| Background : Parkinson’s Disease(PD)is one of the common neurodegenerative disorders.PD is thought to be caused by a combination of ageing,as well as genetic and environmental risk factors.Since the first causative gene SNCA was identified,to date,24 genes with different degrees of genetic evidence have been found to be mutated in monogenic PD.Several large-scale studies have been conducted to screen for PD causative genes in cohorts based on European populations.However,there are few studies about the comprehensiveness and extensibility of systematical study about mutational spectrum of the PD population in mainland China.With the rapid development of next-generation sequencing(NGS),it has provided an effective means for the molecular diagnosis of these PD patients.Considering the high mutation rate of autosomal recessive inheritance pattern(AR),the molecular diagnosis of early-onset ARPD patients is still partially unclear,suggesting that there might be other AR-PD genes that have not been discovered yet.Combining the homozygous mapping regions based on WES data and the variants identified by WES can provide new ideas for the identification of new ARPD pathogenic genes.Objective: To determine the mutational spectrum of familial PD and sporadic early-onset PD(s EOPD)in a mainland Chinese population and the clinical features of mutation carriers.To identify novel candidate AR genes AR-PD families of Chinese mainland population,so that we could expand the spectrum of recessive genes responsible for PD.Methods:1.Performed multiplex ligation-dependent probe amplification assays(MLPA)and WES for 2265 unrelated patients with PD in a mainland Chinese population,including 245 probands from ARPD families,366 probands from families with autosomal-dominant PD(ADPD),and 1652 s EOPD patients(age at onset ≤ 50).2.Compared the frequency of P/LP variants among patients with different AAOs and among different genes separately,in order to determine differences in the molecular diagnosis,and to depict the AAO spectrum for these disease-associated genes in our population.3.Generalized the clinical manifestations of PD patients with certain mutated genes important for PD pathogenesis.4.Using WES data,combined with the analysis of runs of homozygosity regions based on WES data,the detection and analysis of new ARPD pathogenic genes in core ARPD families in mainland China for which molecular diagnosis has not yet been confirmed,and non-core ARPD families and s EOPD patients for which molecular diagnosis has not been confirmed as replication cohort.Results:1.According to standards and guidelines from the American College of Medical Genetics and Genomics,pathogenic/likely pathogenic(P/LP)variants in 24 known PD causative genes occurred more frequently in the ARPD cohort(77 of 245,31.42%)than in the ADPD cohort(11 of 366,3.01%)and the s EOPD cohort(81 of 1652,4.90%).The mutation rates of GBA gene variants in ARPD,ADPD and s EOPD cohorts were 3.68%,7.38% and 7.68%,respectively;and the earlier the age of onset,the higher the mutation rates in ARPD and s EOPD cohorts;2.PRKN was the most frequently mutated gene(n＝105,4.64%),accounting for 58(23.67%)molecularly diagnosed ARPD probands and47(2.85%)molecularly diagnosed s EOPD probands.Functional experiments confirmed that two new P/LP variants of PRKN gene could affect mitophagy,including c.968＿/p.C323＿V324del and c.619-1G>C variants;3.LRRK2 was the most frequently mutated AD gene(n=11,0.49%),accounting for five(1.37%)molecularly diagnosed ADPD probands and six(0.36%)molecularly diagnosed s EOPD probands.Functional experiments confirmed that the new P/LP variant p.N1437 D of LRRK2 gene could lead to an increase in the proportion of LRRK2 kinase.Moreover,ADPD families with SNCA duplication were identified for the first time in the PD population in mainland China;4.Several novel pathogenic/likely pathogenic variants including LRRK2(p.V1447 M and p.Y1645S),ATP13A2(p.R735 X and p.A819D),FBXO7(p.G67E),LRP10(c.322 dup C/p.G109Rfs*51)and TMEM230(c.429 del T/p.P144Qfs*2)were identified in our cohort.5.The AAO of the 169 probands with genetic diagnoses(median,31.5 years)was about 14.5 years earlier than that of patients without molecular diagnoses(i.e.non-carriers,median 46.0 years).Specifically,the age at onset of Parkinson’s disease patients with pathogenic/likely pathogenic variants in ATP13A2,PLA2G6,PRKN,or PINK1 was significantly lower than that of non-carriers,while the age at onset of carriers with other gene pathogenic/likely pathogenic variants was similar to that of non-carriers.6.We found that carriers of PRKN,PLA2G6,and PINK1 P/LP variants had earlier AAOs than non-carriers.PRKN P/LP variant carriers associated with milder motor manifestations.PLA2G6 P/LP-variant carriers showed more severe motor symptoms.We did not find any differences in clinical manifestations between LRRK2 P/LP-variant carriers and non-carriers.Specifically,SNCA duplication carriers showed more severe cognitive dysfunction and depression.Patients with GBA variants showed more severe rapid-eye-movement sleep behavior disorder and autonomic dysfunction.7.In 13 core early-onset consanguineous ARPD families,the possible new AR pathogenic gene variants within the runs of homozygosity regions of the corresponding families were screened,and the analysis indicated that one candidate causative gene was prioritized in each of the six early-onset consanguineous ARPD families,including DAGLB(n = 2)、PD-ARC1,PD-ARC2,PD-ARC4 and PD-ARC5 genes.Combined with the support of genetic evidence in the replication cohort,DAGLB,PD-ARC1 and PD-ARC5 gene was replicated with more genetic support.In addition,and each of the remaining six early-onset consanguineous ARPD families was prioritized with multiple candidate pathogenic genes,among which PD-ARC5,PD-ARC27,PD-ARC21 and PD-ARC22 genes were also replicated with more genetic evidence.8.However,12 core early-onset non-consanguineous ARPD families were not prioritized with any possible causative gene for a recessive inheritance pattern.Conclusion:These results shed insight into the genetic spectrum and genotype-phenotype association profile of PD in mainland China(The mutation rates of ARPD families,ADPD families and s EOPD patients were 35.11%,10.39% and 12.58%,respectively)and expand the existing repertoire of P/LP variants involved in known PD causative genes.DAGLB gene might be a novel PD causative gene;moreover,PD-ARC1,PD-ARC5,PD-ARC27,PD-ARC21 and PD-ARC22 genes could be prioritized as novel candidate PD causative genes. |
