in bone metabolism / 中国骨伤

Osteoporosis is one of the common clinical orthopedic diseases, which can lead to a variety of complications. There are many pathogenic factors in this disease. The latest research found that ATP6V1H is a new gene leading to the occurrence of osteoporosis, and it is likely to become a new target for the future drug treatment of osteoporosis.This paper introduces the biological structure and characteristics of H subunit, summed up the human body caused by loss of ATP6V1H and animal models such as zebrafish, mice bone loss and osteoporosis symptom such as related research reports of the loss, from osteoclast, osteoblast and marrow stromal cell level and the connection between the various subunits further expounds the H subunit regulate bone dynamic balance of mechanism, to explore ATP6V1H in bone developmentand bone related diseases has laid a solid foundation, also provide new ideas for clinical treatment of osteoporosis.

Renal Tubular Acidosis Presenting as Nephrogenic Diabetes Insipidus

Background: Nephrogenic diabetes insipidus (DI) can be primary or secondary to variouscauses. Case Characteristics: One child with Fanconi syndrome with proximal renaltubular acidosis (RTA) due to nephropathic cystinosis, and other with Distal RTA withhearing loss. Observation: Both cases showed features of nephrogenic DI, which resolvedafter treating the primary pathology. Message: Renal Tubular acidosis may causenephrogenic DI.

Morphological characteristics identification and molecular DNA barcoding analysis of Hippocampus spinosissimus / 中国中药杂志

The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.

Molecular identification of Hippocampus trimaculatus and its adulterants based on DNA barcoding / 中草药

Objective: The aim of present study was to establish a DNA barcoding datebase of COI, 16 S rRNA and ATP6 sequences from Hippocampus trimaculatus and identify H. trimaculatus and other adulterants quickly and accurately. Methods: Total genomic DNA was isolated using the muscle tissue from tail of H. trimaculatus. The DNA barcoding genes of COI, 16 S, and ATP6 sequences were amplified by universal primers and PCR products were sequenced from both directions. Sequences assembly and consensus sequence generation were performed using the Codon Code Aligner V4.2. Sequence alignment was performed by using Clustal X software. The Kimura 2-Parameter (K2P) distances were calculated using the MEGA5.0. Identification analyses were performed using neighbor-joining (NJ) methods. Results: The length of the measured sequences of mitochondrial COI, 16 S, and ATP6 were 649 572 and 603-605 bp, respectively. The number of intraspecific variation sites of three genes was 8 bp, 4 bp, and 15 bp, respectively. The average intraspecific K2P genetic distance of COI, 16 S, and ATP6 were 0.002, 0.001, and 0.006. The genetic variation among H. trimaculatus was obviously less than the interspecific genetic variation. The NJ tree showed that H. trimaculatus could be distinguished obviously from other Hippocampus species, which showed high monopholy. Conclusion: Our results indicated that COI, 16 S, and ATP6 sequences are effective DNA barcodes for the identification of H. trimaculatus species and its adulterants, with view to providing the basis data for molecular identification of animal medicinal materials, adulterants and near-source species, and contributing to the clinical medication safety of H. trimaculatus.

Case report and literature review of ATP6V0A2 gene mutation features of the patient with congenital cutis laxa / 天津医药

The incidence of autosomal recessive cutis laxa induced by ATP6VOA2 gene mutation is extremely low in neonates and rarely reported in China.There was one case of ATP6VOA2 gene mutations caused autosomal recessive cutis laxa diagnosed in Tianjin Children's Hospital.This article reviewed the diagnosis and treatment of the patient and reviewed the relevant literature,in order to improve the understanding of the disease.

Mutation analysis of 5 children with primary distal renal tubular acidosis / 中华肾脏病杂志

Objective To analyze the mutations of causal genes in 5 children with primary distal renal tubular acidosis (dRTA),and explore their association of genotype and phenotype,so as to raise the awareness of the disease.Methods The whole exome sequencing was used to identify mutations in these 5 children from 5 families.Results A total of 4 different mutations of ATP6V0A4 gene were found in 2 dRTA children,including a novel heterozygous intron mutation (c.639 + 1G＞ A),a reported heterozygous nonsense variant (c.580C ＞T,p.Arg194*) and 2 novel heterozygous duplications (c.1504dupT,p.Tyr502Leufs*22;c.2351dupT,p.Phe785Ilefs*28).Two novel heterozygous missense mutations of ATP6V 1B 1 gene (c.409C ＞ T,p.Pro 137Ser;c.904C ＞ T,p.Arg302Trp) were identified in the third child,and a heterozygous missense mutation of SLC4A1 gene (c.1765C ＞ A,p.Arg589Ser) previously reported was found in the fourth child.No mutation of the dRTA-related causal genes was found in the fifth child.Furthermore,the mutations of causal genes in each of the first three children were compound heterozygous,which were consistent with the autosomal recessive inheritance pattern,and the variant from the fourth child was de novo.Conclusions The present study has found 7 mutations,including 5 novel variants,which enriches the human gene mutation database (HGMD) and contributes to a better understanding of the disease mechanisms.

