Study On Application Of High Throughput Sequencing Technology To Screen Genetic Mutations In Inherited Polycystic Kidney

Objective: Autosomal recessive polycystic kidney disease is a hereditary fibrous cystic disease of the liver and kidney,with an incidence of 1∶ 40 000 ～ 1∶ 20 000.It is a rare hereditary disease,mainly affecting fetuses and infants,and is one of the common causes of perinatal death.In recent years,with the widespread application of second-generation sequencing technology,sequencing technology has developed rapidly in medicine,making genetic diagnosis of genetic diseases easier and faster.Because genetic diseases have numerous pathogenic gene,clinical diagnosis based on exome sequencing can detect gene mutations related to most diseases at one time,and at the same time perform a comprehensive analysis in combination with clinical phenotypes,so that the clinicians can know the cause of the disease and related factors.Exome sequencing can effectively increase the probability of diagnosis and shorten the time for diagnosis and treatment.In this study,we gave a performance of high throughput sequencing on the peripheral blood of 3 patients who were initially clinically diagnosed ARPKD to screen the mutant genes associated with hereditary polycystic kidney.Methods : Selection in January 2019 to December 2020 in Inner Mongolia medical university affiliated hospital,three patients who were initially clinically diagnosed "Autosomal recessive polycystic kidney".Inclusion criteria: Ultrasound of the bilateral kidneys showed multiple cyst formation and one or more of the following conditions:(1)both parents no renal cyst(especially in age > 30)(2)the clinical,laboratory and imaging evidence of liver fibrosis(3)liver pathology showed abnormal characteristic of bile duct plate(4)brothers and sisters with the pathological or genetic diagnosis of disease(5)parental blood relationship suggests autosomal recessive inheritance.Peripheral blood DNA extraction,target gene capture and database building,high-throughput sequencing and bioinformatics analysis were performed for the eligible patients to screen out the gene mutation sites,which were then verified by first-generation sequencing.Results:(1)One heterozygous splicing variation of BICC1 gene,one heterozygous missense variation of GANAB gene and one heterozygous missense variation of PKD1 gene were detected in Proof 1 sample.The BICC1 mutation c.1858+1G>T detected this time is a classical splicing mutation that can affect RNA splicing,and the frequency of this mutation in the gnom AD East Asian population database is 0.There is no report of this mutation in the relevant literature currently.The GANAB mutation c.224C>T:p.T75 M detected in this study can cause the mutation of the 75 th Threonine of protein translation to methionine,and the frequency of this mutation in the gnom AD East Asian population database is 0.000251.There is no report of this mutation in the relevant literature currently.The PKD1 missense mutation c.2545G>A:p.V849 M detected this time can cause the mutation of valine at the 849 position of protein translation into methionine,and the frequency of this mutation in the gnom AD East Asian population database is 0.000056.There is no report of this mutation in the relevant literature currently.Verification of the above loci by first-generation sequencing indicated that the three variants indeed existed in the protestor.(2)One heterozygous missense variant of PKD1 gene was detected in Proof 2sample.Moreover,the meaningless mutation of PKD1 detected this time,c.1198C>T:p.R400 x,can lead to premature termination of protein translation.The frequency of this mutation in the gnom AD East Asian population database is 0,and it has been reported that this mutation is associated with polycystic kidney.Verification of the meaningless mutation of PKD1 detected this time,c.1198C>T: p.R400 x by first-generation sequencing indicated that the variant indeed existed in the protestor.(3)A heterozygous nonsense variant of PKD1 gene was detected in the Proof 3sample.The heterozygous and meaningless mutation of PKD1 detected this time,c.616C>T:p.Q206 X,can lead to premature termination of protein translation,which is not included in the gnom AD East Asian population database.There is no report of this mutation in the relevant literature currently.Verification of the loci by first-generation sequencing indicated that the variant indeed existed in the protestor.Conclusions:1)Three patients in our study group were clearly diagnosed with autosomal dominant polycystic kidney by whole exon sequencing.Related studies have shown that the majority of autosomal dominant polycystic kidney is inherited from their parents,and about 10% of the patients have newly developed variants.However,all the 3 patients in our study group have no family genetic history of polycystic kidney,so they are considered to be newly developed variants.2)This research group detected PKD1 nonsense variant c.1198C>T:p.R400 X,PKD1 missense variant c.2545G>A:p.V849 M,heterozygous PKD1 nonsense variantc.616C>T:p.Q206 X,BICC1 variant c.1858+1G>T,GANAB variant c.224C>T:p.T75 M.The frequency of these variants in gnom AD East Asian population database was very low or none,and there were few reports in literatures.The mutation spectrum of polycystic kidney gene was enriched,which provided more basis for clinical work.