Part1Function Of DNAH2in Fa Nconi Anemia Pathway Design And Preliminary Validation Of Inherited Bone Marrow Failure Disease Diagnosis Kit

Backgrand:Fanconi anemia (FA) is a rare autosomal recessive or X-linked genetic disease characterized by progressive bone marrow failure, congenital abnormalities and cancer susceptibility. FA is the most frequent inherited bone marrow failure diseases, which has an incidence of about1-5per million live births. To date,15FA genes have been identified in FA pathway and all involved in maintaining genome stability. Identification the subtype of FA is very important because it is associated with diagnosis, clinical treatment and prognosis of FA patients. The traditional complementation test and FA mutation gene sequencing analysis for grouping studies has been used mostly in FA patients, rather than among their families, and in a stepwise method. More complex changes of FA genes in the same patient are plausible as one subtype of FA patients may have different clinical manifestations. In addition,5%of individuals diagnosed with Fanconi anemia have no any mutations of FA genes, which indicates that existence of new mutation genes or cofactors participate in pathogenesis of Fanconi anemia. To gain new insight into FA pathogenesis and the molecular basis underlying the clinical heterogeneity of FA, we carried out exome sequencing and subsequent Sanger sequencing analysis in five FA families. The results showed that all five patients had FANC gene mutations. At the same time, we found DNAH2, DNAH7and UNC13D point mutations occurred at two or three patients simultaneously. We investigated whether these genes involed or not in pathogenesis of FA and the exact mechanism.Objective:1) Screening cofactor genes from mutation genes;2) Studying new mutation genes’effect for FA pathway;3) Providing a new theoretical basis for the pathogenesis of FA.Methods:1) Reducing Decreaseing the mRNA expression of DNAH2, DNAH7and UNC13D genes by RNA interference technique and testing the efficiency by RT-PCR method;2) Screening genes that participate in DNA repair pathway by homologous recombination (HR) technique.3) Studying gene’s effection for ICL repair pathway by immunofluorescence microscopy;4) Studying the gene’s effection for ubiquitination of FACND2by Western blotting;5) Testing cell proliferation by MTS method. Results:1) RT-PCR results showed that the expression level of tested genes are decreased;2) DNAH2and UNC13D participated in HR pathway;3) DNAH2affected ubiquitination of FANCD2;4) DNAH2affected DNA repair by decreasing the accumulation of FANCD2to chromatin;5) DNAH2knockdwon cells are sensitive to DNA crosslink agents (MMC sensitivity results).Conclusion:1) DNAH2and UNC13D participate in HR pathway;2) DNAH2affects ubiquitination of FANCD2and probably participates in pathogenesis of FA;3) DNAH2affects DNA repair by decreasing the accumulation of FANCD2to chromatin. Backgrand:Inherited bone marrow failure syndrome (IBMFS) is a group of hereditary diseases. Typical clinical manifestations are the important diagnosis basis. However, about1/3patients with IBMFS are difficult for diagnosis for lack of specific clinical manifestations. Furthermore, there are false positive results even using some specific experiments, such as MMC and cell cycle tests. In recent years, sequencing related genes that were chosen based on clinical manifestation to diagnosis IBMFS have been used in clinic. However, this method might miss some information and cause false negative results. Furthermore, Sanger sequencing is expensively and difficult to be applied widely. High-throughput sequencing also named as Next-generation sequencing technology is a milestone techenique in genome research field. Single-base cost of the High-throughput technology is cheaper obviously than the Sanger sequencing, which make its possible to be used widely. Recently, High-throughput sequencing capture technology also named Targeted Resequencing has been used widely in clinic, which combined with High-throughput sequencing technologies and microarray technology. This method included three procedures. Firstly, synthesize of oligonucleotide probes by microarray technology, which can bind to specific regions of the genome. Secondly, sequence the probes that bind to the genome by second generation sequencing technology. Finally, analyze the results of samples by bio informatics method. High-throughput sequencing has been used widely to search for candidate genes and disease diagnosis. For difficult diagnosis and expensive sequencing of children with IBMFS in clinic, we try to design IBMFS chip diagnostic kit, captured by High-throughput sequencing technology and to provide comprehensive, fast and economic method for the diagnosis of IBMFS.Objective:1) Design gene chip diagnosis kit for the diagnosis and differential diagnosis of IBMFS;2) Validate the genes obtained from the first part of FA patient study;3) Summarize and analyse the sequencing results, to find similarities and differences among different kinds of IBMFS patients samples.Methods:1) Extract peripheral blood DNA;2) Capture and sequence DNA samples by High-throughput sequencing technology;3) Compare the mutation genes with preliminary validation results which was done in the first part of study shown.Results:2FANCD2gene mutation sites,1DNAH7and1UNC13D gene mutation sites were found in patients with FA, which had been validated in the first part of study. However, there are3mutation sites have not been detected. They are FANCA, FANCD1and FANCD2.Conclusion:IBMF gene chip diagnostic kit has clinical value. But it needs to be optimized for the experimental conditions and bio informatics analysis method before being used as clinical application.