Study On Clinical And Identification Of Molecular Mutation Of Osteogenesis Imperfecta And Genetic Variation Of SOST Gene

Part 1 Study on osteogenesis imperfecta mutation screening of type 1 collagen and SERPINF1 genes and the relationship with phenotypesAims: To elucidate the mutation spectrum of COL1A1 and COL1A2 genes in osteogenesis imperfecta（OI） patients of the Han population and to explore the relationship between the clinical phenotype and pathogenic gene mutations. Using the whole genome sequencing technology to screen new virulence gene of the patients with OI without COL1A1 or COL1A2 gene mutations.Methods:A total of 78 unrelated Chinese OI probands,135 family members and 250 healthy controls were recruited. The clinical characteristics of the probands and family members were recorded and venous blood samples were collected of all the subjects. All the exons and the exon-intron boundaries of the COL1A1 and COL1A2 gene were amplified and directly sequenced in all the probands. The bone mineral density（BMD） of the lumbar spine（1-4） was measured by dual-energy X-ray absorptiometry for measurable OI patients. Using whole-exome sequencing in one OI proband of type VI, we identified one novel mutation in SERPINF1. Using Sanger sequenc to identify mutations of SERPINF1 in 17 OI probands that without mutations of COL1A1 and COL1A2 and we identified the other novel mutation in SERPINF1. Real-time quantitative PCR and detect allelic copy numbers of the both families of SERPINF1 mutations were performed to verify if the deletion existed.Results: There were 44 COL1A1 mutations（56%）, 16 COL1A2 mutations（21%）, 2 SERPINF1 mutations（2%）of 78 probands. The other 16 probands（21%）didn’t identify any virulence genes. A total of 31 novel mutations were observed. The mutation p.Gly257 Arg, p.Gly767 Ser, p.Gly821 Ser in COL1A1 and p.Gly337 Ser in COL1A2 might be at a mutation hotspot for human OI. A total of 32 probands（53%） had familial OI, 27（45%） had sporadic disease, 22（37%） were de novo of the probands of COL1A1 and COL1A2 mutations. A total of 5 family members displayed normal phenotype or close to normal phenotype though they had the same gene mutation as the proband. Both probands of type VI OI displayed one novel missense mutation, c.1067T>A（V356E） and one novel homozygous deletion mutation c.283+473₆43+104del（p.Ala96_Gly215del） of SERPINF1. The heterozygous mutation of the same site of the proband and no mutation of SERPINF1 was found in both families of the probands’ father and mother. The results of allelic copy numbers showed that deletion of SERPINF1 existed in the proband and her mother of family 1. No deletion of SERPINF1 existed in the proband or her mother of family 2.Conclusions: A total of 31 novel mutations were observed in 60 OI probands of COL1A1 and COL1A2 mutations in this study. The mutation p.Gly257 Arg, p.Gly767 Ser, p.Gly821 Ser in COL1A1 and p.Gly337 Ser in COL1A2 might be at a mutation hotspot for human OI. The intrafamilial variation can be explained by germline or somatic mosaicism or nonpenetrance for the mutation. We should pay attention to these probabilities in genetic counseling. Our study shows that the novel missense mutation, c.1067T>A（V356E） and the novel deletion mutation c.283+473₆43+104del（p.Ala96_Gly215del） of the SERPINF1 gene are responsible for OI type VI in some the Han nationality patients. This study provide important data to understand the pathogenic gene mutation spectrum of COL1A1, COL1A2 and SERPINF1 and clinical phenotypes in patients with OI. This study also lay the foundation for future clinical diagnosis and genetic counseling of OI patients.Part 2 Associations of Serum Sclerostin and Polymorphisms in the SOST Gene with Bone Mineral Density and Markers of Bone Metabolism in Postmenopausal WomenAims: The aims of this study were to determine the association of serum sclerostin and single nucleotide polymorphisms（SNPs） within the sclerostin（SOST） gene with bone mineral density（BMD） and markers of bone metabolism in postmenopausal women.Methods:At first 703 healthy postmenopausal women of the Han nationality aged 66.7±9.1 years were recruited. The BMD of the lumbar spine 1-4（L1-4） and left proximal femur were measured by dual-energy X-ray absorptiometry. Serum sclerostin and markers of bone turn over were also measured, including serum intact parathyroid hormone（PTH）, 25-hydroxyvitamin D [25（OH）D], procollagen type 1 N-terminal propeptide（P1NP）, and β-Cross Laps of type I collagen containing cross-linked C-telopeptide（β-CTX）. Ten tagging SNPs（rs1234612, rs1513670, rs1634330, rs1708635, rs2023794, rs7220711, rs74252774, rs851057, rs851058, and rs865429） of the SOST gene were genotyped. In order to obtain sufficient statistical power, we continued to recruit the other 676 healthy postmenopausal women of the Han nationality. A total of 1379 postmenopausal women were observed the associations between polymorphisms of SOST gene and BMD.Results: Serum sclerostin was positively correlated with BMD at lumbar spine, femoral neck, total hip and with serum 25（OH）D（all P<0.01）, but negatively correlated with β-CTX（P<0.01）. The significant relationships between serum sclerostin and BMD and with serum β-CTX persisted even after adjustments for age, BMI and serum 25（OH）D（all P<0.01）. However, there was no correlation between serum sclerostin and age or serum P1 NP. We failed to identify a significant association between the SNP, haplotypes of SOST and BMD, serum sclerostin in 703 postmenopausal women. Five SNPs（rs1513670, rs851057, rs851058, rs1708635 and rs1634330） in the SOST gene were significantly associated with serum P1 NP. When the research object was increased in 1379 cases, the rs2023794 and rs74252774 and the haplotype ACCATTCT of SOST gene were associated with age and body mass index（BMI） adjusted L1-4 BMD（P values were 0.010, 0.007 and 0.007, respectively） even after performing the Bonferroni multiple-significance-test correction. There was a clear trend in these regions that CC genotype of rs2023794 and TT genotype of rs74252774 have higher BMD values than other genotypes. The contributions of rs2023794 and rs74252774 to the phenotypic variation of L1-4 BMD were 0.6 and 0.7%, respectively. However, we failed to find any association between the 10 SNPs and 6 haplotypes of the SOST gene and BMD at hip site in this study.Conclusions: Our results suggested that serum sclerostin was positively correlated with the BMD at lumbar spine, femoral neck, total hip and with serum 25（OH）D but was negatively correlated with serum β-CTX in healthy postmenopausal women of the Han nationality. Five SNPs in the SOST gene were significantly associated with serum P1 NP. The polymorphisms of rs2023794 and rs74252774 and haplotype ACCATTCT in the SOST gene were associated with BMD of the lumbar spine in a large sample of postmenopausal women.