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Allele dependent silencing of COL1A2 using small interfering RNAs
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Metabolic Bone Diseases)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Metabolic Bone Diseases)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Metabolic Bone Diseases)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Metabolic Bone Diseases)
2008 (English)In: International journal of medical sciences, ISSN 1449-1907, Vol. 5, no 6, 361-365 p.Article in journal (Refereed) Published
Abstract [en]

Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI.

Place, publisher, year, edition, pages
2008. Vol. 5, no 6, 361-365 p.
Keyword [en]
COL1A2, allele-preferential silencing, Osteogenesis imperfecta
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-125035PubMedID: 19015742OAI: oai:DiVA.org:uu-125035DiVA: diva2:318323
Available from: 2010-05-07 Created: 2010-05-07 Last updated: 2013-12-05Bibliographically approved
In thesis
1. Osteogenesis Imperfecta: Genetic and Therapeutic Studies
Open this publication in new window or tab >>Osteogenesis Imperfecta: Genetic and Therapeutic Studies
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Osteogenesis imperfecta (OI) is a heterogeneous disease of connective tissue, the cardinal symptom being fractures and severity ranging from mild to lethal. Dominant mutations in collagen I, encoded by COL1A1 and COL1A2, cause >90% of cases.

To delineate genotype-phenotype correlations and pharmaco-genetic response, collagen I was sequenced in 150 unrelated Swedish families and clinical data were collected in Paper I. Mutation type, gene affected, and N- to C-terminal location correlated with phenotype and severity. Bisphosphonate response assessed by calculated yearly change in lumbar spine bone mineral density (BMD) was inversely related to age and BMD at treatment initiation. Mutations associated with a more severe phenotype exhibited an increased response after 2 years; however, all types of OI responded well.

To investigate the effect of naturally occurring variations in collagen I, the only common coding single nucleotide polymorphism (rs42524 in COL1A2) was genotyped in 2004 healthy men in Paper II. Heterozygous genotype was associated with decreased BMD and an increased risk of stroke.

An adolescent with repeated fractures despite a markedly high BMD harbored a unique C-terminal procollagen cleavage-site mutation in COL1A1, which motivated extensive investigations in concert with a similar COL1A2 case in Paper III. The probands were found to have impaired procollagen processing, incorporation of collagen with retained C-propeptide in matrix and increased mineral to matrix ratio, which demonstrates that C-propeptide cleavage is crucial to normal bone mineralization and structure.

Bisphosphonate therapy has insufficient effect in OI, and as classical OI is a dominant disorder severe cases would benefit from silencing of the mutated allele. In Paper IV and V small interfering RNAs (siRNAs) were used to allele-specifically target primary human bone cells heterozygous for I) a coding polymorphism in COL1A2 and II) insertion/deletions in the 3’UTR of COL1A1 and COL1A2. Results were promising with altered allele ratios and decreased mRNA levels in the predicted fashion.

To summarize, this thesis found that collagen I is crucial to bone and connective tissue and that collagen I mutations create markedly diverse phenotypes. Age, BMD and pharmaco-genetic effects influence the response to bisphosphonate therapy in individuals with OI; however, novel approaches are needed. Utilizing allele-specific siRNAs may be a way forward in the treatment of severe OI.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 96 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 936
Keyword
OI, BMD, Genotype, Phenotype, Pharmaco-genetics, Bisphosphonate, Therapy, Gene-therapy, Mutation, Collagen, Collagen type I, Allele-specific silencing, siRNA, RNAi, COL1A1, COL1A2, Stroke, C-propeptide, Mineralization, Heterozygous disadvantage
National Category
Endocrinology and Diabetes
Research subject
Genetics; Medicine; Medical Genetics
Identifiers
urn:nbn:se:uu:diva-208942 (URN)978-91-554-8772-0 (ISBN)
Public defence
2013-11-29, Enghoffsalen, Ingång 50, Akademiska Sjukhuset, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council
Available from: 2013-11-08 Created: 2013-10-11 Last updated: 2014-01-23

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