The Basic Study And Clinical On Familial Hemangioblastoma Of Central Nervous System In Guizhou Province

Objective the epidemiological and clinical features of a familywith CNS HB, introducing the feasibility of diagnostic and treatment, and to explorethe pathogenesis. ethods (1) Detailed epidemiological investigation, physicalexamination and images screening were performed on a family of38members withCNS HB.(2) Medical history, clinical manifestations, imaging data, diagnostic andtreatment methods and follow-up of7CNS HB patients of the family were elaboratedand analyzed.(3) Light microscopy, electron microscopy and immunohistochemicalstaining were applied for pathological observation of tumor tissues in operation on4cases with CNS HB and of brain tissues in operation on6cases with traumatic braininjury(as control).(4)Peripheral blood from38members of the family with CNS HBand30healthy subjects, extracted DNA and RNA, amplified exons1and2of VHLgene by PCR and sequenced. The level of VHL mRNA expression in peripheral bloodcells was determined by real-time PCR.(5) protein extraction and enzyme digestionwere used with acquired tumor tissue of CNS HB and the control group of braintissues, using Label-free proteomics technology for quantitative analysis, biologicalinformation analysis was performed on the differential proteins screened. Results(1)the family members of the existing7cases, age14~53years, average36.5yearsold, explicit rate of10/38(26.3%), the incidence of male more than female (6:4), CNSHB accounted for90%, and multiple HBs accounts for20%; genetic may come fromthe paternal (I2members).(2)7cases of patients were not associated with otherlesions, including6cases of tumors,2cases of cystic tumor;5cases located in thecerebellum, medulla oblongata and besides brainstem,1cases in cervical spinal cord, 1cases in the retina;4patients underwent a total of7microsurgeries, resection ofhemangioblastoma in10. During the follow-up period of8years, in6years aftersurgery, the proband had a recurrence, which was not treated, with continuedfollow-up.1patient received laser treatment of retinal vascular tumor.2cases ofobvious symptoms were followed up and no new lesions was found while the originallesions did not increase. The recurrence rate was42.8%(3/7).(3)The features ofPathology: HE staining showed that tumor was made up of abundant capillaries andinterstitial cells. The cytoplasm was vacuolated and foam. There was no abnormalnuclei of tumor cells. Vimentin, vascular endothelial growth factor receptor, alphainhibin and neuron specific enolase staining in the interstitial cells of HB showedstrong positive expression. CD31, CD34and vimentin showed strong positiveexpression of HB in endothelial cells. However, glial fibrillary acidic protein, angleprotein antigen, epithelial membrane antigen were negative for expression of CNS inHB. Ki-67labeling index, except for1cases of the second operation was2%, were l%.Electron microscope observation: Endothelial cells were flat, naive, large nucleus,pale stained. Weibel-Palade bodies could be seen in cytoplasm. Pericytes located inthe endothelial cells outside, which were near the vascular cavity and volume wassmaller than that of the endothelial cells. Nuclei were oval, less cytoplasm, cell issurrounded by a basement membrane layer completely. Stromal cells were oval,irregular surface, large nuclei, pale staining, cytoplasm was rich in lipid dropletvacuoles.(4) Among the members of this CNS HB family, A→G mutations weredetected at19thnucleotides in5’non coding region of VHL exon1in7patients.Compared with the healthy control group, the expression levels of mRNA of VHLgene decreased significantly. In the family members, the expression levels of mRNAof VHL gene of the attackers (0.062) were more decreased than those without thedisease significantly (0.26).(5)In the CNS HB group, average credible proteins andpeptide fragments were isolated and identified to be385and2195while in the normalgroup, it to be663and3662. By quantitative analysis and difference matching, therewere3differential proteins screened,which were expressed only in CNSHB(vimentin,TPM4and SERPING1).In13proteins expressed both in HB and control tissues,3proteins (HBB, ALB, HBD) were up-regulated in HB tissues and11proteins（such as HSP、ATP、PEBP1、FTL、SOD2、PARK7、KRT1Keratin、Putativeand so on）were down-regulated; There were3proteins (Tubulin beta-4chain,14-3-3,CA1) which were expressed only in control tissues. In the GO function analysis, thebiological process (BP) analysis suggested that positive and negative regulation ofbiological process, response to stimulus, biological adhesion, immune system functionwas significantly improved; metabolic processes, development, signal transduction,cell process function is