【作者】 周芳；

【导师】 毛建华；

【作者基本信息】 浙江大学 ， 儿科学， 2018， 硕士

【摘要】 背景:临床上,儿童先天性肾小管酸中毒(children congenital renal tubular acidosis)以原发性远端肾小管酸中毒(primary distal renal tubular acidosis,dRTA)最常见。原发性远端肾小管性酸中毒是由遗传缺陷导致的,引起尿酸化功能障碍,表现为高氯性代谢性酸中毒。dRTA的主要临床特征是多饮、食欲不振、呕吐、腹泻和/或便秘,以及多尿。慢性酸中毒和继发性改变,如多尿、多饮和呕吐等影响儿童生长发育,导致无法健康成长。但是,总体来说,如果早期诊断和持续碱性药物治疗,dRTA的预后比较良好。而未经治疗,dRTA会导致儿童佝偻病和/或生长发育迟缓和成人骨软化症,甚至进展到肾功能不全。原发性远端肾小管酸中毒可分为常染色体显性遗传、常染色体隐性遗传和散发病例。常染色体显性遗传病人的表型通常到青春期才有临床症状。常染色体隐性遗传病人的症状通常出现较早,导致生长发育迟缓,耳聋等。原发性dRTA可与下列任何一个基因的突变相关:SLC4A1、ATP6V0A4和ATP6V1B1。此外,在大约20%的dRTA患者中,这些相关基因中没有发现突变,这表明其他转运体或通道可能参与肾小管酸中毒的发病过程。二代测序(NGS)覆盖了超过95%的外显子,这些外显子包含了孟德尔遗传中85%的致病突变基因。基因分子诊断对鉴定新的致病基因和研究疾病的发病机制具有重要的意义。目的:通过全外显子测序分析16例中国儿童远端肾小管酸中毒的基因诊断结果,结合33例肾小管酸中毒患儿的临床资料进行分析,便于早期诊断。方法:将2010年1月至2015年9月期间于浙江大学医学院附属儿童医院收治的33例RTA患儿作为研究对象,分析其临床特征及生化指标,其中16例dRTA无血缘关系的患儿参与全外显子测序。采用Sanger测序,探讨这些患儿的基因诊断及表型与基因型的关系。结果:这些肾小管酸中毒患儿的临床和生物学特征包括高氯代谢性酸中毒、生长发育迟缓、低钾血症、肾钙质沉着、肾结石、高钙尿症、低柠檬酸尿症、佝偻病和软骨病。在 15 例 dRTA 患儿中发现了 17 个 SLC4A1、ATP6V1B1、ATP6V0A4、WNK1、CLDN16基因的突变,其中有14个基因突变点以前没有报道过。只有1例患儿未发现致病基因突变。结论:研究的结果证实了中国儿童远端肾小管酸中毒的SLC4A1、ATP6V1B1、ATP6V0A4、WNK1和CLDN16突变的存在,表明了 2个或2个以上的突变中存在复合杂合突变可能是导致远端肾小管酸中毒的发病机制。本研究也表明了全外显子测序技术应用于儿童先天性肾小管酸中毒致病基因的分析是经济、有效的。更多还原

【Abstract】 BackgroundThe primary distal renal tubular acidosis is most common in children with congenital renal tubular acidosis.Primary distal renal tubular acidosis(dRTA)is caused by genetic defects that result in uric acid dysfunction in the presence of hyperchloremic metabolic acidosis.The main clinical features of dRTA are polydipsia,loss of appetite,vomiting,diarrhea and/or constipation,and polyuria.Chronic acidosis and secondary alterations such as polyuria,polydipsia,and vomiting affect growth,leading to failure to thrive.In general,dRTA may have a better prognosis if diagnosed early and subjected continued alkaline treatment.Untreated,dRTA causes rickets and/or growth retardation in children and osteomalacia in adults and gradual deterioration of renal function with time.Primary distal RTA can be transmitted as either an autosomal dominant or an autosomal recessive trait.The autosomal dominant phenotype typically appears in a mild form in adolescence.The autosomal recessive phenotype usually result in growth retardation,and may or not lead to deafness.The parents of children with this variant are unaffected.Autosomal recessive dRTA is associated with mutations in solute carrier family 4 member 1(SLC4A1),ATPase H+ transporting VO subnit A4(ATP6V0A4),and ATPase H+ transporting V1 subunit B1(ATP6V1B1).However,in approximately 20%of patients with dRTA,no mutations were found in these genes,suggesting that defects in other transporters or channels might also be involved in dRTA pathogenesis.Next generation sequencing(NGS)provides coverage of more than 95%of the exons that contain 85%of the disease-causing mutations in Mendelian disorders.Whole-exome sequencing is useful to recognize new pathogenic genes and in evaluating the pathogenesis of a disease.Objective:The aim of the present study was to demonstrate the genetic diagnosis of 16 Chinese children with distal renal tubular acidosis by whole-exome sequencing.Methods:From Jan 2010 to Sept 2015,33 RTA children were hospital in the Children’s Hospital of Zhejiang University School of Medicine.To study clinical and biochemical features of 33 RTA children.16 unrelated patients with distal renal tubular acidosis were recruited to the present study to investigate the possibility of genetic diagnosis and the relationship between phenotype and genotype in these patients.The whole-exome sequencing technique was applied to the 16 patients with primary distal renal tubular acidosis.Sanger sequencing was used to confirm mutation identified by whole-exome sequencing.Results:Clinical and biological features in these children with primary renal tubular acidosis included hyperchloremic metabolic acidosis,impaired growth,hypokalemia,nephrocalcinosis,nephrolithiasis,hypercalciuria,hypocitraturia,and rickets or osteomalacia.Seventeen mutations of the solute carrier family 4 member 1(SLC4A1),ATPase H+ transporting VO subunit A4(ATP6V0A4),ATPase H+ transporting V1 subunit B1(ATP6V1B1),WNK lysine deficient protein kinase 1(WNK1)and the claudin 16(CLDN16)were identified in 15 of the children with dRTA,and 14 of these mutations are novel.Only 1 patient was negative for any mutations in the screening.Conclusion:Our results demonstrated the existence of the SLC4A1,ATP6V1B1,ATP6V0A4,WNK1 and CLDN16 mutations in Chinese children with distal renal tubular acidosis and disclosed that compound heterozygosity at two or more different but related genes can be responsible for the pathogenesis of distal renal tubular acidosis.This study also indicates that whole-exome sequencing technique is a labor and cost effective means of analyzing associated genes of children congenital renal tubular acidosis.更多还原