Study On Pathogenesis Of Fanconi Anemia And Its Related Diseases By High - Throughput Analysis Technique

Objective:Fanconi anemia (FA) is a rare recessive disorder associated with chromosomal fragility, aplastic anemia, congenital abnormalities and a high risk of cancer, including acute myeloid leukemia and many solid tumors. Due to the identification of 15 different FA genes which guard a complex web of interacting proteins that are involved in the recognition or repair of DNA interstrand crosslinks damage, FA patients become ideal models to investigate genetic changes which have been harbored, accumulated and could eventually led to oncogenesis. In this study, we applied whole exome sequencing to reveal some of these genetic alterations with cancer-initiating potentials.Methods:The whole blood cells from 5 diagnosed FA patients and each of their parents have been collected and used for pair ends whole exome sequencing (depth>50X) by Illumina HiSeq 2000. By mapping towards human genome reference data, analyses were performed to reveal FA specific base pair mutations and indel mutations. After filtering out the shared mutations between each patient’s parents respectively, cancer related & DNA damage repair related annotation and pathway enrichment analyses were applied. We choose some of these mutations for traditional PCR validation, and cellular functional experiments in order to identify their roles of oncogenesis.Results:By extracting 15 FA genes’ sequence from whole exome sequencing data of 5 diagnosed FA patients and their parents, traditional PCR validation have been applied to reveal mutation status of these people. We identified that each patient harbored one or more mutations of these 15 FA genes with various mutation types including single nucleotide mutation (missense mutations and nonsense mutations) or indel mutations (frameshift mutation) and compound heterozygous mutations appear to be the majority, rather than widely identified homozygous mutation in foreign FA patients. FANCA mutations were shared by CYH, WFY, WSW, KLP four patients with compound heterozygous mutations, homozygous mutations and frameshift mutations types while FANCD2 and X-linked FANCB mutation has only been identified in patient CYH and WFY with compound heterozygous mutations and homozygous mutation type respectively. Besides, compound heterozygous mutations of FANCM, FANCI and widely recognized breast cancer susceptibility gene BRCA2 were shared by several FA patients simultaneously. Through further investigation, these mutations may not only led to mis-translation or mis-termination of FA protein complex, the mutations located in 31th intron and 15th & 21th exons of FANCA may probably also affect its side splicing. In addition to these 15 known tumor susceptibility genes, we have screened the mutations involved in cancer-associated and DNA damage & repair related pathways after filtering out the dbSNP data and identified the mutations from one or more FA patients in FAM109A, VANGL2, DNAH7, ZNRF4, DNAH2, UNC13D, UBB, ZNF43, ZNF93, LAMA3, ALS2, ACACA genes which involved WNT signaling, ECM-receptor interaction and Insulin signaling pathways et al and also play an important role in Familial hemophagocytic lymphohistiocytosis (FHPL), and Huntington’s disease’s progression in one or more of FA patients. In particular, mutation of ACACA was shared by four FA patients, and this gene participate in both fatty acid biosynthesis & metabolism pathways and cancer related insulin signaling pathways may indicated its important role in mechanism of FA specific bone marrow failure and high risk for multiple cancers.Conclusions:Our study represents the first systematic genetic study on Chinese patients of this rare recessive disorder FA. Unlike the widely reported homozygous mutation in foreign FA patients, we revealed the mutation statuses which include a variety of protein function affected mutation types associated with multiple FA genes in each patient. Through screening of their accumulated mutations of oncogenesis-associated genes, we found these mutations are almost randomly presented in each patient and this may theoretical support the tumor’s multi-hits hypothesis. However, the enriched mutation from 4 patients in ACACA gene which could regulate both bone marrow failure associated lipid metabolism and oncogenesis related insulin signaling pathways may play an important role and server as a new feature in high tumor susceptibility of FA patients. Further investigations on the correlations between these already discovered genetic abnormalities and clinical manifestations & severity in a large cohort of FA patients are needed.Abstract:Fanconi anemia (FA) is a rare recessive disorder associated with chromosomal instability, aplastic anemia, congenital abnormalities and a high risk of cancer, including acute myeloid leukemia and many other solid tumors. Although 15 disease-causing genes of FA have been found over the past several years, the mechanisms of its tumor susceptibility were seldom revealed. By using Gene set enrichment analysis (GSEA) to compare gene expression profiles between 21 FA patients’ myelomonocyte and 11 normal controls in cancer related gene sets, we revealed the significant enrichment in resistance to BCL2 inhibitor gene set, phospholipase c mediated fibroblast growth factors signalling pathways and Insulin & insulin-like growth factors signalling pathways induced cancer genesis gene sets. Further investigation were performed to analyze the enrichment statuses in bioprocess of Gene ontology gene sets and Structural Genomic gene sets and FA specific expression signature was generated. Through above researches, it could be concluded that the high tumor susceptibility of Fanconi anemia patients may closely dependent to their dramatically changes in cancer related growth factors and hormones environment. Thus this study provided new information for investigation of tumor susceptibility and cancer genesis in Fanconi anemia patients.