Silencing of vacuolar ATPase c subunit ATP6V0C inhibits invasion of prostate cancer cells / 北京大学学报(医学版)

Objective:Vacuolar ATPase (V-ATPase) was found within the membranes and internal organelles of a vast array of eukaryotic cells,and was related to various kinds of highly metastatic tumors.LASS2/TMSG1 gene was a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by our laboratory.It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of V-ATPase (ATP6V0C).In this study,To use RNA interference to suppress the expression of ATP6V0C and try to further investigate the molecular mechanism of ATP6V0C in tumor metastasis and its relationship with LASS2/TMSG1 gene.Methods and Results:The expression level of ATP6V0C mRNA and protein in high metastatic potential prostate cancer cell lines (PC-3M-1E8 and PC-3M) was significantly higher than that in low metastatic potential prostate cancer cell lines (PC-3M-2B4 and PC-3),the expression level in PC-3M-1E8 being the highest.Follow-up tests selected PC-3M-1E8 cells for gene silencing.The expression and secretion of MMP-2 and the expression of MMP-9 in ATP6V0C siRNA transfected PC-3M-1E8 cells displayed no obvious change,but the activity of secreted MMP-9 was abated noticeably compared with the controls (P ＜ 0.05).Extracellular hydrogen ion concentration and V-ATPase activity in interference group were both reduced significantly compared with the controls (P ＜ 0.05).The migration and invasion capacity of ATP6V0C siRNA interfered cells in vitro were diminished significantly compared with the controls (P ＜ 0.05).Furthermore,a dramatic reduction of LASS2/TMSG1 mRNA and protein level after transfection of siRNA in PC-3M-1 E8 cells was discovered (P ＜ 0.05).Confocal immunofluorescence showed a vast co-localization of ATP6V0C protein and LASS2/TMSG1 protein in plasma and membrane.The co-localization signals of control group were much stronger than those of interference group.Conclusion:Specific siRNA silencing of ATP6V0C gene inhibits the invasion of human prostate cancer cells in vitro by mechanism of inhibiting V-ATPase activity and then reducing the extracellular hydrogen ion concentration,inhibiting MMP-9 activation and affecting ECM degradation and reconstruction.Meanwhile,ATP6V0C and LASS2/TMSG1 have interaction and it is likely that ATP6V0C functions as a feedback regulator of LASS2/TMSG1.

Sequenciamento total do exoma como ferramenta de diagnóstico de acidose tubular renal distal / Whole-exome sequencing as a diagnostic tool for distal renal tubular acidosis

Resumo Objetivo A acidose tubular renal distal (ATRd) é caracterizada por acidose metabólica devido à excreção renal de ácido prejudicada. O objetivo deste artigo é apresentar o diagnóstico genético de quatro crianças com ATRd com uso do sequenciamento total do exoma. Métodos Selecionamos duas famílias não relacionadas, quatro crianças com ATRd e seus pais, para fazer o sequenciamento total do exoma. A audição foi preservada em ambas as crianças da família um, porém em nenhuma criança da família dois, na qual um par de gêmeas teve perda auditiva severa. Fizemos o sequenciamento total do exoma em dois conjuntos de amostras e confirmamos os achados com o método de sequenciamento de Sanger. Resultados Duas mutações foram identificadas nos genes ATP6V0A4 e ATP6V1B1. Na família um, detectamos uma nova mutação no éxon 13 do gene ATP6V0A4 com uma alteração em um nucleotídeo único GAC → TAC (c.1232G>T) que causou substituição de ácido aspártico por tirosina na posição 411. Na família dois, detectamos uma mutação recorrente do homozigoto com inserção de um par de bases (c.1149_1155insC) no éxon 12 do gene ATP6V1B1. Conclusão Nossos resultados confirmam o valor do sequenciamento total do exoma para o estudo de nefropatias genéticas complexas e permitem a identificação de mutações novas e recorrentes. Adicionalmente, demonstramos claramente pela primeira vez a aplicação desse método molecular em doenças tubulares renais.

Abstract Objective Distal renal tubular acidosis (dRTA) is characterized by metabolic acidosis due to impaired renal acid excretion. The aim of this study was to demonstrate the genetic diagnosis of four children with dRTA through use of whole-exome sequencing. Methods Two unrelated families were selected; a total of four children with dRTA and their parents, in order to perform whole-exome sequencing. Hearing was preserved in both children from the first family, but not in the second, wherein a twin pair had severe deafness. Whole-exome sequencing was performed in two pooled samples and findings were confirmed with Sanger sequencing method. Results Two mutations were identified in the ATP6V0A4 and ATP6V1B1 genes. In the first family, a novel mutation in the exon 13 of the ATP6V0A4 gene with a single nucleotide change GAC → TAC (c.1232G>T) was found, which caused a substitution of aspartic acid to tyrosine in position 411. In the second family, a homozygous recurrent mutation with one base-pair insertion (c.1149_1155insC) in exon 12 of the ATP6V1B1 gene was detected. Conclusion These results confirm the value of whole-exome sequencing for the study of rare and complex genetic nephropathies, allowing the identification of novel and recurrent mutations. Furthermore, for the first time the application of this molecular method in renal tubular diseases has been clearly demonstrated.

