Phenotype And Genotype Analysis And Preimplantation Genetic Diagnosisin Autosomal Dominant Polycystic Kidney Disease Patients

Autosomal dominant polycystic kidney disease(ADPKD)is the most common monogenic hereditary kidney disease in humans and has the incidence of 0.1-0.25%.There are about 12.5 million ADPKD patients around world.It has been the fourth leading cause of end-stage renal diseases(ESRD)and accounts for 5%-10% of all the causes of ESRD.Clinically,ADPKD is characterized by the progressive enlargement of numerous liquid vesicles and the damage to the structure and function of the kidney.About 50% of ADPKD patients may develop ESRD in their 60 s,the treatment for which includes dialysis or kidney transplantation.ADPKD not only cause kidney dysfunction,but induce other extra-renal diseases such as cysts in the liver,pancreas,spleen and genitourinary system,heart valve disease,colonic diverticula and intracranial aneurysms.Thus,ADPKD is a hereditary disease with involvement of multiple systems,significantly threatening the human health.ADPKD may affect all the generations of the ADPKD family and the probability of ADPKD is 50% in the second generation for each gender.It has been confirmed that PKD1 and PKD2 are two pathogenic genes of ADPKD.About 85% of ADPKD patients have PKD1 mutation and remaining 15% have PKD2 mutation.The type of gene mutation is closely related to the clinical manifestations.Patients with PKD1 mutation usually present more serious clinical symptoms and ADPKD progresses more rapidly in them as compared to those with PKD2 mutation.Currently,the genetic test is costly and the detection rate is still at unreliable level.Thus,the clinical diagnosis of ADPKD is mainly dependent on the family history,clinical manifestations and imaging examinations.However,available clinical trials indicate that about 10%-20% of patients who present clinical manifestations of ADPKD have no evident family history.Thus,whether it is reliable to diagnose ADPKD depending on clinical manifestations alone is required to be further validated.With the development of next generation sequencing(NGS),the detection rate of PKD1 mutation and PKD2 mutation increases,but the available databases for genetic diagnosis are still very scarce,and a very few data are from Chinese patients.Previous studies have shown that there is no dominated mutation site in the ADPKD,on the basis of which,together with the “two-hit” theory and “third-hit” theory for the pathogenesis of ADPKD,we speculate that the types and sites of gene mutations as well as the clinical manifestations are different between patients with and without family history of ADPKD.In order to verify this hypothesis,a total of 348 patients with evident family history of ADPKD and 119 patients without family history of ADPKD were reviewed.These ADPKD patients were followed up by hospital visit in the Department of Nephrology of Changzheng Hospital between June 2009 and December 2015.The medical history,family history,medical record,image data and ultrasound findings in parents or other immediate families were systemically reviewed.Results showed the incidence of polycystic liver in ADPKD patients without family history was significantly lower than in those with family history(P<0.01),and ADPKD patients without family history were 2 years older at the definitive diagnosis of disease than those with family history although significant difference was not observed.The incidence of cerebrovascular events in patients without family history was slightly lower than in those with family history(0.84% vs 1.44%),and the incidence of concomitant diabetic mellitus was comparable between them(1.68% vs 1.43%).Two patients with family history developed gastric cancer and prostate cancer,respectively.There were no significant differences in the gender,incidence of hypertension,treatments and disease progression between two groups.Only after we have divided patients into different groups by age,we find more rapidly decrease of estimated glomerular filtration rate(eGFR)in Less than 18 years old age group patients without family history(p<0.05).The patients without family history have smaller base line total kidney volume in 41 to 50 years old age groups than those with family history(p<0.05).A COX regression analysis confirmed that age of patients,age of diagnosis ADPKD,and no family history(NFM)were independent risk factors for predict rapidly increase of total kidney volume(TKV)growth rate.Then,to further investigations of distinctions on pathogenic gene mutations between patients with or without family history.30 patients were selected from each group for the detection of PKD1/2 mutation.Pathogenic mutations were found in 28 patients with family history,and 2 patients had no gene mutation with the detection rate of 93.3%.However,PKD1/2 mutations were observed in only 20 patients without family history.The remaining 10 patients without PKD1/2 mutations were subjected to detection of mutations of TSC,PKHD and HNF-1?,the mutations of which may potentially cause similar clinical and imaging features of ADPKD.PKHD1 mutation was observed in 5 adult patients who,however,had no liver dysfunction and manifestations of hepatic cirrhosis,which indicating complex inheritance patterns such as compound heterozygosity,digenic Inheritance or trans-heterozygosity taking part in genetic inheritance in NFM patients.No mutations were found in remaining 5 patients.Thus,the total detection rate was 83.3%.Of the mutation sites of PKD1/2 in the present study,about 47.9% had never been reported.Eleven new mutation sites were found in patients with family history and 12 new mutations sites in those without family history,in which 4 mutations belong to missense mutation,and will be difficult to identify pathogenicity with no family history.Of note,there was no dominated mutation site among them.Genetic test further confirmed that clinical manifestations are not reliable in the diagnosis of ADPKD in patients without family history.According to the findings from genetic test and existing technical conditions.Of above 60 patients,informed consent was obtained from 6 patients of child-bearing age and their spouses(6 patients had family history and 1 had no family history).Preimplantation genetic diagnosis(PGD)was performed to prevent from the pathogenic genes defect affect their offspring.After ovulation,intracytoplasmic sperm injection(ICSI)was administered.A total of 65 embryos in blastula stage were developed.Screening with PGD yielded 8 healthy embryos without pathogenic gene and with normal chromosomes.Of these embryos,4 were transplanted.Currently,one embryo survived and the patient was at the gestational age of 18 weeks.The fetal development was normal.Genetic test after amniocentesis confirmed that the fetal dose not inherited the pathogenic genes.These findings suggest that PGD technique is safe and reliable.For ADPKD patients without evidence family history,the effectiveness of ovulation may directly determine the reliability and success of PGD.Taken together,our study shows there are differences in the incidence of polycystic liver and disease progression between ADPKD patients with and without family history.Selective patients were subjected for the detection of PKD1/2 mutations with the overall detection rate of 80%.Of patients with family history,the detection rate is 93.3% and new mutation sites account for 39.3%;of patients without family history,the detection rate is 66.7% and new mutations sites account for 60%.Of note,16.7% of patients have non-PKD mutations.Our findings enrich the ADPKD database of Chinese patients.Long range PCR and NGS for genetic test are reliable,have favorable detection rate may be employed for the clinical diagnosis of ADPKD.Nevertheless,the detection rate of PKD mutations is significantly lower in ADPKD patients without family history(p=0.023),and some of them are non-PKD mutations.This suggests that diagnosis of ADPKD depending on clinical manifestations has a poor accuracy,and definitive diagnosis requires the detection of mutations of PKD and other genes that may potentially cause clinical manifestations similar to ADPKD.The incorrect diagnosis of ADPKD might be ascribed to the difference in the clinical manifestations.Finally,PGD technique is employed in the present study for assisted reproduction,and an embryo without pathogenic gene is successfully implanted in a patient,indicating the feasibility of PGD technique in the prevention of ADPKD.Our findings provide evidence for the future multicenter study and confirm the reliability and feasibility of PGD technique in the prevention of ADPKD,which will make great social benefits.