The Study Of Clinical Features Of Early-onset Parkinson’s Disease And Gene Mutation Analysis Of PARKIN In Uyghur Family With Early-onset Parkinson’s Disease

Background Parkinson’s disease(PD), which was first described by James Parkinson in 1817, is a kind of chronic progressive neurodegenerative disorder among elderly people. With life-span extended, the aging phenomenon of population is becoming serious.The incidence of PD is growing and become a great danger to the middle-aged and elderly people. Resting tremor, rigidity, bradykinesia, and postural instability are the main clinical symptoms of the disease.Parkinson disease is a kind of common disease among the aged. There are many people over the age of 60 among the patients. And then, with age increased, the incidence increased. According to statistics, the prevalence of PD is estimated at 1% in people over 60 years of age. It is rare before age 40 years and that is defined as "early-onset Parkinson’s disease"(EOPD) by Quinn. Aproximately 4 to 10% of cases have an onset below age 40, but 18.4% of cases were reported by the Japanese scholars. EOPD is commonly familial aggregation. With compared to late-onset PD(LOPD), EOPD is different at the morbidity, clinical manifestation and prognosis. Wickremaratchi,et al,conducted a case-control study based on 48 cases of EOPD and 123 cases of LOPD, and found that with the LOPD, there ware several characteristics with EOPD at a primary symptom of TD, a slower disease progression, a earlier motor complications,less cognitive decline and a good response to small doses of levodopa. While the primary symptom of LOPD was PIGD(Postural Instability and Gait Disturbance). But there are many debates about the clinical manifestation of EOPD. It is not clear whether there are regional differences.The pathogenesis of PD is still unclear. The present study indicates that there is relate with heredity, environment, infection, aging, oxidative stress, radical damage and lack of nerve growth factor and so on, and the results may be synergistic action of a variety of mechanisms. Among these theories, heredity factor is mostly studied. To the research of gene in familial PD, 24 genetic locus were associated with the PD, including PARk1-16,EIF4G1,GBA,DOCTN1,POLG1,GCH1,TH,SLC6 and ATXN2. PARK2, named parkin also, is mostly studied. In 1997, Parkin gene was first reported among autosome recessive juvenile parkinson’s family in Japan. Other studies indicates that parkin gene paly a great role in familial or sporadic parkinson’s disease.This gene,which locates at 6q25.2-q27, is between D6S305 and D6S253. It includes 12 exons, and the length is 1.5Mb, and code sequence is 1395 bp,which’s open reading frame encode 465 amino acids. Parkin gene mutation is the most common cause of all known recessive inherited PD, and it’s mutation types include deletions, nonsense, missense, polyploidy of exons and so on. Among EOPD, the mutation frequency of Parkin is up to 50% in familial PD. Inherited Parkin gene mutations has obvious diversity. 12 exons of Parkin gene have different degrees of sequence change, of which 4、6、7 are the most common, and they are all in the hotspots of mutation. Some researchers studied the relationship between Parkin gene exon deletion and phenotype by screening 12 family of autosome recessive inherited juvenile parkinsonism from 5 countries. According to their research, they thought different families may have different exon deletions, resulting in different phenotype and may relate to the function of exon. At present, the domestic and foreign scholars have found that some Parkin gene mutation can only explain part of familial PD etiology, and the property or location of a mutation in different populations is different. It shows that different ethnic and regional of PD patients may have different at Parkin gene mutations.The xinjiang uygur autonomous region is located in the northwest of China,1/6 of the national area, more than 20 million population, living 13 different nationalities for example the han, uygur, kazak and mongolian,etc. There are greatly different from the inland at Genetic and cultural traditions, customs and lifestyle, geographical conditions, economic development. Uighurs is the most nation in xinjiang. They had been on the nomdic between south of Lake Baikal with