An ENU-induced splice site mutation of mouse Col1a1 causing recessive osteogenesis imperfecta and revealing a novel splicing rescue.
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ABSTRACT:
SUBMITTER: Tabeta K
PROVIDER: S-EPMC5600972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundOsteogenesis imperfecta (OI), a rare autosomal inheritable disorder characterized by bone fragility and skeletal deformity, is caused by pathogenic variants in genes impairing the synthesis and processing of extracellular matrix protein collagen type I. With the use of next-generation sequencing and panels approaches, an increasing number of OI patients can be confirmed and new pathogenic variants can be discovered. This study sought to identify pathogenic gene variants in a Chinese family with OI I.MethodsWhole-exome sequencing was used to identify pathogenic variants in the proband, which is confirmed by Sanger sequencing and cosegregation analysis; MES, HSF, and Spliceman were used to analyze this splicing variant；qRT-PCR was performed to identify the mRNA expression level of COL1A1 in patient peripheral blood samples; Minigene splicing assay was performed to mimic the splicing process of COL1A1 variants in vitro; Analysis of evolutionary conservation of amino acid residues and structure prediction of the mutant protein.ResultsA novel splicing pathogenic variant (c.3814+1G>T) was identified in this OI family by using whole-exome sequencing, Sanger sequencing, and cosegregation analysis. Sequencing of RT-PCR products from the COL1A1 minigene variant reveals a 132-nucleotide (nt) insertion exists at the junction between exons 48 and exon 49 of the COL1A1 cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which may cause a frameshift resulting in early termination of protein expression. The molecular analysis suggested that the altered amino acid is located at the C-terminus of type I procollagen.ConclusionOur study reveals the pathogenesis of a novel COL1A1 splicing pathogenic variant c.3814+1G>T in a Chinese family with OI I.
| S-EPMC7507304 | biostudies-literature
Project description:BACKGROUND:Osteogenesis imperfecta (OI) is a group of connective tissue disorder caused by mutations of genes involved in the production of collagen and its supporting proteins. Although the majority of reported OI variants are in COL1A1 and COL1A2 genes, recent reports have shown problems in other non-collagenous genes involved in the post translational modifications, folding and transport, transcription and proliferation of osteoblasts, bone mineralization, and cell signaling. Up to now, 17 types of OI have been reported in which types I to IV are the most frequent cases with autosomal dominant pattern of inheritance. CASE PRESENTATION:Here we report an 8- year- old boy with OI who has had multiple fractures since birth and now he is wheelchair-dependent. To identify genetic cause of OI in our patient, whole exome sequencing (WES) was carried out and it revealed a novel deleterious homozygote splice acceptor site mutation (c.1257-2A?>?G, IVS7-2A?>?G) in FKBP10 gene in the patient. Then, the identified mutation was confirmed using Sanger sequencing in the proband as homozygous and in his parents as heterozygous, indicating its autosomal recessive pattern of inheritance. In addition, we performed RT-PCR on RNA transcripts originated from skin fibroblast of the proband to analyze the functional effect of the mutation on splicing pattern of FKBP10 gene and it showed skipping of the exon 8 of this gene. Moreover, Real-Time PCR was carried out to quantify the expression level of FKBP10 in the proband and his family members in which it revealed nearly the full decrease in the level of FKBP10 expression in the proband and around 75% decrease in its level in the carriers of the mutation, strongly suggesting the pathogenicity of the mutation. CONCLUSIONS:Our study identified, for the first time, a private pathogenic splice site mutation in FKBP10 gene and further prove the involvement of this gene in the rare cases of autosomal recessive OI type XI with distinguished clinical manifestations.
| S-EPMC5970456 | biostudies-literature
Project description:Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of pro?1(I) in the culture media being C-terminally processed, pro?1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI.
| S-EPMC3725821 | biostudies-literature
Project description:Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple-helical domains of type I collagen alpha1(I) and type II collagen alpha1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, cartilage-associated protein (CRTAP) and prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis-trans isomerase cyclophilin-B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and 16 with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, "popcorn" epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations.
| S-EPMC2671575 | biostudies-literature
Project description:Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI.
| S-EPMC6817720 | biostudies-literature
Project description:BackgroundOsteogenesis 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.MethodsWe 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.ResultsWe 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.ConclusionOur 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.
| S-EPMC5558703 | biostudies-literature
Project description:Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.
| S-EPMC8307903 | biostudies-literature
Project description:Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.
| S-EPMC4457955 | biostudies-literature
Project description:The heritable disorder osteogenesis imperfecta (OI) is characterized by bone fragility and low bone mass. OI type VI is an autosomal recessive form of the disorder with moderate to severe bone fragility. OI type VI is caused by mutations in the serpin peptidase inhibitor, clade F, member 1 (SERPINF1), the gene coding for pigment epithelium-derived factor (PEDF). Here, we report a patient with OI type VI caused by a novel homozygous intronic variant in SERPINF1 identified by whole-exome sequencing (WES). The mutation was not identified using a low bone mass gene panel based on next-generation sequencing. This variant creates a novel consensus splice donor site (AGGC to AGGT) in intron 4. Analysis of cDNA generated from fibroblasts revealed retention of a 32-bp intronic fragment between exons 4 and 5 in the cDNA, a result of alternative splicing from the novel splice-donor site. As a result, the aberrant insertion of this intronic fragment generated a frameshift pathogenic variant and induced nonsense-mediated decay. Furthermore, gene expression by quantitative PCR showed SERPINF1 expression was dramatically reduced in patient fibroblasts, and PEDF level was also significantly reduced in the patient's plasma. In conclusion, we report a novel homozygous variant that generates an alternative splice-donor in intron 4 of SERPINF1 which gives rise to severe bone fragility. The work also demonstrates clinical utility of WES analysis, and consideration of noncoding variants, in the diagnostic setting of rare bone diseases. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
| S-EPMC6124173 | biostudies-literature
Project description:BackgroundOsteogenesis imperfecta (OI) is a heterogeneous connective tissue disorder characterized by an increased tendency for fractures throughout life. Autosomal dominant (AD) mutations in COL1A1 and COL1A2 are causative in approximately 85% of cases. In recent years, recessive variants in genes involved in collagen processing have been found. Hypodontia (< 6 missing permanent teeth) and oligodontia (≥ 6 missing permanent teeth) have previously been reported in individuals with OI. The aim of the present cross-sectional study was to investigate whether children and adolescents with OI and oligodontia and hypodontia also present with variants in other genes with potential effects on tooth development. The cohort comprised 10 individuals (7.7-19.9 years of age) with known COL1A1/A2 variants who we clinically and radiographically examined and further genetically evaluated by whole-genome sequencing. All study participants were treated at the Astrid Lindgren Children's Hospital at Karolinska University Hospital, Stockholm (Sweden's national multidisciplinary pediatric OI team). We evaluated a panel of genes that were associated with nonsyndromic and syndromic hypodontia or oligodontia as well as that had been found to be involved in tooth development in animal models.ResultsWe detected a homozygous nonsense variant in CREB3L1, p.Tyr428*, c.1284C > A in one boy previously diagnosed with OI type III. COL1A1 and COL1A2 were the only two genes among 9 individuals which carried a pathogenic mutation. We found rare variants with unknown significance in several other genes related to tooth development.ConclusionsOur findings suggest that mutations in COL1A1, COL1A2, and CREB3L1 may cause hypodontia and oligodontia in OI. The findings cannot exclude additive effects from other modifying or interacting genes that may contribute to the severity of the expressed phenotype. Larger cohorts and further functional studies are needed.
| S-EPMC7110904 | biostudies-literature