Publications by authors named "Shireen R Lamandé"

46 Publications

Circadian time series proteomics reveals daily dynamics in cartilage physiology.

Osteoarthritis Cartilage 2021 Feb 19. Epub 2021 Feb 19.

Wellcome Centre for Cell Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, UK. Electronic address:

Objective: Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue.

Methods: We investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis.

Results: Out of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing.

Conclusions: Our circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis.
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http://dx.doi.org/10.1016/j.joca.2021.02.008DOI Listing
February 2021

Resveratrol Promotes Hypertrophy in Wildtype Skeletal Muscle and Reduces Muscle Necrosis and Gene Expression of Inflammatory Markers in Mice.

Molecules 2021 Feb 6;26(4). Epub 2021 Feb 6.

Faculty of Veterinary and Agricultural Science, The University of Melbourne, Parkville, VIC 3010, Australia.

Duchenne muscular dystrophy (DMD) is a progressive fatal neuromuscular disorder with no cure. Therapies to restore dystrophin deficiency have been approved in some jurisdictions but long-term effectiveness is yet to be established. There is a need to develop alternative strategies to treat DMD. Resveratrol is a nutraceutical with anti-inflammatory properties. Previous studies have shown high doses (100-400 mg/kg bodyweight/day) benefit mice. We treated 4-week-old and wildtype mice with a lower dose of resveratrol (5 mg/kg bodyweight/day) for 15 weeks. Voluntary exercise was used to test if a lower dosage than previously tested could reduce exercise-induced damage where a greater inflammatory infiltrate is present. We found resveratrol promoted skeletal muscle hypertrophy in wildtype mice. In dystrophic muscle, resveratrol reduced exercise-induced muscle necrosis. Gene expression of immune cell markers, CD86 and CD163 were reduced; however, signalling targets associated with resveratrol's mechanism of action including Sirt1 and NF-κB were unchanged. In conclusion, a lower dose of resveratrol compared to the dosage used by other studies reduced necrosis and gene expression of inflammatory cell markers in dystrophic muscle suggesting it as a therapeutic candidate for treating DMD.
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http://dx.doi.org/10.3390/molecules26040853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915385PMC
February 2021

Modulating the Mechanical Activation of TRPV4 at the Cell-Substrate Interface.

Front Bioeng Biotechnol 2020 18;8:608951. Epub 2021 Jan 18.

EMBL Australia Node in Single Molecule Science and Cellular and Systems Physiology, Faculty of Medicine, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.

Ion channels activated by mechanical inputs are important force sensing molecules in a wide array of mammalian cells and tissues. The transient receptor potential channel, TRPV4, is a polymodal, nonselective cation channel that can be activated by mechanical inputs but only if stimuli are applied directly at the interface between cells and their substrate, making this molecule a context-dependent force sensor. However, it remains unclear how TRPV4 is activated by mechanical inputs at the cell-substrate interface, which cell intrinsic and cell extrinsic parameters might modulate the mechanical activation of the channel and how mechanical activation differs from TRPV4 gating in response to other stimuli. Here we investigated the impact of substrate mechanics and cytoskeletal components on mechanically evoked TRPV4 currents and addressed how point mutations associated with TRPV4 phosphorylation and arthropathy influence mechanical activation of the channel. Our findings reveal distinct regulatory modulation of TRPV4 from the mechanically activated ion channel PIEZO1, suggesting the mechanosensitivity of these two channels is tuned in response to different parameters. Moreover, our data demonstrate that the effect of point mutations in TRPV4 on channel activation are profoundly dependent on the gating stimulus.
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http://dx.doi.org/10.3389/fbioe.2020.608951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848117PMC
January 2021

Generation of a miR-26b stem-loop knockout human iPSC line, MCRIi019-A-1, using CRISPR/Cas9 editing.

Stem Cell Res 2020 Dec 10;50:102118. Epub 2020 Dec 10.

