| Objective: Primary distal tubular acidosis(dRTA)is a rare genetic disorder caused by the disfunction of secretion of hydrogen ions in the alpha-intercalated cell of the distal nephron.In this study,analysis of disease-causing genes in 6 primary dRTA families were performed to explore the association of genotype and phenotype,and to improve awareness of the disease.Methods: To describe the clinical manifestations of 6 probands from 6 families.The disease-causing genes in 6 probands of 6 pedigree were analyzed via the whole exome sequencing and direct sequencing.Sanger sequencing was performed to valid the potential candidate variants identified by sequencing analysis in 6 probands and their family members.The online software(SIFT,PolyPhen-2 and MutationTaster)and Grantham Matrix scoring system were used to assess the pathogenicity of the missense variants.Sequence alignment of homologous proteins was performed using Vector NTI Advance 11.5-Align.Besides,the splice prediction programs(BDGP,NetGene2,Spliceview and HSF 3.0)was used to analyze the effect of the potential splice site mutations.And the splicing variants were verified in RNA of the patient.Then,we summarized the genotype and phenotype of patients with primary dRTA who were previously diagnosed in our study group,providing a foundation for the genotype-phenotype relationship of the disease.Result:(1)A total of 9 different mutations were identified in 5 patients,including one mutation previously reported in the SLC4A1 gene c.1765C>A(p.Arg589Ser),4 mutations in ATP6V0A4 gene(c.580C>T,p.Arg194*;c.639+1G>A;c.1504 dupT,p.Tyr502Leufs*22 and c.2351 dupT,p.Phe785Ilefs*28),and 4 mutations in ATP6V1B1 gene(c.409C>T,p.Pro137Ser;c.785+1G>A;c.904C>T,p.Arg302 Trp and c.133-134 delTG,p.Cys45Glnfs*37).No any mutations in the known dRTA-related causative genes were found in the patient Ⅵ.(2)The three types of in silico software(SIFT,PolyPhen-2 and MutationTaster)and Grantham Matrix scoring system predicted that both c.904C>T(p.Arg302Trp)of ATP6V1B1 and c.1765C>A(p.Arg589Ser)of SLC4A1 were deleterious.However,c.409C>T(p.Pro137Ser)of ATP6V1B1 was evaluated to be harmful with moderate probability by Grantham Matrix scoring system and PolyPhen-2.However,p.Pro137 Ser was also predicted as deleterious by SIFT and MutationTaster.Sequence alignment demonstrated that the sites of these three missense mutations(p.Pro137 and p.Arg302 of ATP6V1B1,and p.Arg589 of SLC4A1)were highly conserved in all the eight species of their respective homologous proteins.(3)The c.639+1G>A in ATP6V0A4,located at classical splicing donor site,was predicted as a splicing variant resulting in the skipping of exon 8 of ATP6V0A4 gene.The c.785+1G>A of ATP6V1B1 gene,which just located in the 5’-splice donor site,was presumed that this mutation cause the disability of donor site and skipping of exon 8,with the online software BDGP,NetGene2 and Spliceview,respectively.And in vivo,this mutation was verified to really lead to the skipping of exon 8 by the RNA analysis.Besides,HSF 3.0 predicted that the rest of mutations had no significant effect on splicing progress.(4)Genotype–phenotype analysis: A total of 21 patients with primary dRTA were enrolled in the study,including 7 patients with SLC4A1 mutation(33.3%),7 patients with ATP6V0A4 mutation(33.3%),and 5 patients with ATPV1B1 mutation(23.8%).No mutations were found in 2 patients(9.5%).Except for one patient who presented with incomplete dRTA,the others manifested complete dRTA.Sensorineural hearing loss can be seen in the patients harboring the mutations of the ATP6V0A4 and ATP6V1B1 gene.Conclusions: A total of 9 mutations of dRTA-related genes were found in this study,including one mutation in SLC4A1 gene,4 mutations in ATP6V0A4 gene,and 4 mutations in ATP6V1B1 gene.And among of them,there were 5 novel mutations identified that enriched the human gene mutation database.The genotype and phenotype of the primary dRTA diagnosed in the study group were summarized to provide a reference for the exploration of genotype-phenotype correlation of the disease. |