lower; These were associated with the formation of familialCNS HB. Molecular function (MF) analysis revealed that the biological chemotacticactivity function was present only in the tumor group; and the translation regulatoryactivity function lost in the tumor group. Cell component (CC) analysis resultsshowed that the number of proteins in extracellular region part and extracellularmatrix part in CNS HB tissue was significantly higher than that in the control group;while the number of proteins in synapse part and synapse was significantly lower thanthat in the control group. Application of COG analysis (clusters of protein adjacentclass) concluded that: the functions of "the nuclear chromatin structure and dynamics"(class B) and "cell dynamics"(class N) were found only in the CNS HB tissues.However, the functions of "Cell cycle regulation","cell division" and "cellchromosome "(class D),"Coenzyme transport and metabolism"(class H),"Secondarymetabolite biosynthesis" and "transport and catabolism"(class Q) existed only in thecontrol group. So we speculate that the incidence, development and easily repeatedrecurrence of familial CNS HB may be associated with that class B and N wereup-regulated expression, and class D was disturbance, and class H and Q wereabnormal. Meanwhile, the high mobility group protein1, histone H2A, SERPING l,tropomyosin α-4, vimentin were expressed only in CNS HB, which may be involvedin tumor occurrence, development process directly: By promoting cells migration,differentiation and dendritic cells high expression, and regulating protease activity,high mobility group protein1and tropomyosin4possibly accelerated tumor growth,infiltration and recurrence. Some factors promoted the formation of HB such as highexpression of H2A, loss of regulate activity of PCBPl, activation of the hypoxia signal pathway, up regulation of HIF-la, the secretion of EPO and VEGF outward promotedby interstitial cells, and instability of alpha globin mRNA. Significant up-regulationof SERPINGl genes in CNS HB may be one of the causes which contributed to thegrowth of tumor and peritumoral edema. That the loss or disorder of PURA’stranscription, translation regulatory activities in CNS HB resulted in cell proliferation,growth abnormalities, degeneration and necrosis of neuron, inhibited transcriptionactivity of vascular smooth muscle and α-actin gene contributed to the occurrence anddevelopment of tumor. Conclusion (1) Familial hemangioblastoma of central nervoussystem is a heritable VHL disease that was seriously harm to patients and their familymembers, which onseted of a younger age and multiple lesions, easily relapsed.Patients who were questioned for a family history of CNS HB should conduct acensus of the family members, including imaging, gene biology and proteomicsdetection.(2)The right diagnosis of familial hemangioblastomas of the central nervoussystem, which is called as VHL disease, is mainly depended on family history, clinicalmanifestations, MRI detection, pathological examination (α-inhibin, vimentin, VEGF,CD34staining) and detection of VHL gene. Once diagnosed with VHL disease, itmust be lifelong followed-up.(3) The incidence of familial CNS HB is associated withmutation of VHL gene. The expression decreasely of VHL gene mRNA in peripheralblood could be used as one of the disease diagnosis and prognostic indicators.(4)Micro operation resection is the main treatment for CNS HB. And stereotacticradiotherapy can be used as a supplementary treatment. Some with no obvious clinicalsymptoms or small tumor can be closely followed up. The research of biologicalimmune target treatment may be an ideal auxiliary treatment method.(5)In the CNSHB tissues the functions of the biological process of positive and negative regulation,stimulus response, biological adhesion, enzyme regulator activity, structural moleculeactivity, immune system process were significantly up-regulated. The functions ofchemotactic activity, nuclear chromatin structure and dynamics and cell motility onlyexisted in the CNS HB tumor. But the functions of translation activity, cell cyclecontrol, metabolism, development, signal transduction, cell and chromosome divisionwere more down-regulated or lost than that of control group. Some proteins were most closely related to the occurrence and development of CNS HB and were foundfirstly, which were involved in above functions, such as HMGB1, TPM4, H2A,PCBPl, SERPINGl and PURA. Moreover, there was a closely relationship betweenhigh expression of Vimentin and pathogenic of CNS HB, which is concerned with theorigins of CNS HB stem cell. This is similar to those reported in literarures.(6) Theoccurrence of familial CNS HB disease is a complex pathological process thatinvolved in a multiple factors and pathways.