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Osteogenesis Imperfecta: Genetic and Therapeutic Studies
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Metabolic Bone Diseases. (Metabola bensjukdomar)
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 [en]
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: urn:nbn:se:uu:diva-208942ISBN: 978-91-554-8772-0 (print)OAI: oai:DiVA.org:uu-208942DiVA: diva2:655432
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
List of papers
1. Genotype-Phenotype Correlations, Response to Bisphosphonate Treatment and Pharmaco-genetics in 150 Swedish Families with Osteogenesis Imperfecta (Type I, IV and III)
Open this publication in new window or tab >>Genotype-Phenotype Correlations, Response to Bisphosphonate Treatment and Pharmaco-genetics in 150 Swedish Families with Osteogenesis Imperfecta (Type I, IV and III)
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Introduction: Osteogenesis imperfecta (OI) is a rare heterogeneous disorder leading to bone fragility, spanning from mild to lethal in severity. Over 1500 mutations have been described in collagen type I, encoded by COL1A1 and COL1A2. Bisphosphonate treatment is standard of care and published studies clearly show beneficial effects on Bone Mineral Density (BMD) and vertebral geometry. However, information on BMD increase in relation to age and BMD at onset is limited and there are few studies on influence of mutation type on treatment response. In this study Swedish patients with OI types I, IV and III were investigated with respect to genotype-phenotype correlations, BMD response on bisphosphonate treatment, and pharmaco-genetics.

Materials and Methods: 150 families (202 individuals) with OI participated: 137 type I, 40 type IV and 25 type III. Data on phenotype and bisphosphonate treatment were collected and sequencing of COL1A1 and COL1A2 performed.

Results: In 119 families a mutation was detected; in COL1A1 52 quantitative and 35 qualitative mutations were found and in COL1A2 32 qualitative mutations were found. Several unrelated individuals were found to harbor mutations with the same positions and substitutions and only 15 qualitative mutations were novel, supporting the idea of mutational hotspots. Genotype-phenotype analysis confirmed that mutations situated in the a1-chain are associated with a more severe phenotype, blue sclerae are strongly associated with COL1A1 null alleles, qualitative mutations are associated with DI, and for qualitative mutations position relative to N- and C-terminal is correlated to phenotype. A few novel mutations with unconventional locations were found.

Bisphosphonate treatment response was inversely correlated with age (p=<0.0001) and lumbar spine BMD at onset (p=0.006). Mutations associated with a more severe phenotype had an improved response to treatment when analyzing 2-year delta lumbar spine Z-score values; mutations in COL1A1 vs. COL1A2 (p=0.03), qualitative mutations in COL1A1 vs. COL1A2 (p=0.006), serine substitutions in COL1A1 vs. COL1A2 (p=0.007) and qualitative vs. qualitative mutations in COL1A1 (p=0.02) all exhibited this pattern. Bisphosphonate response was not correlated to either OI type or gender.

Conclusions: The genotype-phenotype correlations described here confirm previous reports of influence of chain affected, intrachain location, and mutation type on phenotype. BMD response to bisphosphonate treatment is inversely related to age and BMD at onset. Pharmaco-genetic analyses show an increased response to bisphosphonate treatment for more severe mutations types. This effect is attenuated over time.

Keyword
Osteogenesis imperfecta, OI, Bisphosphonate, Therapy, Genotype, Phenotype, Parmaco-genetics, Mutation
National Category
Endocrinology and Diabetes
Research subject
Medical Genetics; Genetics; Medicine
Identifiers
urn:nbn:se:uu:diva-208940 (URN)
Available from: 2013-10-11 Created: 2013-10-11 Last updated: 2013-12-05
2. Heterozygosity for a coding SNP in COL1A2 confers a lower BMD and an increased stroke risk
Open this publication in new window or tab >>Heterozygosity for a coding SNP in COL1A2 confers a lower BMD and an increased stroke risk
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2009 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 384, no 4, 501-505 p.Article in journal (Refereed) Published
Abstract [en]

Genetic variation plays an important role in osteoporosis and a prime candidate gene is Collagen alpha2(I) (COL1A2). A coding polymorphism (rs42524) in COL1A2 has previously been associated with intracranial aneurysms. Here the effects of this polymorphism have been studied in relation to bone mineral density (BMD) and prevalences of stroke and myocardial infarction (MI). rs42524 was genotyped in elderly men (n = 2004) from the Swedish MrOS cohort. Genotypes were analysed for association to BMD and certain health parameters. Significant associations (overall P < 0.05), were observed between rs42524 genotype and BMD at several skeletal sites. Surprisingly, the heterozygote genotype class exhibited lower BMD than either homozygote group. When subjects were classified as heterozygotes or homozygotes, the heterozygous genotype was found to confer a lower BMD at total hip, femoral neck and trochanter Furthermore, the heterozygote genotype had an increased risk of stroke and MI, with population Attributable Risks being 0.12 and 0.08, respectively.

National Category
Surgery
Research subject
Orthopaedics
Identifiers
urn:nbn:se:uu:diva-111772 (URN)10.1016/j.bbrc.2009.05.006 (DOI)000266689300020 ()19426706 (PubMedID)
Available from: 2009-12-21 Created: 2009-12-21 Last updated: 2017-12-12Bibliographically approved
3. COL1 C-Propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta
Open this publication in new window or tab >>COL1 C-Propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta
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2011 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 32, no 6, 598-609 p.Article in journal (Refereed) Published
Abstract [en]

Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA Z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA Z-score of 0.0 and pQCT vBMD of -1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FTIR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2's bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.

Keyword
osteogenesis imperfecta, C-propeptide, collagen, C-proteinase, mineralization, high bone mass
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-155592 (URN)10.1002/humu.21475 (DOI)000291564000011 ()
Available from: 2011-06-28 Created: 2011-06-27 Last updated: 2017-12-11Bibliographically approved
4. Allele dependent silencing of COL1A2 using small interfering RNAs
Open this publication in new window or tab >>Allele dependent silencing of COL1A2 using small interfering RNAs
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.

Keyword
COL1A2, allele-preferential silencing, Osteogenesis imperfecta
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-125035 (URN)19015742 (PubMedID)
Available from: 2010-05-07 Created: 2010-05-07 Last updated: 2013-12-05Bibliographically approved
5. Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels: a Novel Therapeutic Approach in Osteogenesis Imperfecta
Open this publication in new window or tab >>Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels: a Novel Therapeutic Approach in Osteogenesis Imperfecta
Show others...
2013 (English)In: International Journal of Medical Sciences, ISSN 1449-1907, E-ISSN 1449-1907, Vol. 10, no 10, 1333-1343 p.Article in journal (Refereed) Published
Abstract [en]

Osteogenesis imperfecta, also known as "brittle bone disease", is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3'UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3'UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.

Keyword
osteogenesis imperfecta, OI, allele-specific silencing, siRNA, collagen, COL1A1, COL1A2, indel, insertion/deletion, RNAi, mutation, gene-therapy, therapy
National Category
Endocrinology and Diabetes
Research subject
Genetics; Medical Genetics; Medicine
Identifiers
urn:nbn:se:uu:diva-208936 (URN)10.7150/ijms.5774 (DOI)000324411800011 ()23983594 (PubMedID)
Available from: 2013-10-11 Created: 2013-10-11 Last updated: 2017-12-06Bibliographically approved

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