Murdoch Children's Research Institute, Parkville, Victoria, Australia; Department of Paediatrics, University of Melbourne, Australia. Electronic address:

miR-26b has been implicated in a wide range of human diseases, including cancer, diabetes, heart disease, Alzheimer's disease and osteoarthritis. To provide a tool to explore the importance of miR-26b in this broad context, we have generated and characterized a homozygous miR-26b stem-loop knockout human iPSC line. This gene-edited line exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line will be valuable for studies investigating disease mechanisms and testing therapeutic strategies in vitro.
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http://dx.doi.org/10.1016/j.scr.2020.102118DOI Listing
December 2020

CRISPR/Cas9 editing to generate a heterozygous COL2A1 p.G1170S human chondrodysplasia iPSC line, MCRIi019-A-2, in a control iPSC line, MCRIi019-A.

Stem Cell Res 2020 10 6;48:101962. Epub 2020 Sep 6.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To develop an in vitro disease model of a human chondrodysplasia, we used CRISPR/Cas9 gene editing to generate a heterozygous COL2A1 exon 50 c.3508 GGT > TCA (p.G1170S) mutation in a control human iPSC line. Both the control and COL2A1 mutant lines displayed typical iPSC characteristics, including normal cell morphology, expression of pluripotency markers, the ability to differentiate into endoderm, ectoderm and mesoderm lineages and normal karyotype. These chondrodysplasia mutant and isogenic control cell lines can be used to explore disease mechanisms underlying type II collagenopathies and aid in the discovery of new therapeutic strategies.
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http://dx.doi.org/10.1016/j.scr.2020.101962DOI Listing
October 2020

CRISPR/Cas9 gene editing of a SOX9 reporter human iPSC line to produce two TRPV4 patient heterozygous missense mutant iPSC lines, MCRIi001-A-3 (TRPV4 p.F273L) and MCRIi001-A-4 (TRPV4 p.P799L).

Stem Cell Res 2020 10 3;48:101942. Epub 2020 Aug 3.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To produce in vitro models of human chondrodysplasias caused by dominant missense mutations in TRPV4, we used CRISPR/Cas9 gene editing to introduce two heterozygous patient mutations (p.F273L and p.P799L) into an established control human iPSC line. This control line expressed a fluorescent reporter (tdTomato) at the SOX9 locus to allow real-time monitoring of cartilage differentiation by SOX9 expression. Both TRPV4 mutant iPSC lines had normal karyotypes, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. These iPSC lines, with the parental isogenic control, will be used to study TRPV4 chondrodysplasia mechanisms and explore therapeutic approaches.
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http://dx.doi.org/10.1016/j.scr.2020.101942DOI Listing
October 2020

Generation of a heterozygous COL2A1 (p.R989C) spondyloepiphyseal dysplasia congenita mutation iPSC line, MCRIi001-B, using CRISPR/Cas9 gene editing.

Stem Cell Res 2020 05 11;45:101843. Epub 2020 May 11.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To produce an in vitro model of the human chondrodysplasia, spondyloepiphyseal dysplasia congenita, we used CRISPR/Cas9 gene editing to generate a heterozygous patient COL2A1 mutation in an established control human iPSC line. The gene-edited heterozygous COL2A1 p.R989C line had a normal karyotype, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. When differentiated into cartilage this cell line and the parental isogenic control may be used to explore disease mechanisms and evaluate therapeutic approaches.
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http://dx.doi.org/10.1016/j.scr.2020.101843DOI Listing
May 2020

Generation of a SOX9-tdTomato reporter human iPSC line, MCRIi001-A-2, using CRISPR/Cas9 editing.

Stem Cell Res 2020 01 19;42:101689. Epub 2019 Dec 19.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To develop an iPSC SOX9 reporter line for monitoring differentiation into SOX9 expressing cells such as chondrocytes, cranial neural crest and Sertoli cells, we used gene editing to introduce sequences encoding the tdTomato fluorescent protein into the SOX9 locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. Endogenous SOX9 expression was undisturbed and the tdTomato fluorescent reporter mirrored SOX9 mRNA expression. This iPSC line will be useful for assessing iPSC differentiation into SOX9-expressing cells and enrichment by cell sorting.
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http://dx.doi.org/10.1016/j.scr.2019.101689DOI Listing
January 2020

Expression profiling in exercised mdx suggests a role for extracellular proteins in the dystrophic muscle immune response.

Hum Mol Genet 2020 02;29(3):353-368

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne 3052, Australia.

