Study On Factors Related To The Clinical Feature And Molecular Biology Of Hemorrhagic Moyamoya Disease

Part Ⅰ:Study on Factors Related to the Clinical Feature of Hemorrhagic Moyamoya DiseaseBackground and purposeMoyamoya disease is an uncommon cerebrovascular disease characterized by progressive stenosis or occlusion of bilateral internal carotid artery with formation of abnormal vessel network at the base of the brain.Hemorrhagic moyamoya disease is an important clinical type with a high mortality and morbidity,the prognosis of which is poor.How to prevent rebleeding is the keypoint for the treatment of hemorrhagic moyamoya disease.The best treatment modality for hemorrhagic moyamoya diseas was still unclear.Controversy mainly focused on whether surgical treatment was necessary,optimal operation timing,modality and surgical outcome.Currently,the large sample prospective clinical study on surgical treatment for hemorrhagic moyamoya disease was rare in China.The aim of this study was to identify the clinical and radiological features of hemorrhagic moyamoya disease and whether combined cerebral revascularization could reduce the rebleeding rate and improve the clinical prognosis.MethodsBetween December 2007 and December 2012,211 cases of hemorrhagic moyamoya disease who were hospitalized at Department of Neurosurgery,Huashan Hospital,Fudan University were enrolled in this single-center prospective clinical study.Head CT scan and cerebral angiography were performed for all the patients to analyze the site of hemorrhage,Suzuki stage,AChA-PComA branch extension(Morika grading) and concomitant aneurysms or not. All the patients received surgical treatment,the modality of which was combined cerebral revascularization surgery. Subsequently,we carried out follow-up and recorded the primary end point events(stroke or death within 30 days postoperatively,ipsilateral symptomatic rebleeding beyond 30 days postoperatively) and finally calculated the annual rebleeding rate.Follow-up Angiogram focused on graft patency, Matsushima grading for the effect of cerebral revascularizaton,reduction degree of moyamoya vessels,disappearance of concomitant microaneurysms,improvement of AChA-PComA branch extension.Results1.Among 211 patients with hemorrhagic moyamoya disease,the age of onset ranged from 13 to 58 year,averaged 38.2 years.Female to male ratio was 1.4:1;9 cases(4%) were children(<18 years).The bleeding site included IVH in 94 cases (45%), ICH in 37 cases (18%), ICH with IVH in 75 cases (36%) and SAH in 5 cases (1%).2 cases was discovered with concomitant microaneurysms.Suzuki stage included 0 case with 1,24 cases with 11,83 cases with Ⅲ,90 cases with Ⅳ,10 cases with V and 4 cases with Ⅵ on hemorrhagic side.AChA-PComA branch extension Morika grading included 75 cases with grade 1,132 cases with grade 2 and 4 cases with grade 3 on hemorrhage side;141 cases with grade 1,65 cases with grade 2 and 5 cases with grade 3 on non-hemorrhage side.Morika grading on hemorrhage side was significantly higer than non-hemorrhage side(P<0.05).2.14 of 211 cases were lost during follow up (6.6%).The average follow-up time was 36.1 months.Outcomes of the primary end point events were listed as follows:5 cases with ipsilateral symptomatic rebleeding(2.5%),of which 4 cases died(2.0%) and 1 case with severe disability(0.5%).The annual rebleeding rate was 1.87%/person calculated by Kaplan-Meier method.3.141 of 197 cases were followed up by cerebral angiogram(75%).Graft patency rate was 100%; Matsushima grading:90 cases with Grade A,39cases with grade B and 12 cases with grade C;Reduction degree of moyamoya vessels:28 cases with little decrease(<25%),51 cases with moderate decrease(25%-50%) and 62 cases with significant decrease(>50%);The micoraneurysms both disappeared in the 2 cases.Improvement of AChA-PComA branch extension: 91 cases with improvement,50 cases with unimprovement on surgical side;55 cases with improvement,86 cases with unimprovement on non-surgical side;89 cases with improvement,47 cases with unimprovement on non-rebleeding side;2 cases with improvement,3 cases with unimprovement on rebleeding side;Improvement of AChA-PComA branch extension after combined cerebral revascularization had significant correlation with the reduction of rebleeding rate(P<0.05,OR 2.846,95%CI 1.755-4.615).Whereas, Matsushima grading and reduction degree of moyamoya vessels had no significant differences with the reduction of rebleeding rate(P>0.05).Conclusion1.IVH was the primaruy bleeding site for the patients with hemorrhagic moyamoya disease,followed by ICH with IVH.Suzuki stage III and IV overwhelmed over other stages.2.Combined cerebral revascularization could reduce the rebleeding rate of hemorrhagic moyamoya disease and improve the prognosis.3.Combined cerebral revascularization could lead to the reduction of moyamoya vessels, improvement of AChA-PComA branch extension and disappearance of concomitant microaneurysms by increasing cerebral perfusion,of which the reduction of rebleeding rate was possibly correlated with the improvement of AChA-PComA branch extension and disappearance of concomitant microaneurysms.Part II:Study on Factors Related to the Molecular Biology of Hemorrhagic Moyamoya Disease in Hospital-Based patientsBackground and purposeOnce first episode of bleeding or rebleeding occurred in patients with hemorhhagic MMD,the mortality and morbidity was high and the prognosis was poor.However,the etiology was still unclear.Recently,several genes or locus susceptible to MMD have been identified by genetic studies, among which RNF213 gene was verified to be related to MMD in Japanese population by previous studies.Moreover,R4810K mutation was significantly correlated with MMD.However,the relationship between genotype and clinical phenotype had never been