Research Of The Molecular Mechanism Of Bone Diseases

Brachydactylies (BDs) are a group of inherited malformations characterized by shortening of the digits due to abnormal development of the phalanges and/or the metacarpals. They have been classified on anatomic and genetic basis into five groups, A-E, by Bell in 1951. Group A is further divided into three subgroups, A1, A2, and A3, and usually manifests as autosomal dominant traits. Brachydactyly type A1 (BDA1, MIM 112500) is the first autosomal dominant Mendelian trait in human and first described by Farabee in 1903. According to the reclassified criteria which Fitch used in 1979, BDA1 is characterized by hypoplasia/aplasia of all middle phalanges, sometimes with joint fusions between the middle and the proximal phalanx. In some individuals, the metacarpals were also short. Affected family members might have short stature compared to normal family members. BDA1 often occurred as an isolated malformation. However, others also experienced musculoskeletal abnormalities, scoliosis, nystagmus, and/or developmental delay.Yang et al. mapped the BDA1 locus to chromosome 2q35-36 through linkage analysis of two Chinese families in 2000. Analysis of the function of genes located in this region suggested that Indian Hedgehog gene (IHH) be a good candidate gene for BDA1. Subsequent sequences analysis identified that mutations in the IHH gene were the causal mutations for three Chinese BDA1 families by Gao in 2001. However, IHH mutations account for only a subset of BDA1 cases. Armour et al. found BDA1 is linked to an 11cM critical region on chromosome 5p13.2-13.3 in a four generation BDA1 family of Canadian descent. Kirkpatrick et al. provided evidence that support the existence of a third locus for BDA1. These data suggested that the BDA1 is genetically heterogeneous.We found a large brachydactyly family in Jining city, Shandong province. Clinical and family analysis revealed that the characterization of the cases in this family were consistent with the typical BDA1. To determine the causal gene of this BDA1 family, we carried out linkage analysis and mutation detection.1 Family Study A large family from Shandong province, China, was recruited in this study. Twenty six individuals (14 affected, 12 unaffected) were included in this study. Affected and unaffected individuals were physically examined, and six selected individuals (II-5, III-3, III-4, ffl-9, 111-12(?)111-13) were radiographed.The defects found in the affected individuals of this family were confined to the hands and feet. The hands of the affected family members appeared to be broad and showed shortening of all digits. X-ray examination revealed anomalies of hand and foot bones, but the defects were mainly confined to the middle phalanges and metacarpals. The severity of BDA1 was varied among the affected members of the family. Individual III-4 is one of the most severe case, whose middle phalanges were all absent or fused to the distal phalanges and both the third and forth metacarpals were shortened in both hands. Another affected individual, III-3, also has all the middle phalanges missing and shows shortened metacarpals. But her shortened metacarpals were limited to the first and third metacarpals, and her proximal 1^st phalange was shortened, too. To our knowledge, the previously reported BDA1 patients were not known to have severely shortened first metacarpals. Other affected members examined by radiography had shortened middle phalanges, though with varying numbers.2 Linkage Analysis and Mutation DetectionTwo known disease loci located in 2q35-2q36 and 5pl3.2-pl3.3, respectively. So we selected polymorphic microsatellite markers from these two regions for linkage analysis. Result of genotyping D5S1470 located in 5p13.2-p13.3 showed that patients in this BDA1 family carried different alleles and excluded that the causal gene for this BDA1 family is in this region. Using a set of markers spanning the IHH gene, we were able to demonstrate that BDA1 was linked to chromosome 2q35-q36. Complete linkage was shown for markers D2S2250, D2S433, D2S163, and D2S2359, and recombination event was observed between D2S434 and D2S2250 in individual 111-13. These results suggested IHH gene may be the best candidated gene for this BDA1 family.Four pairs of primers were designed to amplify all three exons and exon/intron boundaries of the IHH gene. Agarose gel analysis showed the sizes of PCR products were consistent with the expectation and indicated that there were no large insert or deletion mutitons