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disorder caused by mutations in the DMD gene that leads to the absence or severe reduction of dystrophin protein in muscle. The mdx mouse, also dystrophin deficient, is the model most widely used to study the pathology and test potential therapies, but the phenotype is milder than human DMD. This limits the magnitude and range of histological damage parameters and molecular changes that can be measured in pre-clinical drug testing. We used 3 weeks of voluntary wheel running to exacerbate the mdx phenotype. In mdx mice, voluntary exercise increased the amount of damaged necrotic tissue and macrophage infiltration. Global gene expression profiling revealed that exercise induced additional and larger gene expression changes in mdx mice and the pathways most impacted by exercise were all related to immune function or cell-extracellular matrix (ECM) interactions. When we compared the matrisome and inflammation genes that were dysregulated in mdx with those commonly differentially expressed in DMD, we found the exercised mdx molecular signature more closely resembled that of DMD. These gene expression changes in the exercised mdx model thus provide more scope to assess the effects of pre-clinical treatments. Our gene profiling comparisons also highlighted upregulation of ECM proteins involved in innate immunity pathways, proteases that can release them, downstream receptors and signaling molecules in exercised mdx and DMD, suggesting that the ECM could be a major source of pro-inflammatory molecules that trigger and maintain the immune response in dystrophic muscle.
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http://dx.doi.org/10.1093/hmg/ddz266DOI Listing
February 2020

Identification of Two Independent Variants in Dogs with Ehlers-Danlos Syndrome.

Genes (Basel) 2019 09 21;10(10). Epub 2019 Sep 21.

Murdoch Children's Research Institute, Parkville, Victoria 3052, Australia.

The Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis. Clinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole-genome sequencing revealed de novo mutations of in both cases, confirming the diagnosis of the classical form of EDS. The heterozygous p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in α1(V) mRNA nonsense-mediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, ultrastructural analysis of the dermis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed 'collagen cauliflowers', consistent with mutations underlying classical EDS. In the second case, DNA sequencing demonstrated a p.Gly1571Arg missense variant in the gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of the collagen V triple helix and thus affect protein stability and/or integration of the mutant collagen into the collagen V/collagen I heterotypic dermal fibrils. This is the first report of genetic variants in the gene causing the clinical presentation of EDS in dogs. These data provided further evidence of the important role of collagen V in dermal collagen fibrillogenesis. Importantly, from the clinical perspective, we showed the utility of DNA sequencing, combined with the established clinical criteria, in the accurate diagnosis of EDS in dogs.
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http://dx.doi.org/10.3390/genes10100731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826881PMC
September 2019

Extra-skeletal manifestations in mice affected by -dependent autosomal dominant osteopetrosis type 2 clinical and therapeutic implications.

Bone Res 2019 11;7:17. Epub 2019 Jun 11.

1Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy.

Autosomal dominant osteopetrosis type 2 (ADO2) is a high-density brittle bone disease characterized by bone pain, multiple fractures and skeletal-related events, including nerve compression syndrome and hematological failure. We demonstrated that in mice carrying the heterozygous mutation, whose human mutant homolog affects patients, the clinical impacts of ADO2 extend beyond the skeleton, affecting several other organs. The hallmark of the extra-skeletal alterations is a consistent perivascular fibrosis, associated with high numbers of macrophages and lymphoid infiltrates. Fragmented clinical information in a small cohort of patients confirms extra-skeletal alterations consistent with a systemic disease, in line with the observation that the gene is expressed in many organs. ADO2 mice also show anxiety and depression and their brains exhibit not only perivascular fibrosis but also β-amyloid accumulation and astrogliosis, suggesting the involvement of the nervous system in the pathogenesis of the ADO2 extra-skeletal alterations. Extra-skeletal organs share a similar cellular pathology, confirmed also in vitro in bone marrow mononuclear cells and osteoclasts, characterized by an impairment of the exit pathway of the protein product, ClC7, through the Golgi, with consequent reduced ClC7 expression in late endosomes and lysosomes, associated with high vesicular pH and accumulation of autophagosome markers. Finally, an experimental siRNA therapy, previously proven to counteract the bone phenotype, also improves the extra-skeletal alterations. These results could have important clinical implications, supporting the notion that a systematic evaluation of ADO2 patients for extra-skeletal symptoms could help improve their diagnosis, clinical management, and therapeutic options.
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http://dx.doi.org/10.1038/s41413-019-0055-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559989PMC
June 2019

The use of simultaneous reprogramming and gene correction to generate an osteogenesis imperfecta patient COL1A1 c. 3936 G>T iPSC line and an isogenic control iPSC line.