the irtysh and the balkhash lake in north and northwest of China in the 3rd century BC, named "jingle". They gradually blend of turkic and han people, therefore they have obvious difference with the han nationality. With the increasing of population aging, the uygur PD patients also increases. At present,there is only data about the parkin gene mutation and polymorphism among the familial PD of the han patients.Whether the parkin mutation is relate with xinjiang uygur familial patients and the parkin gene is different from the han nationality or other races, it still need further study. It is of great significance for the pathogenesis and prevetion strategy of PD to develop the molecule research of xinjiang uygur familial PD patients,describe the characteristics of xinjiang uygur parkin gene,explore the difference among different races and its influencing factors,especially the molecular characteristics.Purpose 1)To investigate and find the clinical characteristic of EOPD and provide for reference of clinical diagnosis and treatment.2)To investigate the characteristic of Parkin gene mutation and lay the foundations for further studies on nosogenesis of Uygur familial EOPD.Methods Part I: A case-control study was conducted. The clinical characteristics of 160 PD patients, based on UK-Parkinson’s Disease Society Brain Bank criteria, collected by our group were reviewered retrospectively, which patients were coming from clinic service and hospitalization on xinjiang people hospital from January in 2011 to December in 2013. The inclusion criteria included: bradikinesia, rigidity, rest tremor, postural instability, unilateral onset, progressive disorder, excellent response to L-dopa.The exclusion criteria incorporated: history of repeated strokes, history of repeated head injury, history of definite encephalitis, oculogyric, neuroleptic treatment at onset of symptoms or the use of dopamine depletion agent, MPTP exposure, negative response to large doses of levodopa, other neurological symptoms and signs.According to Quinn’s criteria, a total of 160 PD patients were divided EOPD and LOPD. According to Hoehn&Yahr, a total of 160 PD patients were divided Hoehn&Yahr 1-2.5 stage and Hoehn&Yahr 3-5 stage. Clinical data included the history, neurological physical examination, blood routine examination, brain MRI, UPDRS score, Hoehn&Yahr score, SDA,SAS Scale and Mo CA scale, etc. Comparing to EOPD and LOPD with the clinical characteristic, Hoehn&Yahr scales, UPDRS scale, Mo CA scales, SDA,SAS, levodopa effect and clinical type. Comparing to Hoehn&Yahr 1-2.5 stage and Hoehn&Yahr 3-5 stage with the clinical material and the scales, we analysis the related factors with the severity of the disease by logistic regression.Part II : To investigate a PD family of long lived Tuo Ke Xun county in Xinjiang, based on UK-Parkinson’s Disease Society Brain Bank criteria, there are 15 members of 3 generations in this family, 3 people are diagnosed PD(age on onset <40years), other 12 people have no symptoms of PD.Extract genomic DNA collected 10 cases of family members of peripheral blood, blood DNA extraction kit is utilized to extract genomic DNA. According to the specific primer sequences and amplification conditions, amplify parkin gene 3-7 exons. PCR amplification conditions was as follows: 34 cycles for 95℃ denaturation for 2 min, 55℃ annealing for 30 s, 72℃ extension for 30 s, 95℃ denaturation for 20s;and then 55 ℃ denaturation for 30 s, 72 ℃ extension for 7 min. The products was electrophoresed on 2% agarose gel and all mutated alleles were notarized by DNA direct sequencing of PCR product from a second amplification. To detect the parkin gene mutation of the Uighurs in xinjiang by DNA extraction and exon sequencing technology. TO explore the relationship of parkin gene mutation and the Uighurs EOPD.Part III: A field investigation was conducted to this early-onset PD family in January 2014. The data include general information and clinical symptom, specialist neurological signs, UPDRS score, Hoehn & Yahr score, SDS,SAS and Mo CA score and so on. And the family members of positive signs were compared with the survey in 2012 to understand the progress of the clinical symptoms of PD patients.The study protocol was approved by the ethics Committe of xinjiang uygur autonomous region. All participants