of IHH gene. The mutation screen limited to the DNA from one affected (IV-3) and one unaffected individual (IV-2) with the direct sequencing of PCR products identified a heterozygous G-to-A transition (G298A) in exon 1, which results in an aspartate to asparagine substitution (D100N). To detect the G298A mutation in all family members and controls, a modified primer was used to create a restriction endonuclease (Sal 1) site to distinguish 298G and 298A alleles. By using PCR-RFLP analysis, the G298A transition was identified in all affected individuals. Moreover, the G298A allele was not detected among 200 chromosomes from unrelated normal Chinese controls.3 Haplotype AnalysisThe IHH G298A mutation had previously been identified in six Caucasian families with BDA1, and McCready et al. provided the evidence that support the existence of a common ancestral allele between Farabee's family and Drinkwater's families.To discriminate either a common founder mutation or a de novo mutation contributed to this Chinese kindred, we constituted the disease-associated haplotype with microsatellite markers D2S2250, D2S433, D2S163, and D2S2359 that are tightly linked to the IHH gene and five intragenic SNPs described by McCready of this Chinese family. Then the disease-related haplotype of this Chinese BDA1 family was compared with the common haplotype reported for the Farabee-Drinkwater families.A comparison of the haplotypes from different families indicated our patients carried different alleles compared to the patients from Farabee-Drinkwater's families at three microsatellite markers (D2S2250, D2S433, and D2S163) and two intragenic SNPs (SNPe and SNPd). Another three intragenic SNPs (SNPa, SNPb, and SNPc) were not informative in our family and ethnic-matched controls. We then concluded that the IHH gene G298A mutation in this Chinese BDA1 family had different origin from those in Caucasian families. It looked like the IHH gene G298A mutation occurred more than once historical.In conclusion, we identified a BDA1 family through clinical research and family analysis. By using linkage analysis, we excluded the linkage of the causal gene with 5p13.2-p13.3 and confirmed the linkage with 2q35-2q36. Combined candidate gene analysis and mutation detection, we identified a heterozygous missense mutation G298A in exon 1 of IHH gene, which results in an aspartate to asparagine substitution (D100N) was the causal mutation of this BDA1 family. Comparision of disease-related haplotypes from this family with the Farabee-Drinkwater families' revealed that a de novo G298A mutation, not a fouder mutation, caused the BDA1 of this family. Sequence analysis around IHH gene 298G showed that it maybe a mutational hot spot due to spontaneous deamination of a 5-methylcytosine on the noncoding strand of the gene. As a mutational hot spot, we predict that the IHH G298A mutation will be identified in additional BDA1 families. Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporosis results from a failure to acquire optimal peak bone mass during growth and/or to maintain bone mass in later years, which means a disruption of the fine equilibrium between the activity of osteoblasts responsible for bone deposition and osteoclasts for bone resorption.Low-density lipoprotein receptor related protein 5, LRP5, is a member of low-density lipoprotein receptor family. LRP5 have multiple functions by banding and internalizing different ligands. Recent studies have indicated that the loss-of-function mutations in LRP5 gene cause Osteoporosis-Pseudoglioma syndrome, an autosomal recessive disorder characterized by severe juvenile-onset osteoporosis and congenital or juvenile-onset blindness. The gain-of-function mutations in LRP5 gene lead to increased bone mass and osteopetroses.To further understand the role of LRP5 in bone formation, we used mouse bone marrow stromal cell line ST2 as a study model. We transfected ST2 cells with the constructs expressing Lrp5 gain-of-function muatation and LRP5-RNAi, respectively. We analyzed the effect of LRP5 status on ST2 cells proliferation and differentiation in the stable-transfected cells. With Suppression Subtractive Hybridization (SSH) technique, we constructed the subtractive cDNA libraries of ST2 cells stable-expressed Lrp5 gene with gain-of-function muatation and Lrp5-RNAi construct, respectively. The LRP5-related genes could be screened from these subtractive cDNA libraries.1. Generation and transfection of the construct expressing the Lrp5 gene with G171V mutationFusion-PCR method was used to