Study On The Molecular Mechanism Of Fanconi Anemia By Whole Exome Sequencing

ObjectiveFanconi anemia is a rare genetic disease, characterized by bone marrow failure, congenital malformations, cancer, and hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C. FA has been found 15 susceptibility genes, all involved in maintaining genome stability. But 1/3 patients cannot find Fanc genes mutation, and gene knockout mice have no obvious FA phenotype, suggesting the existence of new FA genes and more susceptibility genes may have cooperative activity. The main objective is to find Fanc gene mutations and new gene mutations, to clarify their synergy effect in causing bone marrow failure.MethodsThe exome of 5 patients of Fanconi anemia and their parents were Sequenced using two-terminal Illumina HiSeq 2000. The sequencing depth was 50×. By bioinformatics analysis, single nucleotide variation insertion?deletion in Fanc gene will be got by comprising with the human genome database, new mutations in other genes with growth will be got by comparising with their parents' exome. Combining high-throughput sequencing and traditional molecular biology, cell biology, animal models and other studies, ultimately clarify the mechanism of the synergy between genes mutation and bone marrow failure.ResultsFive patients were diagnosed by Clinical manifestations, single-cell gel electrophoresis and MMC test. By exome sequencing data analysis all 5 patients showed some of mutations in 15 known Fanconi anemia genes. The gene mutation types included Single nucleotide mutations (missense mutation and a nonsense mutation), or insertion deletion (frameshift mutation). Single nucleotide mutations included homozygous mutations, heterozygous mutations, complex heterozygous mutation (one gene has multiple mutations), resulting in errors or termination of protein translation. Fanc mutations have different types, ways and numbers, and have no correlation with clinical manifestations and severity, suggesting that the mechanism of FA caused by Fanc mutation is complex. For example, patients WFY have 3 genes mutation, the BRCA2 and FANCA had homozygous mutations, while in FANM gene find complex heterozygous mutations. In the mean while patients WSW only have the GG heterozygous deletion in FANCA causing a frameshift mutation, and the mutation was inherited from his father, but his father have no clinical manifestations of FA, suggesting that he may have unknown mutations. In addition to Fanc genes, comparing with the database, and remove the SNP inherited from their parents,137-170 novel mutations were found per patient, Some gene mutations are shared by many patients, so FA patients continue to accumulate mutations due to defects in DNA crosslink repair during postnatal growth. Analysis of the Regulation by KEGG analysis found that Fanconi anemia mutations involve in the network of various synthetic pathways, apoptosis pathway, ubiquitination pathway, the calcium signaling pathway, WNT signaling pathway, Notch signaling pathway, insulin signaling pathway, B cell receptor signaling pathway, mTOR signaling through, MAP K signaling pathway, VEGF signaling pathway and T cell receptor signals.Conclusion1) Fanc mutations have different types, ways and numbers, and have no correlation with clinical manifestations and severity, suggesting that the mechanism of FA caused by Fanc mutation is complex.2) 137-170 novel mutations were found per patient, some gene mutations are shared by many patients, so FA patients continue to accumulate mutations due to defects in DNA crosslink repair during postnatal growth.3) The Fanconi anemia gene mutations affect a variety of anabolic pathways, apoptosis pathway and ubiquitination pathway. ObjectiveIdentifying of unknown MLL fusion partner gene, find the Coordination mechanism between the MLL fusion gene and the synergy gene.MethodsGot pure leukemia cells by Flow sorting. Using whole genome sequencing, whole transcriptome sequencing and epigenetic modification, detect unknown MLL fusion partner gene, identify the genetic differences between monozygotic twins including SNVs, small InDels, structural variations and alternative splicing. The results were PCR amplified and sequenced, and test the mutation in larger sample of patients with acute leukemia. The genes that may participate in leukemia will be verified functionally, and ultimately determine their role in the development of leukemia.ResultsAfter comprehensive clinical manifestations with bone marrow morphology, flow leukemia immunophenotyping and FISH results, the patients were diagnosed as 11q23/MLL acute myeloid leukemia. STR-PCR analysis identified the two sisters are monozygotic twins. Using Solid pair-ended whole-genome sequencing and Solexa pair-end the whole transcriptome sequencing, the data were analysised by bioinformatics software, the sick sister genome and transcriptome found two fusion genes MLL-NRIP3 and NF1-AARSD1, and MLL-NRIP3 have 3 types of splice variants, while healthy sister did not find any fusion gene. Comparing the twins'genome and transcriptome data with the database,23 mutations were found. Divided by type of mutation, of which one is nonsense mutations,4 are splice sites,18 are missense mutations. Divided by ways of mutation,5 cases are homozygous mutations, and 18 are heterozygous mutations.Conclusion1) After comprehensive clinical manifestations with bone marrow morphology, flow leukemia immunophenotyping and FISH results, the patients were diagnosed as 11q23/MLL acute myeloid leukemia.2) The sick sister genome and transcriptome found two fusion genes MLL-NRIP3 and NF1-AARSD1, and MLL-NRIP3 have 3 types of splice variants, while healthy sister did not find any fusion gene. 3) Comparing the twins'genome and transcriptome data with the database,23 mutations were found. ObjectiveIdentification of the mutation gene in a family X-linked sideroblastic anemiaMethodsEach ALAS2 exon was PCR amplified and sequenced, analysis whether there are mutations. If a known gene mutation is not detected, then using DNA sequencing to find shared mutation, the mutant gene will be sequenced in other 2 patients and 4 cases of specific gene carriers. Detected the gene sequence in three cases who may be potential earners.Results and conclusionsTo determine the nature of the mutation in the ALAS2 gene causing family XLSA, genomic DNAs were isolated from two affected patients, and each ALAS2 exon was PCR amplified and sequenced. No nucleotide changes were found by sequencing each of the regional, including intron/exon boundaries,1 kb of 5'flanking and the 8th intron sequence. The expression level of ALAS2 gene is normal. No nucleotide changes were found by sequencing each of exon of ABC7 gene.Speculated that the causative gene mutation is unknown.