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Specific Aims

Specific Aims. To establish animal models for investigating the role of COL2A1 gene mutations in osteonecrosis To establish cell models for investigating the role of COL2A1 gene mutations in osteonecrosis To identify genetic risk factors associated with sporadic ANFH.

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Specific Aims

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  1. Specific Aims • To establish animal models for investigating the role of COL2A1 gene mutations in osteonecrosis • To establish cell models for investigating the role of COL2A1 gene mutations in osteonecrosis • To identify genetic risk factors associated with sporadic ANFH

  2. Col2a1 Transgenic Mouse Model G2306A G3665A Gly717Ser Gly1170Ser Human Mouse BC052326 Gly1102Ser Gly649Ser G3425A G2066A Transgene constructs Wildtype Mutant *hG1170S

  3. Col2a1 Knock-in Mouse Model • Total 13.2 Kb • 5’arm (exon 39 to 47): 3.3 Kb • pGK-Puro: 1.6 Kb • 3’arm (exon 48 to 52): 3.0 Kb • G to A transition was created by site-directed mutagenesis • TK: 2.2 Kb

  4. Non-Mendelian Ratio in Mouse Breeding

  5. Litter 2. Pedigree for Col2a1 Knock-in Mice (E15.5)

  6. Litter 5. Pedigree for Col2a1 Knock-in Mice (E17.5)

  7. Head E17.5 WT KI/KI dorsal ventral

  8. Thorax E17.5 WT KI/KI dorsal ventral

  9. E17.5 WT KI/KI Tail Piggy tail Leg

  10. Litter 6. Pedigree for Col2a1 Knock-in Mice (E 18.5) • Abnormal appearance (exencephaly-like, spina bifida-like) was observed in three heterozygotes. #1 #8

  11. Variable Phenotypes of Col2A1 Knock-in Mice • Reduced number of KI/KI adult and live born • Fetal demise occurred after 18.5 d.p.c. in KI/KI • Minimum/little phenotype of KI/+ live born • Developmental anomalies in KI/+ or KI/KI: • cleft palate in KI/KI • skull defect (exencephaly) in KI/KI or KI/+ • spine defect (spina bifida) in KI/KI or KI/+

  12. COL2A1 Mutation in Pediatric Cases of Osteonecrosis

  13. Variable Expression of Disease Phenotype • Modifier genes • Stochastic • randomly determined; having a random probability distribution or pattern that may be analyzed statistically but may not be predicted precisely.

  14. Mutation of type I Collagen Gene • 成骨不全症(Osteogenesis imperfecta，OI)，是一種先天性遺傳疾病，出現在男女的比例上大約相同，這種疾病會造成第一型膠原纖維缺陷，使骨骼忍受外力衝擊的能力較正常人差，即使是輕微的碰撞，也會造成嚴重的骨折，因此這類的病患被稱為「玻璃娃娃」。

  15. Procollagen Suicide http://www.biochemsoctrans.org/bst/027/0015/bst0270015f09.htm

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