XXIII ed FECTS and ISMB Joint Meeting, August 25 th –29 th , 2012, Katowice, Poland Connective Tissue Disorders P3.1 Oral presentation Cell therapy of a patient with type III osteogenesis imperfecta caused by G23569A (p.Gly517Asp) COL1A2 gene mutation and unstable collagen type I Aleksandra Auguściak-Duma 1,5 , Marcin Majka 2 , Magdalena Janeczko 3 , Jolanta Goździk 4 , Danuta Jarocha 2 , Joanna Witecka 1,5 , Marta Lesiak 1,5 , Halina Koryciak-Komarska 1,5 , Aleksander L. Sieroń 1,5 , Jacek Józef Pietrzyk 3 1 Department of General and Molecular Biology and Genetics, Medical University of Silesia, Katowice, Poland; 2 Department of Transplantation, UJCM, Krakow, Poland; 3 Department of Pediatrics and Medical Genetics, Chair of Pediatrics, UJCM, Krakow, Poland; 4 Department of Clinical Immunology, UJCM, Krakow, Poland; 5 Network of CoE BioMedTech Silesia, Poland e-mail: Aleksandra Augusciak-Duma <aaugusciak@sum.edu.pl> This report presents clinical case of the newborn girl diag- nosed with osteogenesis imperfecta type III, with multiple bone fractures, extreme shortness and limbs deformities, treated at the age of 4 and 6 weeks with transplantation of allo- genic bone marrow derived mesenchymal stem cells. The diagnosis of osteogenesis imperfecta was confrmed by the bio- chemical analysis of collagen type I recovered from culture medium of cultivated skin fbroblast, which revealed its un- stable triple helical structure at temperature about 2°C low- er than normal. Subsequent sequencing of both genes en- coding procollagen type I revealed a substitution G23569A in COL1A2 gene causing missense mutation changing glycine at position 517 to aspartate. To obtain skin fbro- blasts for procollagen type I production and DNA isola- tion the skin biopsy was used. The donor of mesenchymal stem cells was the girl’s father. She received two intravenous infusions of suspended cultured mesenchymal cells in 16 days apart without any side effects. An analysis of procolla- gen type I secreted to the culture medium by bone marrow- derived mesenchymal stem cells obtained from the patient, 3 months following transplantation revealed its normal triple helix stability. During the subsequent two years of follow up two new bone fractures were noted. Currently a two- year-old girl’s presents extreme growth and weight defciency. The motoric development is also retarded, but the patient constantly improves and makes progresses. Acknowledgement The work was in part fnanced from Institutional grant KNW-1-017/10 awarded to ALS. Some of the equipment used in this work was purchased using EU funds (European Regional Development Fund) within the Sec- toral Operational Program "Increase of Economic Competitiveness." No. WKP 1/1.4/3/2/2005/103/223/565/2007/U and Silesian Bio-Farma Center for Biotechnology, Bioengineering and Bioinformatics Project no POIG.02.01.00-00-166/08 THE OPERATIONAL PROGRAMME IN- NOVATIVE ECONOMY FOR 2007-2013. Priority Axis 2. R&D Infra- structure. P3.2 Oral presentation Mutations in FKBP10, a collagen PPIase, extend the recessive phenotype associated with type XI OI, and cause diminished collagen cross-linking in matrix Aileen M. Barnes 1 , MaryAnn Weis 2 , Wayne A. Cabral 1 , Elena Makareeva 3 , Edward L. Mertz 3 , William Paton 4 , Geraldine Duncan 5 , Carlos Trujillo 6 , Sergey Leikin 3 , David R. Eyre 2 , Sherrie J. Bale 7 , Joan C. Marini 1 1 NICHD/NIH, Bone and Extracellular Matrix Branch, Bethesda, MD, United States; 2 University of Washington, Orthopaedic Research Laboratories, Seattle, United States; 3 NICHD/NIH, Section on Physical Biochemistry, Bethesda, MD, United States; 4 Alaska Native Medical Center, Anchorage, AK, United States; 5 Christchurch Hospital, Christchurch, New Zealand; 6 Dr. Erfan & Bagedo General Hospital, Genetics Unit, Jeddah, Saudi Arabia; 7 Gene Dx, Gaithersburg, MD, United States e-mail: Aileen Barnes <barnesai@mail.nih.gov> Recessive osteogenesis imperfecta (OI) is caused by de- fects in genes whose products interact with type I collagen for modifcation and/or folding. Recently, mutations in FKBP10, encoding the ER-resident molecular chaperone and isomerase FKBP65, have been shown to cause both re- cessive OI and Bruck Syndrome (OI with contractures). We identifed a homozygous frameshift mutation in FKBP10 in a 5-generation Palestinean pedigree, associated with mod- erately severe recessive OI, as well as an Alaskan pedigree with Kuskokwim Disease (OMIM% 208200), character- ized by congenital contractures and osteopenia, caused by an in-frame deletion in FKBP10. The Kuskokwim pedigree is the frst description of a predominantly contracture dis- order caused by FKBP10 mutations. The child with moderate type XI OI has a homozygous FKBP10-null mutation (c.1271_1272delCCinsA). FKBP10 transcripts in proband fbroblasts are 4% of control; with absent FKBP65 protein on Western blot. The Alaskan pedigree has an in-frame FKBP10 deletion of a single resi- due (c.875_877del, p.Tyr293del) in the 4 th PPIase domain of FKBP65 which leads to normal to increased levels of FKBP10 transcripts (79–167%) and residual protein (~5%) on Western blot. Both pedigrees show minimal changes in type I collagen migration on gel electrophoresis with a slight increase (~15%) in hydroxylysine levels. On matrix deposition of cultured fbroblasts, the mature cross-linked collagen fraction was decreased by >95% in the null mu- tation and by 45–85% in the Kuskokwim pedigree. Mass spectrometry revealed that hydroxylation of the telopep- tide lysine involved in collagen cross-linking was <1% in the FKBP10-null mutation and 2–10% in the Kuskokwim, compared to 58–59% hydroxylation of collagen secreted from normal fbroblasts. The lack of cross-linking af- fected the organization of matrix. The FKBP10-null cells deposited a very sparse (~10% of control) and disorgan- ized matrix, as seen on immunofuorescence microscopy. The Kuskokwim pedigree cells deposited a normal amount of matrix, yet the matrix was still disorganized, compared to the orderly fbrils seen in control. The decrease in the amount of matrix was confrmed in both pedigrees by Ra- man microspectroscopy: the FKBP10-null cells had ~30% of control matrix, while 60–95% of control matrix was