Study On Full Exon Sequencing Of Focal Nodular Hyperplasia Of Liver

BackgroundFocal nodular hyperplasia (FNH) is the second most common benign liver tumour only after liver haemangiomas. It is most often found in females between the ages of 30 to 50 with the female-to-male ratio between 8:1 to 12:1. Most cases were asymptomatic and discovered incidentally on routine abdominal imaging. Meanwhile, it was difficult to determine whether the symptoms are directly attributable to the lesions. The classic histopathological description of FNH was a non-encapsulated nodule with a central fibrous region, from which there were several radiating septa dividing into the nodules of hyperplastic hepatocytes. The central fibrous regions usually contained abnormal vessels with different sizes, as well as proliferating bile ductules. Atypical FNH accounted for 20% approximately, because of no central fibrous scar. The subtypes comprised of the telangiectatic FNH (tFNH), the mixed hyperplastic and adenomatous forms, and cytologic atypia. The most accepted etiological theory considered a vascular malformation as the trigger event. There was no documented case of transforming into a malignant lesion by histologically proven. Therefore it was regarded with no malignant potential. Most of the literatures using with clonal analysis suggested the polyclonal origin of FNH. However recent pathological series showed that 75-100% of tFNH lesions were monoclonal in nature, whereas 100% of typical FNH lesions polyclonal and 100% of hepatocellular adenoma lesions monoclonal. There is a dilemma for differentiating the atypical FNH from hepatocellular adenoma (HCA) and fibrolamellar subtype of hepatocellular carcinoma (FLHCC) in clinic.Next generation sequencing (NGS) is a high-throughput sequencing technology. Advantages of NGS are high sensitivity, high sequencing efficiency and relatively low cost. Whole exome sequencing is based on the platform of NGS technology and can comprehensively screen for mutations of whole exome which is the important coding domain of genes. Due to application of whole exome sequencing technology, oncology research has made a great breakthrough.ObjectiveThe purpose is to explore FNH related mutations by screening whole exome, to understand functions of the related genes in etiology, pathogenesis and biologic behaviors of FNH and to solve difficulties in differentiation from other hepatic lesions.Materials and Methods1. Selection of FNH patients for exome sequencingWe chose five patients who underwent hepatic lesion resection in Department of Hepatobiliary Surgery of Chinese Academy of Medical Sciences Cancer Hospital. The histologic diagnoses were all FNH. The resected tissue and blood samples were prepared by the rules.2. Workflow of exome sequencingAccording to the workflow of whole exome sequencing, we finished purification of gDNA from tissue or blood samples, Sanger sequencing of HLA gene to ensure paired samples, individual library preparations, hybridizations, captures, pooling and sequencing samples and bioinformatics analysis.3. Data analysisWe studied hotspot gene researches and biologic function of these genes by reviewing literatures. Based on the characteristics of gene function and mutation frequency, we selected candidate genes for validation.4. ValidationWe performed Sanger sequencing to verify mutations in candidate genes.ResultsBio-informatics analysis result of whole exome sequencing showed 158 mutations in five FNH tissue samples. ARID2, EGFR, TRPS1 and DAXX were selected as candidate genes and five mutations underwent validation because two mutations of TRPS1 were discovered separately in two samples. Sanger sequencing result showed that four mutations were valid, including missense mutation in ex on 14 of ARID 2, frame shift deletion in exon 6 of EGFR, missense mutation in exon 3 and nonsense mutation in exon 5 of TRPS1.ConclusionMutations of TRPS1 were discovered in two samples which meant TRPS1 might have a high mutation frequency in FNH and that TRPS1 might play a role in pathogenesis of FNH. Interestingly, anti-fibrosis function of TRPS1 was reported in several researches while classic histopathological description of FNH is central fibrous scar. The relationship of TRPS1 and FNH remains to be investigated in further researches.