High-Throughput Sequencing Of Young And Middle Aged Parkinson’s Disease Patients And The Interpretation Of Sequence Variants

Background:Parkinson’s Disease(PD)is the second neurological degenerative disease.Typical clinical manifestations include bradykinesia,muscular rigidity,rest tremor,and postural and gait impairment.Besides,non-motor symptoms can also be observed,such as constipation,pain and rapid eye movement disorders.Studies have shown that about 10%of primary Parkinson’s disease is associated with genetic factors,and there are currently more than 20 genes associated with PD.The onset age of such PD patients is generally earlier,but specific clinical phenotypes are limited to identify it from sporadic PD patients.Therefore,it is important to conduct the gene detection in PD patients of young and middle age.Objective:This study was designed to detect the variation of related pathogenic genes in young and middle-aged patients by conducting the latest high-throughput targeting sequencing technique,and standardize the interpretation of sequence variants under the ACMG guidelines:(1)To establish the standardized interpretation and diagnosis strategy of PD genetics diagnosis;(2)To study the distribution of mutation genes in young and middle-aged patients with PD;(3)To discover new mutations of potential pathogenic significance.Subjects and Methods:1.Subjects:To collect PD patients admitted to Qilu Hospital of Shandong University from September 2014 to December 2016.Finally,109 young and middle-aged PD patients were enrolled,including 51 males and 58 females.2.Research methods:(1)Inclusion criteria:Patients were diagnosed according to the diagnostic criteria of the UK brain bank of Parkinson’s disease,age ≤ 65 years old.This study has been approved by the Qilu Hospital Ethics Committee of Shandong University,and all clinical subjects have signed the informed consent.(2)Clinical data collection:The diagnosis of PD was independently assessed by two experts in Parkinson’ disease.Besides,clinical basic information of every patient was collected,including gender,age,and onset age.(3)High throughput sequencing of PD-related genes:design and sample sequencing of PD-related gene by high-throughput capture chips;(4)Data analysis:Classification of pathogenic genes,gene frequency calculation and interpretation of variants according to ACMG guidelines.Results:1.Among the 109 patients with PD,18 cases(16.5%)were involved in PD related gene mutations,including 10 cases of LRRK2(9.17%),5 cases of PARK2(4.59%),1 case of LRRK2 + PARK2(0.92%),1 case of HTRA2(0.92%)and 1 case of PINK1(0.92%).2.The mean onset ages of patients without gene mutation group,LRRK2 mutation group,PARK2 pathogenic mutant group and PARK2 heterozygous mutation group was 40.3 years old,45 years old,33 years old and 41.5 years old,respectively.3.LRRK2(p.G2385R)In the tested samples,the gene frequency was the highest(3.67%);followed by PARK2(P.G135R,2.75%),PARK2(p.M309L,1.38%),LRRK2(P.A419V,0.92%),PARK2(P.R217W,0.92%),PARK2(p.V231L,0.92%),PARK2(p.E309X,0.92%)and PINK1(p.L519fs,0.92%).4.A total of 22 non-reported mutations were found,among which 5 mutations had a significant potential pathogenicity,including PARK2(c.838 + 2T>C,splicing),PARK2(p.R217W),PARK2(p.S223X),LRRK2(c.3777 + 1G>A,splicing)and PARK2(p.E309X).Conclusion:1.High-throughput targeting sequencing combined with multiplexed probe amplification technology,following the interpretation of sequence variants according to the ACMG guidelines,is of great significance in the genetic diagnosis of Parkinson’s disease;2.The proportion of mutant PD subtype was 16.5%among the young and middle aged patients.Besides,LRRK2 mutant subtype and PARK2 subtype were 9.17%and 4.59%,respectively.The most common mutations were LRRK2(p.G2385R)and PARK2(p.G135R).3.For the newly discovered mutation sites,pathogenicity need to be further verified,based on the analysis of pathogenicity using ACMG.