New insights on the clinical variability of FKBP10 mutations

To date 45 autosomal recessive disease-causing variants are reported in the FKBP10 gene. Those variant were found to be associated with Osteogenesis Imperfecta (OI) for which the hallmark phenotype is bone fractuers or Bruck Syndrome (BS) where bone fractures are accompanied with contractures. In addition, a specific homozygous FKBP10 mutation (p.Tyr293del) has been described in Yup'ik Inuit population to cause Kuskokwim syndrome (KS) in which contractures without fractures are observed. Here we present an extended Palestinian family with 10 affected individuals harboring a novel homozygous splice site mutation, c.391+4A > T in intron 2 of the FKBP10 gene, in which the three above mentioned syndromes segregate as a result of skipping of exon 2 and absence of the FKBP65 protein. At the biochemical level, Hydroxylysyl pyridinoline (HP)/lysyl pyridinoline (LP) values were inversely correlated with OI phenotypes, a trend we could confirm in our patients. Our findings illustrate that single familial FKBP10 mutations can result in a phenotypic spectrum, ranging from fractures without contractures, to fractures and contractures and even to only contractures. This broad intrafamilial clinical variability within one single family is a new finding in the field of bone fragility

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results: We evaluated two suitable RNA isolation kits (theRNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5x dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention

b3galt6 Knock-out zebrafish recapitulate β3GalT6-deficiency disorders in human and reveal a trisaccharide proteoglycan linkage region

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human beta 3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study, therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention

Characterization of a col1a1a haploinsufficient zebrafish model for Osteogenesis Imperfecta type I

Introduction: ‘Osteogenesis Imperfecta (OI) is heritable fragile bone disorder, in most cases caused by autosomal dominant mutations in the genes encoding the type I collagen alpha chains. Animal models have proved indispensable for unraveling molecular mechanisms in OI pathogenesis. The zebrafish has recently shown to be a useful vertebrate organism to model OI both at the phenotypic and molecular level. Two different zebrafish mutants, harboring missense mutations in the triple helical domain of col1a1a, encoding the α1 chain of the type I collagen protein, have been described. They display generalized reduced bone density and misshapen bones with evidence of fractures. However, until now, no zebrafish mutants carrying a nonsense mutation in col1a1a have been described. Such mutant, displaying a quantitative defect of collagen type I synthesis, could serve as a model for the mild OI type I. We have characterized a zebrafish mutant that is heterozygous for a nonsense mutation in the col1a1a gene and evaluated this mutant as a possible model for OI type I. Methodology: The described col1a1a nonsense mutant was generated by the Zebrafish Mutation Project (ZMP). The bone was visualized using alizarin red staining. Adult zebrafish were scanned with a Triumph µCT scanner to determine bone densities. To assess osteoblast activity, embryos were treated with the osteoblast stimulating agent Retinoic Acid (RA) or DMSO from 4 days post fertilization (dpf) to 8 dpf and stained with alizarin complexone in order to evaluate the ossification in the trunk region. Results: Heterozygous col1a1a zebrafish mutants were phenotyped both at larval and adult stages. Larvae displayed no abnormalities in bone structure and geometry or delayed ossification at 8 dpf. The fin folds, appeared to have an overall normal appearance at 3 dpf. Adult mutant fish appeared to have the same body length as their wild type siblings. Bone staining and µCT-scanning revealed no skeletal abnormalities or reduced Bone Mineral Density (BMD). Quantification of type I collagen by western blotting revealed a 25% decrease of the α1(I) chain, in both larval and adult mutants. Osteoblast activity in mutant embryos was similar to wild type embryos as assessed by RA treatment. Discussion: . Thorough phenotypic analysis of a zebrafish mutant carrying a heterozygous nonsense mutation in the col1a1a gene did not reveal bone fractures or other skeletal malformations. Moreover, no decrease in bone density or osteoblastic activity could be detected Protein analysis in the heterozygous mutants revealed a decrease of only 25 % of the α1(I) chain, a reduction that is considered to be insufficient to give rise to a skeletal phenotype. This reduction of only 25% can be explained by the existence of a paralogue of col1a1a, col1a1b, also encoding for the α1(I) chain Therefore, we are currently creating a double mutant where both col1a1a and col1a1b paralogues are knocked out with the expectation that this will be a more relevant model for OI type I

Characterization of a COL1A1A haploinsufficient zebrafish model for Osteogenesis Imperfecta type I