introduce G171V mutation using the wild Lrp5 expression constructs as the template. The resulting fragments containing the G171V mutation was used to replace the corresponding fragment from the wild type cDNA, and resulting insert was cloned into pcDNA3.1C. vector and named as pcDNA3.1C-GFLrp5. The construct was confirmed by PCR, restriction enzyme digestion and direct sequencing. The pcDNA3.1C-GFLrp5 Construct was transfected into ST2 cells. Cell-based immunofluorescence and Western Blotting analysis showed that pcDNA3.1C-GFLrp5 expressed in ST2 cells and transported to the membrane.To inhibit the Lrp5 expression, we transfected the Lrp5-RNAi constructs, mu6/LRP5 to ST2 cells. The efficiency RNAi targeting was confirmed by cotransfectiong cells with mu6/LRP5 and pcDNA3.1C-GFLrp5. The decreasing in the expression of the pcDNA3.1C-GFLrp5 construct was observed with the anti-myc antibody by cell-based immunofluorescence and Western Blot.2. Effects of stable expressing pcDNA3.1C-GFLrp5 construct and Lrp5-RNAi construct mu6/LRP5 on proliferation and differentiation of ST2 cellsST2 Cells, a clone of stromal cells, were isolated from the bone marrow of BC8 mice, capable of the potential ability to differentiate into various mesenchymal cell lineages. However, when subcultured with medium supplemented with ascorbic acid, the ST2 cells developed an osteoblastic phenotype. We transfected pcDNA3.1C-GFLrp5 construct and the Lrp5 gene RNAi mu6/LRP5 construct into the cultured ST2 and selected with G418. The cells stable-expressiong Lrp5 G171V mutation expression construct and Lrp5 RNAi expression construct were obtained, and named as S-LGF and S-LLF, respectively.MTT assays revealed that the cells proliferation of S-LGF was significantly higher than that of S-LLF cells measured at different time points: cultured for 24 hours, 48 hours and 72 hours. This suggested that the exogenous Lrp5 G171V mutant protein can notably enhance the proliferation of ST2 cells compared with ST2 cells in which Lrp5 were silenced. The more bone-like nodules were observed in the S-LGF than S-LLF cells by von Kossa's staining when cells cultured for 28 days under conditioned medium supplemented with ascorbic acid. The difference was much more significantly when cells cultured for 40 days.3. Construction and screening of the S-LGF and S-LLF subtractive cDNA librariesSuppression subtractive hybridization (SSH) is the combination of suppression PCR and subtractive hybridization technique. This technique could clone the unknown sequence of differentially expressed genes. The method is characterized with high efficiency, low false positive rate, targeting sequence enrichment, and low complexity of the experimental results. Using the S-LGF mRNA as the tester and the S-LLF mRNA as driver, we constructed the forward subtracted cDNA library. The reverse subtracted cDNA library was constructed by using the S-LLF mRNA as the tester and the S-LGF mRNA as driver.Combining PCR amplification with cultured E. coli as template and the restriction endonuclease digestion, we screened the forward and reverse subtracted cDNA libraries. We found 105 clones with insert fragment out of 200 random selected clones from the forward subtracted cDNA library and 97 out of 200 from the reverse subtracted cDNA library. According to the patterns of restriction endonuclease digestion, 60 and 61 clones with different inserts from the forward and reverse subtracted cDNA libraries were selected for sequencing, respectively.4. The differentially expressed sequencesWe obtained 51 cDNA fragments differentially expressed in the S-LGF cells by comparison analysis with nucleotide sequences in GenBank. There are 44 cDNA fragments which were highly homologous with the known genes. Three cDNA fragments belong to the RIKEN cDNA gene. While other 4 cDNA fragments were not homologous with any known genes, which may be from novel genes. Similarly, 58 differentially expressed cDNA fragments in the S-LLF cells were obtained. Among them, 43 cDNA fragments were from known homologous genes and eight were RIKEN cDNA genes. Also, seven differentially expressed cDNA fragments were not matched to known homologous sequence.These differentially expressed sequences will be confirmed by RT-PCR and real-time PCR. Further analysis of these differentially expressed genes may lead to identification of new genes related with Lrp5 controlling osteoblast proliferation and function and eventually shed new light on the molecular mechanisms and the new drugs development for the treatment of osteoporosis.