Gene Mutation Screening Of A Family With Paget’s Disease Of Bone And Candidate Genes Fuction Study

Paget’s disease of bone(PDB) is a chronic progressive metabolic disease of bonecharacterized by focal areas of increased and disorganized bone turnover, resulting inbone pain, deformity, pathologic fracture and other complications. Bone lesion cantypically affect one or multiple bones in the axial and weight bearing regions of theappendicular skeleton. The histological hallmarks include the presence of giantmultinucleated osteoclasts that are actively resorbing bone in bone lesion. PDB isdivided into sporadic PDB and familial PDB. It is common in the United Kingdomand other countries in western Europea and north America. The cause of PDB is notvery clear, but it is generally believed that environmental and genetic factors affect theincidence of PDB. The PDB’s prevalence of the immigrants from high prevalenceareas is significantly higher than that of natives, such as New Zealand, Australia andSouth Africa. The prevalence differences among races and familial PDB at the sametime demonstrate the important role of genetic factors in the pathogenesis of PDB.Many patients of PDB have positive family history. Familial PDB is usuallyautosomal dominant inheritance, incomplete penetrance. In recent years, with thecontinuous development of molecular genetics techniques, there are more furtherstudies of the genetic etiology of PDB. Recent study found that the gene mutationsabout RANK/RANKL and NFkB signaling pathway such as SQSTM1/p62genemutations are associated with abnormal activation of osteoclasts and PDB’soccurrence. However, these mutations are not found in more than half cases, suggesting that other gene variants may be involved in its genesis and development.ObjectiveBased on the detection of known susceptibility genes on the pedigree of PDB,using genome-wide scan and sequencing analysis, we screened all patients of thefamily and identified two gene mutations in ASB16and FKBP5. We did somefunction research about the mutations. The purpose is to explore its role in thepathogenesis of PDB, and lay the foundation for an in-depth understanding of thegenetic mechanisms of its incidence.Methods1. SQSTM1and TNFRSF11A gene mutation screening: Genomic DNA wasextracted from the blood samples of the patients and unaffected members of thepedigree. SQSTM1and TNFRSF11A genes were determined as candidate genes byreviewing literature. Primers for all the exons of the two genes were designed anddirect sequencing were performed after Polymerase chain reaction(PCR) to detectmutation. Then results were compared with GenBank through software Sequencher5.0Demo.2. Research on the promoter activity of ASB16: The G/C mutation was identified at5’end+27of ASB16through whole-exome sequencing on patients’ genome. Toobtain the genotype frequency of target gene site by screening normal samples. Thecandidate promoter sequence of ASB16was determined through promoter predictionsoftware. The gene fragment of ASB16after PCR was inserted into pMD-18T cloningvector. The wild and mutant constructed cloning vector were identified by colonyPCR screening and sequencing analysis. The target gene fragment was separatedafter digesting the recombinant cloning plasmid pMD-18T/ASB16by NheⅠ andHind Ⅲ restriction enzyme digestion, inserted into luciferase reporter vectorpGL3-basic. The positive recombinant plasmid pGL3/ASB16and internal controlplasmid pRL-TK were co-transfected into HEK293cells by liposome. The activity ofASB16candidate promoter was observed by detecting the luciferase activity of recombinant luciferase reporter vector in cells after48h. The result was analyzed bystatistical software SPSS17.0.3. Cloning and expression of the human FKBP5gene and purification: The G/Cmutation was identified in cDNA of FKBP5through whole-exome sequencing onpatients’ genome. Primers were designed according to the target gene sequence andrestriction enzyme sites and His-Tag was added. The wild and mutant target genefragment of FKBP5after PCR was respectively inserted into pMD-18T cloning vector.The target gene fragment was separated after digesting the recombinant cloningplasmid pMD-18T/FKBP5by Nco Ⅰ andBamHⅠ restriction enzyme digestion,inserted into expression vector pET-11d. The wild and mutant positive recombinantplasmid was respectively transformed into E.coli BL21, the monoclonal colony waspicked and cultured to logarithmic phase, and then induced by IPTG. The recombinantprotein was identified by SDS-PAGE and Western blot. The human protein FKBP5was purified by Ni2+-NTA affinity chromatography under non-denaturing conditions,then was analyzed by SDS-PAGE.Results1. No mutation but14previously reported single nucleotide polymorphisms(SNP)were identified in the SQSTM1and TNFRSF11A genes.2. Result of research on the promoter activity of ASB162.1the genotype GC and GG frequency of ASB16target site is respectively2.74%and97.26%in screened normal samples.2.2The amplification product of ASB16candidate promoter can be seen a brightelectrophoretic bands at463bp, confirming to the design. The candidate promoterfragment was connected with linear cloning vector pMD-18T, then positiverecombinant cloning plasmids were identified by colony PCR screening and DNAsequencing. We have successfully obtained the wild and mutant cloning vectorpMD-18T/ASB16. The wild and mutant candidate promoter fragment wasrespectively inserted into luciferase reporter vector pGL3-basic, then positive recombinant reporter plasmids were identified by colony PCR screening and DNAsequencing. We have successfully obtained the wild and mutant luciferase reportervector pGL3/ASB16.2.3Detection of the luciferase activity results shows: Compared with the negativecontrol group, the luciferase activity increased in the recombinant luciferase reportervectors pGL3/ASB16groups (P <0.05). There is no significant difference betweenwild group and mutant group(P>0.05)3. Cloning and expression of the human FKBP5gene and purification3.1The amplification product of FKBP5can be seen a bright electrophoretic bandsat1400bp, confirming to the design. The wild and mutant FKBP5gene fragment wasrespectively connected with linear cloning vector pMD-18T, then positiverecombinant cloning plasmids were identified by colony PCR screening and DNAsequencing. We have successfully obtained the wild and mutant cloning vectorpMD-18T/FKBP5. The wild and mutant candidate promoter fragment wasrespectively inserted into expression reporter vector pET-11d, then positiverecombinant reporter plasmids were identified by colony PCR screening andrestriction analysis. We have successfully obtained the wild and mutant prokaryoticexpression vector pET-11d/FKBP5.3.2The wild and mutant recombinant expression vector pET-11d/FKBP5wasrespectively transformed into competent strain BL21and induced by ITPG., thenanalyzed by SDS-PAGE. The result showed there was a thick protein band in the52kDa after induced, which was consistent with the size of the target protein., andmost of the target protein expressed in soluble form in the supernatant of bacterialultrasound. Western blot show that recombinant proteins can specificaly react withboth the anti-FKBP5polyclonal antibody and anti-His-Tag monoclonal antibody,which indicating that the specificity of the human protein FKBP5. The protein FKBP5was purified by Ni2+-NTA affinity chromatography under non-denaturing conditions,then was analyzed by SDS-PAGE. The result show that the purity of recombinantprotein FKBP5acounts to more than85%. Conclusion1. The PDB in this pedigree is not associated with any of the SNPs in SQSTM1and TNFRSF11A gene. The PDB in this pedigree may be caused by genes other thanSQSTM1and TNFRSF11A.2. A new SNP in ASB16is identified. The wild and mutant luciferase reportervector pGL3/ASB16is successfully constructed. The candidate promoter of ASB16have certain activity. There is no difference between wild and mutant group in ASB16promoter activity. It is suggested that the new SNP is not associated with theincidence of the PDB.3. The wild and mutant prokaryotic expression vector pET-11d/FKBP5issuccessfully constructed. The wild and mutant human protein FKBP5with high puritywas obtained. Lay the foundation for further biological activity research andfunctional studies of the protein.