Genotüübi ja fenotüübi korrelatsioonidel põhinev Osteogenesis Imperfecta perekonnasisene ja -vaheline varieeruvus

Väitekirja elektrooniline versioon ei sisalda publikatsiooneOsteogenesis Imperfecta (OI) on haruldane geneetiline haigus, mis on tuntud ka kui “habraste luude haigus”. Patsientide fenotüübid varieeruvad kergest osteopeeniast kuni raskete või isegi letaalsete juhtudeni. Umbes 90% juhtumitest on haiguse peamiseks sihtmärgiks I tüüpi kollageen. Käesoleva doktoritöö eesmärgiks oli tuvastada genotüüpide-fenotüüpide korrelatsioone OI perekondades perekonnasisese ja -vahelise varieeruvusega. Me uurisime OI patsientide kliinilisi tunnused ja kollageeni patogensete variantide spektrit. Analüüsisime de novo kollageeni-OI juhtumeid ja genotüübi-fenotüübi korrelatsioone Eesti, Ukraina ja Vietnami OI patsientidel. Lisaks viisime läbi mittekollageeni-OI patsientide geneetilise sõeluuringu harvaesineva OI vormi (tüüp V) tuvastamiseks. Lõppeesmärgi saavutamiseks analüüsisime perekondade siseseid ja perekondade vahelisi varieeruvusi kollageeni patogensete variantidega OI perekondades. Eestis tuvastasime 90%-l OI patsientidest I tüüpi kollageeni patogensed variantid. Enamus neist põhjustasid kollageeni kvantitatiivse defekti. Ukraina OI patsientidest 64% kandsid I tüüpi kollageeni patogense variandi ja nende hulgas oli kvalitatiivsete ja kvantitatiivsete kollageeni defektide arv peaaegu võrdne. Pooled tuvastatud OI patogensetest variantidest olid varem kirjanduses kirjeldamata. Samuti ei olnud 57%-l patsientidel varasemat OI esinemist perekonnas teada ja haigus ilmnes de novo. Tuvastasime viis perekonda, kellel esineb tüüpi V OI, kuid esinesid varieeruvad fenotüübid, mis mitmel juhul sarnanesid klassikalise kollageen-OI fenotüüpidega. Perekondade sisene ja vaheline varieeruvus korreleerus kollageeni defekti tüübi ja geeniga. Kvalitatiivse kollageeni defektiga patsientidel oli suurem fenotüübiline varieeruvus võrreldes nendega, kellel esines kvantitatiivne kollageeni defekt. Doktoritöö tulemused laiendavad arusaamist Osteogenesis Imperfecta geneetikast, etioloogiast, patogeneesist ja “haprade luude” fenotüübi seostest. Käesolev uuring aitab kaasa OI diagnoosimisele ja edasistele OI-uuringutele, mille eesmärk on parandada OI patsientide elukvaliteeti.Osteogenesis Imperfecta (OI) is a rare genetic disorder, also known as a “brittle bone disease”. Patients’ phenotypes range from mild bone fragility to severe or even lethal cases. In up to 90% of cases the main target of the disorder is collagen type I. Current thesis aimed to identify genotype-phenotype associations in OI families with inter- and intrafamilial phenotypical variability. We have investigated clinical characteristics and described spectrum of collagen-related pathogenic variants in OI patients. We have analyzed de novo cases among collagen-related OI patients and genotype-phenotype correlations in OI patients of Estonian, Ukrainian and Vietnamese origin. Further, we have performed genetic screening of non-collagen OI patients for a rare OI form - type V. Finally, inter- and intrafamilial diversity in collagen-related OI families was analyzed. In Estonian population 90% of patients harbored collagen pathogenic variants. Out of them, majority had quantitative defect of the collagen I. Among Ukrainian OI patients, 64% had collagen I pathogenic variants, with almost equal amount of qualitative and quantitative collagen defects among them. Almost half of all identified OI pathogenic variants were undescribed previously. 57% of patients did not have OI family history and the disorder appeared as de novo. We have identified five families with OI type V with variable phenotypes, which mimicked classical collagenous OI types. Identified inter- and intrafamilial variability correlated with type of the collagen defect and affected gene. So that, patients who had qualitative collagen defect had higher phenotype variability compared to those who suffered from quantitative collagen defect. Results of current thesis broaden understanding of Osteogenesis Imperfecta genetics, etiology, pathogenesis and development of a “brittle bone” phenotype. Current work contributes into OI diagnosis, family planning and further OI research with aim to improve quality of life for OI patients.https://www.ester.ee/record=b5259878~S

Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients

Background Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country. Methods We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5′UTR and 3′UTR regions, to identify causative OI mutations. Results We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains. Conclusion Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products

Transcriptional landscape analysis identifies differently expressed genes involved in follicle-stimulating hormone induced postmenopausal osteoporosis

Osteoporosis is a disorder associated with bone tissue reorganization, bone mass, and mineral density. Osteoporosis can severely affect postmenopausal women, causing bone fragility and osteoporotic fractures. The aim of the current study was to compare blood mRNA profiles of postmenopausal women with and without osteoporosis, with the aim of finding different gene expressions and thus targets for future osteoporosis biomarker studies. Our study consisted of transcriptome analysis of whole blood serum from 12 elderly female osteoporotic patients and 12 non-osteoporotic elderly female controls. The transcriptome analysis was performed with RNA sequencing technology. For data analysis, the edgeR package of R Bioconductor was used. Two hundred and fourteen genes were expressed differently in osteoporotic compared with non-osteoporotic patients. Statistical analysis revealed 20 differently expressed genes with a false discovery rate of less than 1.47 × 10(−4) among osteoporotic patients. The expression of 10 genes were up-regulated and 10 down-regulated. Further statistical analysis identified a potential osteoporosis mRNA biomarker pattern consisting of six genes: CACNA1G, ALG13, SBK1, GGT7, MBNL3, and RIOK3. Functional ingenuity pathway analysis identified the strongest candidate genes with regard to potential involvement in a follicle-stimulating hormone activated network of increased osteoclast activity and hypogonadal bone loss. The differentially expressed genes identified in this study may contribute to future research of postmenopausal osteoporosis blood biomarkers

RNA sequencing analysis reveals increased expression of interferon signaling genes and dysregulation of bone metabolism affecting pathways in the whole blood of patients with osteogenesis imperfecta

Background Osteogenesis imperfecta (OI) is a rare genetic disorder in which the patients suffer from numerous fractures, skeletal deformities and bluish sclera. The disorder ranges from a mild form to severe and lethal cases. The main objective of this pilot study was to compare the blood transcriptional landscape of OI patients with COL1A1 pathogenic variants and their healthy relatives, in order to find out different gene expression and dysregulated molecular pathways in OI. Methods We performed RNA sequencing analysis of whole blood in seven individuals affected with different OI severity and their five unaffected relatives from the three families. The data was analyzed using edgeR package of R Bioconductor. Functional profiling and pathway analysis of the identified differently expressed genes was performed with g:GOSt and MinePath web-based tools. Results We identified 114 differently expressed genes. The expression of 79 genes was up-regulated, while 35 genes were down-regulated. The functional analysis identified a presence of dysregulated interferon signaling pathways (IFI27, IFITM3, RSAD12, GBP7). Additionally, the expressions of the genes related to extracellular matrix organization, Wnt signaling, vitamin D metabolism and MAPK-ERK 1/2 pathways were also altered. Conclusions The current pilot study successfully captured the differential expression of inflammation and bone metabolism pathways in OI patients. This work can contribute to future research of transcriptional bloodomics in OI. Transcriptional bloodomics has a strong potential to become a major contributor to the understanding of OI pathological mechanisms, the discovery of phenotype modifying factors, and the identification of new therapeutic targets. However, further studies in bigger cohorts of OI patients are needed to confirm the findings of the current work

Mutation analysis of the <i>COL1A1</i> and <i>COL1A2</i> genes in Vietnamese patients with osteogenesis imperfecta

BackgroundThe genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI.MethodsGenomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish's osteogenesis imperfecta mutation database.ResultsThe sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G > A (p.Gly821Ser) in four unrelated patients and one, c.2005G > A (p.Ala669Thr), in two unrelated patients.ConclusionOur data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required

COL1A1/2 Pathogenic Variants and Phenotype Characteristics in Ukrainian Osteogenesis Imperfecta Patients