analyzed.With an increasing number of MMD patients detected in China,hemorrhagic MMD was one of the important clinical types.So far, the gentetic studies of hemorrhagic MMD have never been reported.We designed this case-control study to validate whether RNF213 R4810K was related with hospitalized patients with hemorrhagic MMD and further carried the genomic sequencing of RNF213 gene to look for the susceptible gene or locus with hemorrhagic MMD.MethodsThe study enrolled 170 patients with MMD and 507 healthy adults at Department of Neurosurgery,Huashan Hospital,Fudan University from January 2007 to December 2011.All the blood samples were collected.Genotyping of R4810K mutation in RNF213 gene was performed with unlabeled probe high resolution melting assay.Statistical analysis was carried out for the difference of allele and genotype frequency between MMD group and control group,genotype and clinical phenotype.Subsequently,genomic sequencing of RNF213 gene was performed to look for the susceptible gene or locus with hemorrhagic MMD.ResultsR4810K mutation was identified in 22 of 170 MMD cases (13%),including 21 heterozygotes and 1 homozygote.Two of 507 healthy controls (0.4%) were heterozygous R4810K carriers. The R4810K mutation greatly increased the risk for MMD (OR=36.7,95% CI:8.6-156.6, P=6.1E-15).Further analysis between genotype and phenotype found R4810K mutation significantly correlated with ischemia (OR=5.4,95% CI:1.8-16.1, P=0.001). Genomic sequencing covering RNF213 exon 40 to exon 68 also identified eight other non-R4810K variants:P4007R, Q4367L, A4399T, T4586P, L4631V, E4950D, A5021V and M5136I,of which A4399T mutation rate was highest(4/20).Genotyping of A4399T by the same method revealed that the polymorphism was found in 28/170 cases (16.5%) and 45/507 controls (8.9%),which was associated with MMD (OR=2.0,95% CI:1.2-3.3, P=0.004),especially with hemorrhage (OR=2.8,95% CI:1.2-6.5, P=0.014).Conclusion1.RNF213 R4810K was associated with ischemic MMD,but not with hemorrhagic MMD.2.Genomic sequencing identified eight new variants:P4007R, Q4367L, A4399T, T4586P, L4631V, E4950D, A5021V and M5136I. Among them A4399T was significantly associated with hemorrhagic MMD, which implied A4399T might be a susceptible locus with hemorrhagic MMD.3.Identification of novel variants in the RNF213 gene by genomic sequencing further highlights the genetic heterogeneity of MMD.Part III:Study on Factors Related to the Molecular Biology of Hemorrhagic Moyamoya Disease in Community-Based PopulaitonBackground and purposeGenetic study of hemorrhagic MMD based on hospitalized patients have found that RNF213 A4399T was the susceptible genetic locus with hemorrhagic MMD.Howevere,the human subjects were inpatients from the hospital.Sporadic MMD predominated in China nowadays.lt was still unclear that whether RNF213 A4399T was associated with hemorrhagic MMD in community-based population and the susceptible gene with hemorrhagic MMD in community-based population.The purpose of this study was to elucidate whether RNF213 A4399T was associated with hemorrhagic MMD in community-based population and meanwhile carried out the whole-exome sequencing study to look for the susceptible gene with hemorrhagic MMD.MethodsBetween January 2013 and October 2013, a community-based epidemiological survey was conducted in Shenhe,Dayuan and Baima Villages,Ciwu Town,Zhuji City,Zhejiang Province on the basis of the characteristic regional distribution of hemorrhagic MMD.All the volunteered villagers received questionnaire survey,blood sampling and head MRA examination.HRMA was used to perform the genotyping of RNF213 A4399T.3D-TOFMRA was applied for screening MMD. The epidemiological and clinical features of hemorrhagic MMD in this community were summarized and statistical analysis was made for the relationship between RNF213 A4399T and hemorrhagic MMD.Meanwhile,the blood samples of hemorrhagic MMD in this community were collected for the whole-exome sequencing study to identify the susceptible gene with hemorrhagic MMD.Results1.596 villagers participated in the epidemiological survey on hemorrhagic MMD.537 questionnaires were valid(90%).Finally 537 villagers were enrolled in the community-based epidemiological study.2.The average age of the 537 villagers was 51 years,with 352 females(65.5%).14 villagers were diagnosed with sporadic MMD,all of whom were found in Shenhe Village.The average age was 52 years with 8 females(57.1%).9 cases were hemorrhagic MMD,1 case was ischemic MMD and 4 cases were asymptomatic MMD.3.RNF213 A4399T mutation was detected in 6 of 14 cases with MMD including one homozygote and five heterozygotes,9 of 253 normal controls including nine heterozygotes.Moreover,RNF213 A4399T mutation was detected in 5 of 9 cases with hemorrhagic MMD,1 of 4 asymptomatic MMD.One case with ischemic MMD detected no RNF213 A4399T mutation.RNF213 A4399T was significantly associated with hemorrhagic MMD in the community-based population(P<0.05).4.Blood samples from 10 cases with MMD and the healthy first-degree relatives as controls were collected and the whole-exome sequencing was conduted.10 cases with MMD consisted of 7 hemorrhagic type,l ischemic type and 2 asymptomatic type.The outcome identified 4 MMD susceptible genes:RNF213、HLA-DRB5、 KRTAP5-1 and SYNE1.Among them,RNF213 A4399T mutation was detected in 4 of 7 hemorrhagic MMD,but no mutation of RNF213 A4399T was detected in the 10 healthy controls.Conclusion1.RNF213 A4399T was associated with hemorrhagic MMD in community-based population.2.Whole-exome sequencing identified 4 MMD susceptible genes:RNF213、 HLA-DRB5、KRTAP5-1 and SYNE 1.Among them,RNF213 A4399T was associated with hemorrhagic MMD.3.The relationship between HLA-DRB5,KRTAP5-1,SYNE1 and MMD was still unknown which was expected for further research in the future.