| Part1, next-generation sequencing identifies the causative mutations in MFS patients.Objective:Marfan syndrome (MFS) is known as an autosomal dominant heterogeneous disorder of connective tissues, characterized by early development of thoracic aneurysms/dissections together with defect of the ocular and skeletal systems. Loss-of-function mutations in the fibrillin-1encoded gene FBN1is major cause of this disorder, and some causative mutations of the transforming growth factor beta receptor2gene TGFBR2can also present similar symdrome. Using targeted semiconductor sequencing, we attempted to confirmed genotypes of the clinical indentified MFS patients, the purposes of which were to validate the clinical diagnosis, and to test the coverage rate and accuracy of FBN1and TGFBR2genes with the rapid and cost-effective measure.Methods:Two unrelated MFS patients were involved in the study, with confirmed clinical diagnosis according to revised Ghent nosology. Written consents were gave from them to participate in this study. Peripheral blood samples were acquired and maintained in anticoagulation manner. A MFS-specific targeted resequencing panel including was designed by on-line tool Ion AmpliSeq Designer; Genomic DNA was isolated from anticoagulated whole blood leukocytes. Ion torrent adapter-ligated libraries of MFS patients were prepared. Subsequent emulsion PCR and enrichment of the sequencing beads of the pooled libraries was performed using the OneTouch2system. Sequencing was performed on Ion PGM. Raw data were processed with the Ion Torrent platform-specific software Torrent Suite v4.2.1and analyzed by various bioinformatic on-line tools.Results:By targeted sequencing of the two candidate MFS genes in the probands of the two pedigrees, an average output of589714mapped reads and89.87%on target per sample were achieved. The average readdepth in target region was794folds. Nineteen known or novel variants were detected, of which16were assigned in one patient and15in another patient. Of these19variants, twelve are annotated as noncoding and2are annotated as synonymous, whereas5are non-synonymous, including3missense mutations,1nonsense mutation and1frameshift-insertion. Conclusion:It is practicable of targeted sequencing FBN1and TGFBR2genes of MFS on Ion torrent PGM with specific designed panel. The measure is rapid and accurate. Part2, sanger-based sequencing validates the next-generation resequecing resluts and filles the gap.Objective:Sanger-based sequencing was performed to fill in the technical gaps of uncovered regions of the two targeted genes. All mutations detected by next generation sequencing were validated by using of Sanger-based sequencing. The results of Sanger-based sequencing and next generation sequencing were compared, by which the accuracy of next generation sequencing on multi-exon genes of FBN1and TGFBR2were tested. All pathogenic and putative likely pathogenic novel mutations were validated by direct Sanger sequencing in400matched healthy controls and family member of proband.Methods:The clinical information was collected from MFS probands and their consanguineous relatives. The patients characteristics were normally recorded in manner of questionnaire form, and a completed pedigree tree was drawn for each family respectively. The patients and their relatives were carefully examed and scored according to revised Ghent nosology. Seven milliliter of periphery blood sample was obtained from every participant, and with400anonymous healthy adult controls the genomic DNA samples were simultaneously prepared. The written forms of consents were given from all participants. Sanger sequencing was performed on uncovered regions and for mutations validation. After sequencing procedure, the software Chromas3.0was employed to switch raw data into visible form, and then Dnaman v6.0.40was employed for comparing the sequencing results. The detected mutations were validated, and genotype-phenotype relationships were analyzed.Results:By Sanger sequencing, no mutation was indentified in uncovered regions of genes FBN1and TGFBR2. The mutations detected by next generation sequencing were all confirmed by Sanger sequencing results. We identified two pathogenic mutations in four MFS patients of the two pedigrees, including a novel frameshift insertion p.G2120fsX2160and a reported nonsense mutation p.Arg529X (rs137854476) in FBN1gene. Moreover, we revealed a rare probably-benign Chinese-specific polymorphism c.913A>G, p.Thr305Ala in FBN1gene.Conclusion:The availability of next generation sequencing for MFS causative genes FBN1 and TGFBR2were validated by Sanger sequencing. The potential MFS patients in our study were confirmedly diagnosed by causative mutations in gene FBNl. Part3, moderate hypothermic circulation arrest in the total aortic arch replacentment for patients with type A aortic dissectionObjective:type A aortic dissection is the most dangerous complicate of MFS. We employed the technique of total aortic arch replacement plus elephant trunk procedure to correct the involved aorta. We have also made some changes of the manner, e.g. elevation of the hypothermic temperature during circulatory arrest period to a relative moderate level (nasopharyngeal temperature20.1-28℃) with low flow of antegrade cerebral perfusion. We conduct this study to assess the risk factor of operation manner, and to evaluate the outcome of operation for MFS patients.Methods:from2010to2013, the perioperative clinical data of99patients were collected continuously. The patients were assigned into two subgroups according to the hypothermic levels during circulatory arrest, data of the two groups were compared. Categorical variables were compared using the χ2test or Fisher’s exact test. Continuous variables were compared using an unpaired t-test. Stepwise multivariable logistic regression was used to assess the potential risk factors of30-day mortality and neurological adverse events (temporary neurological dysfunction and permanent neurological dysfunction). Adjusting parameters and variables of known clinical importance for the binary dependent variables (age, gender, hypertension, diabetes, emergency surgery, body mass index, duration of operation, preoperative LVEF%, hepatitis B virus, duration of CPB, circulatory arrest, coronary artery disease, preoperative shock, preoperative dialysis, circulatory arrest over40minutes, hypothermia groups, consumption of red blood cells (RBC) and fresh frozen plasma) were included in the multivariate logistic regression models.Results:All involved patients underwent treatment of total arch replacement plus elephant trunk procedure. Bentall procedure was performed for33patients, which with the proportion of84.6%was the main manner for aortic root repair. Morbidity of temporary neurologic dysfunction was17.2%. Seventeen patients died in30days postoperation. Mean operation duration was508.8±80.0min, mean cardiopulmonary bypass time was227.9±37.3min, mean cross clamp time was127.6±23.6min, circulatory arrest time was29.9±6.0min. With regard to the patients of MFS, all patients were cured discharged. One patient suffered from TND after operation. By multi-variable analysis, we indentified deep hypothermia during circulatory arrest was independent risk factor for neurological adverse events (P=0.031; OR=0.385;95%CI=0.162-0.919). During the fellow-up period of1-4years, one patient was reintervened for recurred type B aortic dissection, no patients died.Conclusion:the total aortic arch replacement combined with elephant trunk procedure can be safely and effectively performed on MFS patients. The moderate hypothermia manner could decrease the morbidity of postoperative neurological adverse events. |
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