Osteogenesis imperfecta (OI) is a hereditary bone disorder caused by defects of type I collagen. Although up to 90% of patients harbor pathogenic variants in the COL1A1/2 gene, which codes for collagen α1/2 chains, the spectrum of OI genotypes may differ between populations, and there is academic controversy around OI genotype-phenotype correlations. In the current study, 94 Ukrainian OI families were interviewed. Clinical and genealogical information was collected from patients in spoken form, and their phenotypes were described. To identify the spectrum of collagen I pathogenic variants, COL1A1/2 mutational analysis with Sanger sequencing was performed on the youngest affected individual of every family. Of the 143 patients investigated, 67 (46.85%) had type I OI, 24 (16.78%) had type III, 49 (34.27%) had type IV, and III (2.10%) had type V. The mean number of fractures suffered per patient per year was 1.32 ± 2.88 (type I 0.50 ± 0.43; type III 3.51 ± 6.18; type IV 1.44 ± 1.77; and type 5 0.77 ± 0.23). 87.23% of patients had skeletal deformations of different severity. Blue sclera, dentinogenesis imperfecta, and hearing loss were present in 87%, 55%, and 22% of patients, respectively. COL1A1/2 pathogenic variants were harbored by 60 patients (63.83%). 27 pathogenic variants are described herein for the first time. The majority of the pathogenic variants were located in the COL1A1 gene (76.19%). Half (49.21%) of the pathogenic variants were represented by structural variants. OI phenotype severity was highly correlated with type of collagen I defect. The current article presents an analysis of the clinical manifestations and COL1A1/2 mutational spectrum of 94 Ukrainian OI families with 27 novel COL1A1/2 pathogenic variants. It is hoped that this data and its analysis will contribute toward the increased understanding of the phenotype development and genetics of the disorder

IFITM5 pathogenic variant causes osteogenesis imperfecta V with various phenotype severity in Ukrainian and Vietnamese patients

Background Osteogenesis imperfecta (OI) covers a spectrum of bone fragility disorders. OI is classified into five types; however, the genetic causes of OI might hide in pathogenic variants of 20 different genes. Often clinical OI types mimic each other. This sometimes makes it impossible to identify the OI type clinically, which can be a risk for patients. Up to 90% of OI types I–IV are caused by pathogenic variants in the COL1A1/2 genes. OI type V is caused by the c.-14C > T pathogenic variant in the 5′UTR of the IFITM5 gene and is characterized by hyperplastic callus formation and the ossification of interosseous membranes. Results In the current study, we performed IFITM5 5′UTR region mutational analysis using Sanger sequencing on 90 patients who were negative for COL1A1/2 pathogenic variants. We also investigated the phenotypes of five patients with genetically confirmed OI type V. The proportion of OI type V patients in our cohort of all OI patients was 1.48%. In one family, there was a history of OI in at least three generations. Phenotype severity differed from mild to extremely severe among patients, but all patients harbored the same typical pathogenic variant. One patient had no visible symptoms of OI type V and was suspected to have had OI type IV previously. We also identified a case of extremely severe hyperplastic callus in a 15-year-old male, who has hearing loss and brittleness of teeth. Conclusions OI type V is underlined with some unique clinical features; however, not all patients develop them. The phenotype spectrum might be even broader than previously suspected, including typical OI features: teeth brittleness, bluish sclera, hearing loss, long bones deformities, and joint laxity. We suggest that all patients negative for COL1A1/2 pathogenic variants be tested for the presence of an IFITM5 pathogenic variant, even if they are not expressing typical OI type V symptoms. Further studies on the pathological nature and hyperplastic callus formation mechanisms of OI type V are necessary

The clinical features of osteogenesis imperfecta in Vietnam

Purpose Osteogenesis imperfecta (OI) has not been studied in a Vietnamese population before. The aim of this study was to systematically collect epidemiological information, investigate clinical features and create a clinical database of OI patients in Vietnam for future research and treatment strategy development. Method Participants underwent clinical and physical examinations; also medical records were reviewed. Genealogical information was collected and family members’ phenotypical manifestations recorded. Cases were classified according to the Sillence classification. Results In total, 146 OI patients from 120 families were studied: 46 with OI Type I, 46 with Type III and 54 with Type IV. Almost patients had skeletal deformations. One hundred and forty-two had a history of fractures, 117 blue sclera, 89 dentinogenesis imperfecta and 26 hearing loss. The total number of fractures was 1,932. Thirty-four patients had intra-uterine fractures and nine had perinatal fractures. Surgery was performed 163 times in 58 patients; 100 osteosyntheses and 63 osteotomies. Bisphosphonate treatment was used in 37 patients. The number of affected individuals and predominance of severe forms of OI indicate that the disease is under diagnosed in Vietnam, especially in cases without a family history or with mild form of OI. Deformities appeared in all patients with different severity and localisation, affecting mostly the lower limbs. OI medical and surgical treatment rates are low and in most cases surgery was performed due to fractures. Conclusions Compared to previous studies, our results indicate a lower OI prevalence and greater severity of symptoms in the Vietnamese population when compared with other areas. Further investigation, improved diagnosis and treatment are needed to increase the patients’ quality of life