Introduction: Animal models for OI have proved indispensable for unraveling molecular mechanisms in OI pathogenesis. The zebrafish has recently shown to be a useful vertebrate organism to model OI both at the phenotypic and molecular level. Forward genetic screens for skeletal dysplasia in adult zebrafish have identified mutations in bmp1a and col1a1a that accurately model OI. Two different zebrafish mutants, harboring missense mutations in the triple helical domain of col1a1a have been described. They display generalized reduced bone density and misshapen bones with evidence of fractures. However, until now, no zebrafish mutants carrying a nonsense mutation in col1a1a have been described. Such mutant, displaying a quantitative defect of collagen type I synthesis, could serve as a model for the mild OI type I. We have characterized a zebrafish mutant that is heterozygous for a nonsense mutation in the col1a1a gene and evaluated this mutant as a possible model for OI type I. Methodology: The described col1a1a nonsense mutant was generated by the Zebrafish Mutation Project (ZMP). The bone was visualized in live zebrafish embryos using alizarin complexone and in fixed fish using alizarin red. Adult zebrafish were scanned using a Triumph µCT scanner and the data was analyzed using AMIDE software in order to determine bone densities. To assess osteoblast activity, embryos were treated with the osteoblast stimulating agent Retinoic Acid (RA) or DMSO from 4 days post fertilization (dpf) to 8 dpf and stained with alizarin complexone in order to evaluate the ossification in the trunk region. To quantify type I collagen a western blot was performed using protein lysates prepared form whole embryos at 4 dpf and from adult fish by caudal fin amputation. Results: Heterozygous col1a1a zebrafish mutants were phenotyped both at larval and adult stages. Larvae displayed no abnormalities in bone structure and geometry or delayed ossification at 8 dpf. The fin folds, appeared to have an overall normal appearance at 3 dpf. Adult mutant fish appeared to have the same body length as their wild type siblings. Bone staining and µCT-scanning revealed no skeletal abnormalities or reduced Bone Mineral Density (BMD). Quantification of type I collagen by western blotting revealed a 25% decrease of the α1 chain in both larval and adult mutants. Osteoblast activity in mutant embryos was similar to wild type embryos as assessed by RA treatment. Discussion: We have characterized a zebrafish mutant carrying a nonsense mutation in the col1a1a gene. Phenotypic analysis of heterozygous mutants, both in larvae and adults, didn’t show a decrease in BMD, presence of bone fractures or other skeletal malformations. Moreover, osteoblast activity is not affected as shown by RA treatment. Western blotting for type I collagen revealed a decrease of only 25 % of the α1 chain, a reduction that might be insufficient to give rise to a skeletal phenotype. Moreover, bone defects tend to be milder when modeled in zebrafish, as the mechanical load on the skeleton is less due to the aqueous environment. Also, the paralogues col1a1a and col1a1b might have redundant functions, explaining the absence of a bone phenotype in the col1a1a mutant. Therefore, we are creating a double mutant where both col1a1a and col1a1b paralogues are knocked out with the expectation that this will be a more relevant model for OI type I. References: Asharani et al., Am J Hum Genet. 2012 Apr 6;90(4):661-74 Fisher et al., Dev Biol. 2003 Dec 1;264(1):64-7

A zebrafish model for Bruck Syndrome caused by PLOD2 mutations

Introduction: Bruck syndrome, which exhibits the bone fragility of OI and joint contractures, is caused by recessive mutations in either PLOD2 or FKBP10. PLOD2 is encoding the lysyl-hydroxylase 2 (LH2) enzyme, which is a collagen telopeptide lysyl hydroxylase (ref). Defective LH2 causes underhydroxylation of lysine residues within the telopeptides of type I collagen, resulting in aberrant cross-linking of bone collagen. Although the molecular role of PLOD2 has been documented, no animal models for this disease have been reported. To further elucidate the function of PLOD2 in vertebrate skeletal development, we generated a zebrafish model Method: The described plod2 nonsense mutation was generated by the Zebrafish Mutation Project (ZMP). Adult homozygous mutant zebrafish were scanned using a Triumph µCT scanner and the data was analyzed using AMIDE software in order to determine bone densities. Furthermore, the bone in fixed mutants was stained using alizarin red. Results: Preliminary results indicate that homozygous plod2 mutant zebrafish have a shortened body axis and malformed craniofacial structures. Detailed bone phenotyping using µCT and alizarin red staining revealed malformations of the skeleton including the presence of platyspondyly. Bone density measurements did not reveal a decrease in bone density. Discussion: We generated a zebrafish mutant carrying a homozygous nonsense mutation in plod2. Although preliminary, bone phenotyping revealed severe bone abnormalities with a shortened body axis and the presence of platyspondyly as the most remarkable findings, features that are also regularly detected in Bruck Syndrome patients. Therefore, this zebrafish mutant is a promising model for further unraveling the pathogenetic mechanisms leading to Bruck Syndrome. References: Ha-Vinh R, Alanay Y, Bank RA, Campos-Xavier AB, Zankl A, Superti-Furga A, Bonafe´ L (2004) Phenotypic and molecular characterization of Bruck syndrome (osteogenesis imperfecta with contractures of the large joints) caused by a recessive mutation in PLOD2. Am J Med Genet A 131:115–120

Abnormal Bone Collagen Cross‐Linking in Osteogenesis Imperfecta/Bruck Syndrome Caused by Compound Heterozygous PLOD2 Mutations

ABSTRACT Bruck syndrome (BS) is a congenital disorder characterized by joint flexion contractures, skeletal dysplasia, and increased bone fragility, which overlaps clinically with osteogenesis imperfecta (OI). On a genetic level, BS is caused by biallelic mutations in either FKBP10 or PLOD2. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of cross‐linking lysine residues in fibrillar collagen telopeptide domains. This modification enables collagen to form chemically stable (permanent) intermolecular cross‐links in the extracellular matrix. Normal bone collagen develops a unique mix of such stable and labile lysyl‐oxidase–mediated cross‐links, which contribute to bone strength, resistance to microdamage, and crack propagation, as well as the ordered deposition of mineral nanocrystals within the fibrillar collagen matrix. Bone from patients with BS caused by biallelic FKBP10 mutations has been shown to have abnormal collagen cross‐linking; however, to date, no direct studies of human bone from BS caused by PLOD2 mutations have been reported. Here the results from a study of a 4‐year‐old boy with BS caused by compound heterozygous mutations in PLOD2 are discussed. Diminished hydroxylation of type I collagen telopeptide lysines but normal hydroxylation at triple‐helical sites was found. Consequently, stable trivalent cross‐links were essentially absent. Instead, allysine aldol dimeric cross‐links dominated as in normal skin collagen. Furthermore, in contrast to the patient's bone collagen, telopeptide lysines in cartilage type II collagen cross‐linked peptides from the patient's urine were normally hydroxylated. These findings shed light on the complex mechanisms that control the unique posttranslational chemistry and cross‐linking of bone collagen, and how, when defective, they can cause brittle bones and related connective tissue problems. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research