Stem Cell Res 2019 07 4;38:101453. Epub 2019 May 4.

Murdoch Children's Research Institute, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Australia. Electronic address:

To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we used a simultaneous reprogramming and CRISPR-Cas9 genome editing method to produce an iPSC line with the heterozygous patient mutation (COL1A1 c. 3936 G>T) along with an isogenic gene-corrected control iPSC line. Both IPSC lines had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This osteogenesis imperfecta mutant and isogenic iPSC control line will be of use in exploring disease mechanisms and therapeutic approaches in vitro.
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http://dx.doi.org/10.1016/j.scr.2019.101453DOI Listing
July 2019

Generation of a heterozygous COL1A1 (c.3969_3970insT) osteogenesis imperfecta mutation human iPSC line, MCRIi001-A-1, using CRISPR/Cas9 editing.

Stem Cell Res 2019 05 23;37:101449. Epub 2019 Apr 23.

Murdoch Children's Research Institute, University of Melbourne, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Australia. Electronic address:

To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we have used gene editing to produce a facsimile of the patient heterozygous COL1A1 mutation in an established control iPSC line. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line and the isogenic parental iPSC line will be of use in exploring osteogenesis imperfecta disease mechanisms and therapeutic approaches in vitro.
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http://dx.doi.org/10.1016/j.scr.2019.101449DOI Listing
May 2019

A recurrent COL6A1 pseudoexon insertion causes muscular dystrophy and is effectively targeted by splice-correction therapies.

JCI Insight 2019 03 21;4(6). Epub 2019 Mar 21.

Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland, USA.

The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI-related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.
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http://dx.doi.org/10.1172/jci.insight.124403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483063PMC
March 2019

Genetic Disorders of the Extracellular Matrix.

Anat Rec (Hoboken) 2020 06 6;303(6):1527-1542. Epub 2019 Mar 6.

Musculoskeletal Research, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville Victoria, Australia.

Mutations in the genes for extracellular matrix (ECM) components cause a wide range of genetic connective tissues disorders throughout the body. The elucidation of mutations and their correlation with pathology has been instrumental in understanding the roles of many ECM components. The pathological consequences of ECM protein mutations depend on its tissue distribution, tissue function, and on the nature of the mutation. The prevalent paradigm for the molecular pathology has been that there are two global mechanisms. First, mutations that reduce the production of ECM proteins impair matrix integrity largely due to quantitative ECM defects. Second, mutations altering protein structure may reduce protein secretion but also introduce dominant negative effects in ECM formation, structure and/or stability. Recent studies show that endoplasmic reticulum (ER) stress, caused by mutant misfolded ECM proteins, makes a significant contribution to the pathophysiology. This suggests that targeting ER-stress may offer a new therapeutic strategy in a range of ECM disorders caused by protein misfolding mutations. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
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http://dx.doi.org/10.1002/ar.24086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318566PMC
June 2020

Effect of rapamycin on bone mass and strength in the α2(I)-G610C mouse model of osteogenesis imperfecta.

J Cell Mol Med 2019 03 30;23(3):1735-1745. Epub 2018 Dec 30.

Orthopaedic Research and Biotechnology Unit, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.

