Analysis Of PRRT2Gene Mutation And Genotype-phenotype Correlation In A Cohort Of Paroxysmal Kinesigenic Dyskinesia Cases And Using Exome Sequencing To Indentified The Disease-causing Gene Of Gordon Holmes Syndrome

Background：Paroxysmal kinesigenic dyskinesia (PKD) is one of the mostcommon paroxysmal movement disorders. It is often induced by sudden stand up, turnaround, move and identified by the following criteria: kinesigenic trigger of theattacks, short duration of attacks (<1min), no loss of consciousness or pain duringattacks, exclusion of other organic diseases, and normal neurologic examinations It4has been found that genetic factor is one of the main reason for PKD, currently Itbelieves that the number of PKD patients who have a positive family history accountsfor about60%of total patients, and often compiled with a autosomal dominantinheritance pattern, the other patient was sporadic.Recently, mutations in the PRRT2gene were identified to be the causative factor of PKD. Although growing evidencesupports the contribution of PRRT2mutations to the development of PKD, evidenceof PRRT2mutation frequence and the genotype to phenotype correlation in PKD isstill lacking. In this study, we enrolled a cohort of PKD patients to screen for PRRT2gene mutation, and investigate the genotype to phenotype correlation.Objectives:1. To investigate the frequence of PRRT2gene mutations in the recruit PKD patients and the information of the indentified PRRT2gene mutation;2. To investigate genotype-phenotype correlation in the recruit PKD casesMethods:1. Patients given a clinical diagnosis of PKD were recruited through the Departmentof Neurology of the First Affiliated Hospital of Zhengzhou University from Dec2011to July2013. Clinical evaluations were performed by two independentresearchers and patients provided informed consent upon recruitment. Weextracted DNA from blood samples of the included subjects using a standardprotocol.2. The coding and flanking intronic regions of the PRRT2gene were amplified in3separate fragments. Sanger sequencing was performed to identify PRRT2mutations. The mutations indentified by this study were also tested in normalcontrols.3. Clinical characteristics were compared between PKD patients with PRRT2mutations and patients without PRRT2mutations. The patients were followed upfor6months to observe the response to carbamazepine.4. Variables were compared across the two groups using the two-sample Student’st-test for quantitative variables, the chi-square for categorical data, Fisher’s exacttest for categorical data where one or more of the cells had an expected frequencyof5, and the Wilcoxon rank-sum test for continuous non-para-metric data. Theentire analysis was conducted using the statistical software Statistical Package forthe Social Science (SPSS)17.0for Windows. All tests were two-tailed andinterpreted at the5%level of significance.Results:1. The study recruits30participants including19male patients and11femalepatients. In the all included patients,11had a positive family history come from5pedigrees the other19patients were sporadic. The mean disease onset age is14.33±3.92.2. We found that9patients from4pedigrees and6sporadic patients were positive for PRRT2mutations and all harbored a hot–spot c.649dupC (p.P217fsX7)heterozygous mutation, we also indentified a synonymous mutation1c.011C/T（p.Gly337Gly）in one sporadic patient whereas the other14patients werenegative for PRRT2gene mutations.3. Analysis of clinical-genetic associations revealed that patients who carried thec.649dupC (p.R217Pfs*8)mutation had a significantly earlier age of onsetcompared with those who did not (P=0.01). In addition, PKD attacks werebilateral in the majority of the PRRT2mutation carriers, whereas all of thenon-PRRT2mutation carriers in this study had unilateral attacks (P=0.001).Compared to the non-PRRT2mutation carriers, PRRT2mutation carriers had ahigher frequency of PKD attacks (P=0.039). In particular, there was nosignificant difference for the presence of an aura preceding the attacks betweengroups with and without mutations. After six months of follow up,p.R217Pfs*8–positive patients showed dramatic improvement with completeabolition of dyskinetic episodes with carbamazepine, while only7of the18patients without PRRT2mutations showed a response to the antiepileptic drug.Conclusion:1..PRRT2gene mutations are response for most of the familial PKD cases and partof the sporadic cases. The hot–spot c.649dupC (p.P217fsX7) heterozygousmutation account for93.75%of the total PRRT2mutations and we alsoindentified a synonymous mutation1c.011C/T（p.Gly337Gly.2. Our study indicated that positivity for PRRT2mutation is a predictor of younger ageof onset and more frequent of attacks in PKD patients. Interestingly, the presenceof PRRT2mutations also predicted a good response to carbamazepine therapy,especially at low dose. Therefore, genetic testing shows potential clinical significance for guiding the choice of medication for individual PKD cases. Background：Hereditary ataxia (Hereditary ataxia, HA) is a group of hereditaryneurological disorder characterized by progressive cerebellar atrophy and cerebellarataxia. Gordon Holmes syndrome (GHS) is a rare condition of hereditary ataxiacharacterized by ataxia with hypogonadism with an autosomal recessive inheritancemodel, male patients often showed hypoplastic external genitalia, gonads na ve andinfertility, female patients often showed no menstrual cramps and hypoplasia ofsecondary sexual characteristics after puberty. Despite almost100years of clinicalrecognition, less than20families had been reported in the world and there is still littleunderstanding of the pathophysiological mechanisms or underlying genetic causes ofGHS. Recently, mutations in RNF216and OTUD4were reported in GHS patients.Objectives:In our previous work, we collected the first Gordon Holmes pedigree in Chinesepopulation, this study was design to use exome sequencing technology to furtherexplore causative gene in this Gordon Holmes syndrome family. Methods:1In this study. we collected clinical data for all family members with twoneurology specialist’s detailed physical examination, all family members agreeon that the registration of clinical data, collect blood samples and regularfollow-up.2We used whole-exome capture+high-throughput sequencing technology andfollowed bioinformatic analysis in the two patients and one normal control toinvestigate the candidate disease-causing mutation.3We then used Sanger sequencing technology to repeat the result of exomesequencing and validated the candidate disease-causing gene in cohort of relevantpatients and healthy controls.Results:1In our pedigrees, the two female patients showed progressive ataxia withcerebellar atrophy, hypogonadism, and mild cognitive dysfunction. A youngerbrother of the two patients was normal, complied with an autosomal recessiveinheritance.2Each of the three sample generated about8Gb data with average sequencingdepth of more than90%, the target sequence coverage of over99%in the exomesequencing. Each sample was found harbored about90,000SNP and2000indels.After filter (1) publication database (2) recessive genetic model analysis,(3) IBDanalysis,(4) SNP pathogenic predicting, we found STUB1gene mutationc.737C-T (p.T246M) mutation was the most likely causative locus in thispedigree.3We also validated the candidate disease-causing gene STUB1in a cohort ofataxia patients without hypogonadism and Gordon Holmes syndrome patients butdidn’t find any STUB1mutation. It suggests that Gordon Holmes syndromedisease has genetics heterogeneity and additional genetic factors likely remain tobe identified in other GHS patient populations. Conclusion:STUB1gene c.737C-T (p.T246M) mutation is the most likely causativemutation in this pedigree, combined with subsequent functional in vitro and animalstudies, the results suggest that: STUB1gene is the third disease-causing gene ofGordon Holmes syndrome.