Identification of causative mutations in patients with Leigh syndrome and MERRF by mitochondrial DNA-targeted next-generation sequencing

PURPOSE: Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make their exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mitochondrial DNA (mtDNA) mutations in 2 Korean families with myoclonic epilepsy with ragged-red fibers (MERRF) and Leigh syndrome, respectively. MATERIALS AND METHODS: Whole mtDNAs were sequenced by the method of mtDNA-targeted next-generation sequencing (NGS). RESULTS: Two causative mtDNA mutations were identified from the NGS data. An m.8344A>G mutation in the tRNA-Lys gene (MT-TK ) was detected in a MERRF patient (family ID: MT132), and an m.9176T>C (p.Leu217Pro) mutation in the mitochondrial ATP6 gene (MT-ATP6) was detected in a Leigh syndrome patient (family ID: MT130). Both mutations, which have been reported several times before in affected individuals, were not found in the control samples. CONCLUSION: This study suggests that mtDNA-targeted NGS will be helpful for the molecular diagnosis of genetically heterogeneous mitochondrial diseases with complex phenotypes.

Medullary Sponge Kidney Detected in the Pediatric Age

PURPOSE: Medullary sponge kidney (MSK) is a rare congenital disease characterized by diffuse ectasia or dilatation of precalyceal collecting tubules. MSK incidence and prevalence in the general population is uncertain and only a few patients are reported especially in the pediatric age. There has been increasing reports of patients with MSK who have other malformative disorders. Also several case reports concerning about etiological association of some genes. METHODS: Collaborative study through nation-wide survey was done to investigate the incidence and etiological association of some genes such as GDNF gene, ATP6V1B1, ATP6V0A4 gene in developing MSK in Korean children. RESULTS: Four cases of MSK who have various other malformative disorders were collected. There are no mutations of GDNF gene, ATP6V1B1, ATP6V0A4 gene in all patients. CONCLUSION: MSK is one of the very rare diseases in pediatric age. The etiological association of GDNF gene , ATP6V1B1, ATP6V0A4 gene in developing MSK in Korean children is not proved.

Mutation analysis of ATP6V0A4 and ATP6V1B1 gene in autosomal recessive distal renal tubular acidosis children / 中华肾脏病杂志

Objective To analyze and identify the mutations of ATP6V0A4 and ATP6V1B1 gene in autosomal recessive distal renal tubular acidosis (rdRTA) children,and study the association of genotype and phenotype. Methods Genome DNA was amplified by PCR.Mutations of ATP6V0A4 and ATP6V1B1 gene in 3 children from 3 families were examined by direct sequencing.One hundred unrelated healthy subjects were selected to evaluate all mutations found in this study. Results A novel homozygous nonsense mutation was identified in ATP6VOA4 gene in one child, and a novel heterozygous nonsense variant and a frame-shift alteration were found in another child.No mutation of both genes was found in the third child.Conclusions Study of mutant genes of rdRTA in Chinese patients is helpful to understand the association in genotype and phenotype and increase the level of cognition and treatment to this disease.

Genetic studies in a family with distal renal tubular acidosis and sensorineural deafness.

Distal renal tubular acidosis (RTA) with sensorineural deafness is a rare entity, inherited in an autosomal recessive manner. It is caused by mutations in the ATP6V1B1 gene, leading to defective function of H+-ATPase pump in the distal nephron, cochlea and endolymphatic sac. We report two siblings with distal RTA and sensorineural deafness having mutation C>T in the first coding exon of the gene, resulting in a non functional protein. The parents were found to be carriers for the mutation.

Regulation of Vacuolar H+-ATPase c Gene Expression by Oxidative Stress

By using differential display, we identified one of the genes encoding the multi-subunit complex protein V-ATPase, c subunit gene (ATP6L), and showed alterations of the gene expression by oxidative stresses. Expression of the ATP6L gene in Neuro-2A cells was increased by the treatment with H2O2 and incubation in hypoxic chamber, implying that the expression of the ATP6L gene is regulated by oxidative stresses. To examine mechanisms involved in the regulation of the gene expression by oxidative stresses, the transcriptional activity of the rat ATP6L promoter was studied. Transcription initiation site was determined by primer extension analysis and DNA sequencing, and promoter of the rat ATP6L and its deletion clones were constructed in reporter assay vector. Significant changes of the promoter activities in Neuro-2A cells were observed in two regions within the proximal 1 kbp promoter, and one containing a suppressor was in -195 to -220, which contains GC box that is activated by binding of Sp1 protein. The suppression of promoter activity was lost in mutants of the GC box. We confirmed by electrophoretic mobility shift and supershift assays that Sp1 protein specifically binds to the GC box. The promoter activity was not changed by the H2O2 treatment and incubation in hypoxic chamber, however, H2O2 increased the stability of ATP6L mRNA. These data suggest that the expression of the ATP6L gene by oxidative stresses is regulated at posttranscriptional level, whereas the GC box is important in basal activities of the promoter.