signed infromed consent.Results Part I The study included 160 PD patients. PD Patients were diagnosed in 160 with 30 EOPD cases, with 130 LOPD cases. There were 10 cases of male and 20 cases of female in the EOPD group; Age of the EOPD group was 39.10±5.74 years(mean ±standard deviation);Age at onset was 35.33±4.19 years;Disease duration was 3.77±2.45years; There were 90 cases of male and 40 cases of female in the LOPD group; Age of the LOPD group was 70.44±7.11 years; Age at onset was 63.71±6.10 yeases; Disease duration was 6.74±3.32 yeases; To compare with LOPD, primary symptom of EOPD is tremor(53.3%,p=0.001), Hoehn&Yahr scales is H-Y 1-2.5stage(66.67% p=0.01) and UPDRS scale is lower(p=0.000), levodopa effect is good(p=0.03), and clinical type of EOPD is TD(p=0.01). The cranial MRI examination in early-onset PD is normal mostly, and late onset PD patients have a 52.2% abnormal performance, considering a merger with age, course of the disease and other diseases. But there is no significant difference in SAS,SDS and Mo CA(p>0.05).The logisitic regression ananlysis of multifactors to the clinical features of PD patients. We found the relationship between H-Y 1-2.5 stage and the age, age at oneset, disease duration, primary symptoms and EOPD/LOPD. In this study, it is found that EOPD is a independent predictor to severity of PD.Part IIa Uyghur family, with 15 members of three generations, showing autosomal recessive inheritance was studied. The family pedigree was painted after our investigation of genetic characteristics of the family(see Fig. 1). The proband and 2 siblings were diagnosed with PD. Blood samples were collected from 10 subjects, including three affected cases(6,8,9) and seven unaffected cases(1,3,7,10,11,14,15). Three affected case consist with the diagnostic threshold for early onset Parkinson disease. Proband was a young man, 35 years old, three years history of PD, with manifestations of resting tremor Right limb,rigidity,gait dysfunction and Face stiff,and head computed tomography(CT) and Magnetic Resonance Imaging(MRI) scan,blood routine examination,Coagulation weren’t detected abnormal. Blood Copper was lower, but ceruloplasmin and Liver ultrasound were normal, and K-F ring was negative, Madopar test is positive. Characteristics of Another two patients same as Proband. To further confirm the mutation of parkin gene in this family, after PCR amplification, mutations in extron 3 to 7 were detected by direct sequencing. The result show that two point mutation were detected on extron 7 of parkin gene in this family,and extron 3 to 6 was’ t found any mutation. The two point mutations are heterozygous C924>T(R275W) and G951>C(G284R). In this family, NO.6, NO.8, NO.9 and NO.15 were detected the mutation G951>C(G284R), and NO.1, NO.6, NO.8, and NO.14 carried the mutation C924>T(R275W). We didn’t found any mutation of parkin gene among the other family membersPart III As follow up this family, there was 3 PD patients at the second generation at January,2014. The phenotype of parents and the next generation is normal. Number 6 patients had a more obvious neurological positive signs in on and off stage, main in muscle tension and tremor. Number 8 patient had a more higher truck muscle tension in on and off stage to 2 years ago.Number 9 patient had more higher muscle tension and obvious tremor in on and off stage,UPDRS score, Hoehn&Yahr score, anxiety, depression scale and Mo CA scale have a worse performance in on and off stage.Conclusion Part I In this study,EOPD is aproximately 18.75% of all cases, but 18.4% of cases were reported by the Japanese scholars.It is seem. We found that EOPD is the independent factors on the severity of PD.Part II The study first found that composite heterozygous mutation G951> C(G284R) and C924> T(R275W) mutation in seventh exon may be related with Uighur earlyonset familial PD in the parkin gene.Part III Through the follow-up of this pedigree, we found that clinical symptoms of PD patients have significantly increased in the past 2 years, but the neuropsychologyl worsen more obvious. In terms of movement disorders, it is mainly worse in sports,walk, and tremors. For cognitive function, three PD patients had no significant changes of Mo CA score. According to UPDRSⅣscore, the incidence of dyskinesia increased significantly.