Osteogenesis imperfecta (OI) is commonly caused by heterozygous type I collagen structural mutations that disturb triple helix folding and integrity. This mutant-containing misfolded collagen accumulates in the endoplasmic reticulum (ER) and induces a form of ER stress associated with negative effects on osteoblast differentiation and maturation. Therapeutic induction of autophagy to degrade the mutant collagens could therefore be useful in ameliorating the ER stress and deleterious downstream consequences. To test this, we treated a mouse model of mild to moderate OI (α2(I) G610C) with dietary rapamycin from 3 to 8 weeks of age and effects on bone mass and mechanical properties were determined. OI bone mass and mechanics were, as previously reported, compromised compared to WT. While rapamycin treatment improved the trabecular parameters of WT and OI bones, the biomechanical deficits of OI bones were not rescued. Importantly, we show that rapamycin treatment suppressed the longitudinal and transverse growth of OI, but not WT, long bones. Our work demonstrates that dietary rapamycin offers no clinical benefit in this OI model and furthermore, the impact of rapamycin on OI bone growth could exacerbate the clinical consequences during periods of active bone growth in patients with OI caused by collagen misfolding mutations.
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http://dx.doi.org/10.1111/jcmm.14072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378195PMC
March 2019

Collagen VI disorders: Insights on form and function in the extracellular matrix and beyond.

Matrix Biol 2018 10 22;71-72:348-367. Epub 2017 Dec 22.

Musculoskeletal Research, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Vic, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Vic, Australia.

Mutations in the three canonical collagen VI genes, COL6A1, COL6A2 and COL6A3, cause a spectrum of muscle disease from Bethlem myopathy at the mild end to the severe Ullrich congenital muscular dystrophy. Mutations can be either dominant or recessive and the resulting clinical severity is influenced by the way mutations impact the complex collagen VI assembly process. Most mutations are found towards the N-terminus of the triple helical collagenous domain and compromise extracellular microfibril assembly. Outside the triple helix collagen VI is highly polymorphic and discriminating mutations from rare benign changes remains a major diagnostic challenge. Collagen VI deficiency alters extracellular matrix structure and biomechanical properties and leads to increased apoptosis and oxidative stress, decreased autophagy, and impaired muscle regeneration. Therapies that target these downstream consequences have been tested in a collagen VI null mouse and also in small human trials where they show modest clinical efficacy. An important role for collagen VI in obesity, cancer and diabetes is emerging. A major barrier to developing effective therapies is the paucity of information about how collagen VI deficiency in the extracellular matrix signals the final downstream consequences - the receptors involved and the intracellular messengers await further characterization.
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http://dx.doi.org/10.1016/j.matbio.2017.12.008DOI Listing
October 2018

Nutraceuticals and Their Potential to Treat Duchenne Muscular Dystrophy: Separating the Credible from the Conjecture.

Nutrients 2016 Nov 9;8(11). Epub 2016 Nov 9.

Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.

In recent years, complementary and alternative medicine has become increasingly popular. This trend has not escaped the Duchenne Muscular Dystrophy community with one study showing that 80% of caregivers have provided their Duchenne patients with complementary and alternative medicine in conjunction with their traditional treatments. These statistics are concerning given that many supplements are taken based on purely "anecdotal" evidence. Many nutraceuticals are thought to have anti-inflammatory or anti-oxidant effects. Given that dystrophic pathology is exacerbated by inflammation and oxidative stress these nutraceuticals could have some therapeutic benefit for Duchenne Muscular Dystrophy (DMD). This review gathers and evaluates the peer-reviewed scientific studies that have used nutraceuticals in clinical or pre-clinical trials for DMD and thus separates the credible from the conjecture.
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http://dx.doi.org/10.3390/nu8110713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133099PMC
November 2016

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

Stem Cell Reports 2016 09 1;7(3):518-526. Epub 2016 Sep 1.

Murdoch Childrens Research Institute, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, VIC 3050, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032031PMC
September 2016

Diagnosis and etiology of congenital muscular dystrophy: We are halfway there.

Ann Neurol 2016 07 25;80(1):101-11. Epub 2016 May 25.

Institute for Neuroscience and Muscle Research, Kids Research Institute, Children's Hospital at Westmead, Westmead, New South Wales, Australia.

Objective: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and next generation sequencing (NGS) technologies.

Methods: A total of 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy, and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS.

Results: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan, or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39 of 123). Of 85 patients presenting in the past 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43 of 85). The diagnoses within the cohort were heterogeneous. Forty-five of 59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11 of 59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome, and 2 had microdeletions. Within the cohort, 5 patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1), and 7 had variants in TTN or RYR1, large genes that are technically difficult to Sanger sequence.

