Genome-wide DNA Methylation,RNA And Exome Sequencing Studies Of Pituitary Adenomas

Background and ObjectivePituitary adenomas(PAs)are commonly occurring neoplasms that cause a variety of neurological and endocrine effects.Although alterations of the hypothalamic hormonal influence,heredity and somatic mutations,and epigenome modifications were contributed to the etiology of PAs,the pathophysiologic mechanisms driving tumorigenesis and invasion of sporadic PAs remain unknown.In this study,we explored the genome and epigenome alterationsin PAs,including 1)Genome-scale DNA methylation studies to investigate associations among methylation statuses and PA invasion and histopathology subtype;2)A high-throughput RNA-Sequenigto exploregene fusion and gene expression in different subtypes of PAs;3)A genome-wide exome sequencing for gene mutation detection,in order to find out the altered genes which may account to PA tumorigenesis and development.MethodsTwenty-four sporadic pituitary macroadenomas,including six functional PAs and eighteen nonfunctional PAs were enrolled in this study.More specifically,these included 17 nonfunctional adenomas,five somatotroph adenomas,one corticotroph adenoma,and one silent corticotroph adenoma,which were collected from LAC-USC hospital and Keck medical school hospital,and were assigned dichotomized Knosp invasion scores.Whole genome DNA and RNA were purified and examined using Illumina HGM450 methylation array to investigate genome-wide DNA methylation Pattems.RNA sequencing,and whole exome sequencing using Illumina Hi-seq 2000 platform.Subsequently,MethyLight and Sanger sequencing were used for DNA methylation and mutation validation.ResultsThis study detected the genome and epigenome genetics for PA tumorigenesis and illustrated that 1)PA samples clustered into subgroups according to functional status.Compared with hormonally-activePAs,nonfunctional PAs exhibited global DNA hypermethylation(mean beta-value 0.47 versus 0.42,P=0.005);the most significant site of differential DNA methylation was within the promoter region of the potassium voltage-gated channel KCNAB2(FDR=5.11 x 10-10).Two enhancer-region CpGs in invasion-associated genes(FLT1 and SLIT3)were significantly hypomethylated in invasive compared to noninvasive nonfunctional PAs.Pathway analysis of promoter-associated CpGs showed that nonfunctional PAs are most closely associated with the ion-channel activity signal Pathway(FDR<0.25).DNA hypermethylation tended to be negatively correlated with the gene expression.2)Gene fusion is rarely detectable in PAs.3)Recurrent insertion mutation in exome 5 of the ZNF gene could be found in PAs.ConclusionWe firstly applied infinium HumanMethylaiton450 BeadChip to detect genome-wide DNA methylation in PA patients.This study displayed the profile of DNA methylation of PA.And prelimitly illustrated the significant difference between functional and non-functional PAs,and the non-functional PA was hypermethylated in the genome-wide when compared with the functional PA,and we found that DNA hypermethylation of KCNAB2 and downstream ion-channel activity signal pathways may contribute to the endocrine-inactive status of nonfunctional PAs.In this study,we proceeded a deep detection for the difference DNA methylation analysis between invasive and non-invasive PAs,and found that there was no significant DNA methylation difference between invasive and non-invasive PAs,which may imply that the weak association of DNA methylation with invasive phenotype.Meanwhile,in this study we proceeded the gene enrichment analysis between the functional and nonfunctional PAs,and illustrated that the genes enriched in ion-channel activated pathway were hyper-methylated in non-functional PAs,and informed that DNA methylation played an important role in the pathway regulation in non-functional PAs and resulted in different founction phenotypes of PA.The RNA-Seq study illustrated that DNA hypermethylation was negativeassociated with mRNA expression in PA,and displayed the difference expression level of mRNA between functional and non-functional PAs.Meanwhile,we used the RNA-Seq data to detect gene fusion in PAs,and found that it was rare in the begnin tumors of PA,but we confirmed a long intergenic transcription between geen PBXIP1 and PMVK,which illustrated the specific genetic characters of PA.Meanwhile,this is the first time to use whole exome sequencing for PA novel mutation detection,and we firstly found the TGTG insertion mution in gene ZNFwas recurrent in PAs.In summary,this study completed the genome study in PA in genome-scale DNA methylation,RNA expression,gene fusion and gene mutation,which provided the basic genetic theories for PAs etiology research and furtherclinical diagnosis and therapy.