Mutation Screening Of Pathogenic Genes In A Large Pedigree With Hereditary Gingival Fibromatosis

Background and ObjectiveHereditary gingival fibromatosis(HGF),a rare benign oral disorder with unknown etiology,is mostly family-aggregated,so it is also known as familial gingival fibromatosis.The main manifestations are diffuse and progressive hyperplasia of the gums.The conditon can involve the gingival margin,gingival papilla,and attached gums of the full mouth.The hyperplastic gums can cover part or the entire crown,frequently accompanied by retained deciduous teeth,delayed permanent germination,teeth dislocation and malocclusion,which seriously affects the patient's chewing,beauty and pronunciation,and makes the patient feel inferior.Clinically,it is characterized by benign gingival enlargement with normal color and firm consistency and non-hemorrhagic symptomatic diseases.The incidence of HGF has been described as one in 175 000,both genders being equally affected.An autosomal dominant pattern of inheritance is frequent,although some pedigrees also support an autosomal recessive inheritance.A few cases showed sporadic cases with no pedigree and the penetrance rate is variable.Until now,five subtypes related to HGF have been described,including 2p21-p22(GINGF1),5q13-q22(GINGF2),2p22.3-p23.3(GINGF3),11p15(GI-NGF4),4q12(GINGF5),corresponding to different candidate susceptibility.However,only two genes were clearly associated with the disease:SOS1 and REST.These studies fully show that this rare condition is genetically heterogeneous and currently.Therefore,this study intends to find the pathogenic genes for the physiological development of gingival tissues by surveying a large pedigree of HGF and analyzing the clinical characteristics of patients,mutation detection of reported pathogenic genes,whole exome sequencing and co-segregation of pedigrees to find pathogenic genes.It provides new clues for the physiological development of gingival tissues,and provides new theoretical basis and new research targets for the clinical diagnosis and treatment,genetic counseling and prevention of HGF.Materials and MethodsPart I:HGF pedigree investigation and Glinical characteristics of patientsThere are 48 members in 4 generations in this pedigree,and 19 patients were diagnosed by clinical examination.The patient's general health status and oral specialty examination were conducted,and the history of medication was asked to exclude gingival hyperplasia caused by the drug and systemic diseases.By collecting the gingival tissue removed by the patient during gingival resection treatment.HE and Massion staining were performed to observe the histological changes of the patient and confirm the pathological diagnosis.Part II:Reported mutation detection of pathogenic genesBy phenol/chloroform method collected peripheral venous blood of pedigree members,then extracted genomic DNA and designed primers by NCBI.Using PCR to amplify all exon regions of SOS1 and REST genes,and directly sequence in both directions.By Using DNA Star-Seqman software to compare each sequencing result(with the gene exon sequence queried on the UCSC website),and then the sequencing peak map is checked for abnormal changes.Part?:Whole exome sequencing of HGF pedigree samplesThe whole genomic DNA of ?-6,?-6 and ?-10(?-? represents the generation number of the pedigree)were extracted in the HGF pedigree.After testing the DNA concentration,purity and integrity,it was handed over to Novogene(Beijing)Biological Co.,Ltd.for high-throughput exome sequencing.This technology platform uses the Aglient SureSelect exon targeting sequence enrichment system to capture the entire exon sequence,and then high-throughput sequencing of the capture library using Illumina HiSeq2000.ResultsClinical features of HGF patients:Most patients have slightly protruding lips,and the hyperplastic gums touch each other when they are biting.The permanent teeth were completely erupted along with arrange disorder due to widely progressively hyperplasia of the gums.The full-mouth dentition has obvious hyperplasia in the posterior tooth area,and the gums appear nodular hyperplasia to the buccal and lingual side.But the anterior teeth hyperplasia of adolescent patients is relatively heavy.Clinically,it is benign gingival enlargement with normal consistency and non-hemorrhagic symptomatic illness.A pink gingiva with marked stippling can be seen to cover almost all the tooth.Pathological features of patients with HGF:HE staining of gingival tissue showed thatthe squamous epithelium thickened,and the nail process was significantly extended into the lamina propria.There are a lot of dense and hyperplastic collagen fiber bundles in connective tissue,and there are few components of fibroblasts and blood vessels.Massion staining showed that the patient's gingival connective tissue was filled with thick,neatly arranged collagen fiber bundles,and its collagen content was significantly richer than normal people.Mutations screening of pathogenic genes:The template DNA concentration of the whole blood sample extracted by phenol/chloroform method is greater than 200ng/?l,and A260/A280 is between 1.8 and 2.0.The concentration and purity of genomic DNA meet the requirements of subsequent experiments.The specific primers of all exon regions of SOS1 and REST genes were designed for mutation verification.The result that no mutations were found in the reported pathogenic sites.In addition,SOS1 and REST genes were also excluded from the whole exome sequencing results.We screened more than ten candidate disease-causing genes were confirmed to be single nucleotide polymorphisms(SNPs)by co-segregation and verification of the pedigree.Conclusions1.According to the clinical manifestations and pathological changes,it is not difficult to diagnose the hereditary gingival fibromatosis.However,it is genetically heterogeneous greatly complicates the study of pathogenic mechanisms.2.The SOS1 and REST genes are currently pathogenic genes that have been successfully cloned and identified.However,they are not "mutation hot spots" in this pedigree.3.The disease-causing gene of this pedigree may not be located in the known gene coding region,but in the highly repetitive sequence,flanking sequence or intron of the gene.