Interpretation: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. Ann Neurol 2016;80:101-111.
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http://dx.doi.org/10.1002/ana.24687DOI Listing
July 2016

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

J Biol Chem 2016 Feb 14;291(7):3197-208. Epub 2015 Dec 14.

From the Department of Paediatrics, University of Melbourne, Parkville 3052, Australia, the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia,

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.
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http://dx.doi.org/10.1074/jbc.M115.704817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751368PMC
February 2016

Aberrant mitochondria in a Bethlem myopathy patient with a homozygous amino acid substitution that destabilizes the collagen VI α2(VI) chain.

J Biol Chem 2015 Feb 22;290(7):4272-81. Epub 2014 Dec 22.

From the Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia, Department of Paediatrics, University of Melbourne, Parkville 3010, Australia,

Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.
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http://dx.doi.org/10.1074/jbc.M114.632208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326835PMC
February 2015

Activating internal ribosome entry to treat Duchenne muscular dystrophy.

Nat Med 2014 Sep;20(9):987-8

Murdoch Childrens Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Melbourne, Victoria, Australia.

Mutations in the DMD gene, encoding dystrophin, cause the most common forms of muscular dystrophy. A new study shows that forcing translation of DMD from an internal ribosome entry site can alleviate Duchenne muscular dystrophy symptoms in a mouse model.
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http://dx.doi.org/10.1038/nm.3677DOI Listing
September 2014

Nonsense-mediated mRNA decay of collagen -emerging complexity in RNA surveillance mechanisms.

J Cell Sci 2013 Jun 31;126(Pt 12):2551-60. Epub 2013 May 31.

Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Australia.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mRNA surveillance system that degrades mRNA transcripts that harbour a premature translation-termination codon (PTC), thus reducing the synthesis of truncated proteins that would otherwise have deleterious effects. Although extensive research has identified a conserved repertoire of NMD factors, these studies have been performed with a restricted set of genes and gene constructs with relatively few exons. As a consequence, NMD mechanisms are poorly understood for genes with large 3' terminal exons, and the applicability of the current models to large multi-exon genes is not clear. In this Commentary, we present an overview of the current understanding of NMD and discuss how analysis of nonsense mutations in the collagen gene family has provided new mechanistic insights into this process. Although NMD of the collagen genes with numerous small exons is consistent with the widely accepted exon-junction complex (EJC)-dependent model, the degradation of Col10a1 transcripts with nonsense mutations cannot be explained by any of the current NMD models. Col10a1 NMD might represent a fail-safe mechanism for genes that have large 3' terminal exons. Defining the mechanistic complexity of NMD is important to allow us to understand the pathophysiology of the numerous genetic disorders caused by PTC mutations.
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http://dx.doi.org/10.1242/jcs.120220DOI Listing
June 2013

Autosomal dominant congenital spinal muscular atrophy: a true form of spinal muscular atrophy caused by early loss of anterior horn cells.

Brain 2012 Jun;135(Pt 6):1714-23

Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Locked Bag 4001, Westmead, New South Wales, 2145, Australia.

Autosomal dominant congenital spinal muscular atrophy is characterized by predominantly lower limb weakness and wasting, and congenital or early-onset contractures of the hip, knee and ankle. Mutations in TRPV4, encoding a cation channel, have recently been identified in one large dominant congenital spinal muscular atrophy kindred, but the genetic basis of dominant congenital spinal muscular atrophy in many families remains unknown. It has been hypothesized that differences in the timing and site of anterior horn cell loss in the central nervous system account for the variations in clinical phenotype between different forms of spinal muscular atrophy, but there has been a lack of neuropathological data to support this concept in dominant congenital spinal muscular atrophy. We report clinical, electrophysiology, muscle magnetic resonance imaging and histopathology findings in a four generation family with typical dominant congenital spinal muscular atrophy features, without mutations in TRPV4, and in whom linkage to other known dominant neuropathy and spinal muscular atrophy genes has been excluded. The autopsy findings in the proband, who died at 14 months of age from an unrelated illness, provided a rare opportunity to study the neuropathological basis of dominant congenital spinal muscular atrophy. There was a reduction in anterior horn cell number in the lumbar and, to a lesser degree, the cervical spinal cord, and atrophy of the ventral nerve roots at these levels, in the absence of additional peripheral nerve pathology or abnormalities elsewhere along the neuraxis. Despite the young age of the child at the time of autopsy, there was no pathological evidence of ongoing loss or degeneration of anterior horn cells suggesting that anterior horn cell loss in dominant congenital spinal muscular atrophy occurs in early life, and is largely complete by the end of infancy. These findings confirm that dominant congenital spinal muscular atrophy is a true form of spinal muscular atrophy caused by a loss of anterior horn cells localized to lumbar and cervical regions early in development.
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http://dx.doi.org/10.1093/brain/aws108DOI Listing
June 2012

Mutations in TRPV4 cause an inherited arthropathy of hands and feet.

Nat Genet 2011 Oct 2;43(11):1142-6. Epub 2011 Oct 2.

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia.

Familial digital arthropathy-brachydactyly (FDAB) is a dominantly inherited condition that is characterized by aggressive osteoarthropathy of the fingers and toes and consequent shortening of the middle and distal phalanges. Here we show in three unrelated families that FDAB is caused by mutations encoding p.Gly270Val, p.Arg271Pro and p.Phe273Leu substitutions in the intracellular ankyrin-repeat domain of the cation channel TRPV4. Functional testing of mutant TRPV4 in HEK-293 cells showed that the mutant proteins have poor cell-surface localization. Calcium influx in response to the synthetic TRPV4 agonists GSK1016790A and 4αPDD was significantly reduced, and mutant channels did not respond to hypotonic stress. Others have shown that gain-of-function TRPV4 mutations cause skeletal dysplasias and peripheral neuropathies. Our data indicate that TRPV4 mutations that reduce channel activity cause a third phenotype, inherited osteoarthropathy, and show the importance of TRPV4 activity in articular cartilage homeostasis. Our data raise the possibility that TRPV4 may also have a role in age- or injury-related osteoarthritis.
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http://dx.doi.org/10.1038/ng.945DOI Listing
October 2011

TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families.

Orphanet J Rare Dis 2011 Jun 9;6:37. Epub 2011 Jun 9.

Genetic Health Services Victoria and Murdoch Childrens Research Institute, Parkville, Victoria, Australia.

Background: The TRPV4 gene encodes a calcium-permeable ion-channel that is widely expressed, responds to many different stimuli and participates in an extraordinarily wide range of physiologic processes. Autosomal dominant brachyolmia, spondylometaphyseal dysplasia Kozlowski type (SMDK) and metatropic dysplasia (MD) are currently considered three distinct skeletal dysplasias with some shared clinical features, including short stature, platyspondyly, and progressive scoliosis. Recently, TRPV4 mutations have been found in patients diagnosed with these skeletal phenotypes.

Methods And Results: We critically analysed the clinical and radiographic data on 26 subjects from 21 families, all of whom had a clinical diagnosis of one of the conditions described above: 15 with MD; 9 with SMDK; and 2 with brachyolmia. We sequenced TRPV4 and identified 9 different mutations in 22 patients, 4 previously described, and 5 novel. There were 4 mutation-negative cases: one with MD and one with SMDK, both displaying atypical clinical and radiographic features for these diagnoses; and two with brachyolmia, who had isolated spine changes and no metaphyseal involvement.

Conclusions: Our data suggest the TRPV4 skeletal dysplasias represent a continuum of severity with areas of phenotypic overlap, even within the same family. We propose that AD brachyolmia lies at the mildest end of this spectrum and, since all cases described with this diagnosis and TRPV4 mutations display metaphyseal changes, we suggest that it is not a distinct entity but represents the mildest phenotypic expression of SMDK.
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http://dx.doi.org/10.1186/1750-1172-6-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135501PMC
June 2011

Collagen VI microfibril formation is abolished by an {alpha}2(VI) von Willebrand factor type A domain mutation in a patient with Ullrich congenital muscular dystrophy.

J Biol Chem 2010 Oct 21;285(43):33567-76. Epub 2010 Aug 21.

Departments of Paediatrics, Murdoch Childrens Research Institute, University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia.

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.
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http://dx.doi.org/10.1074/jbc.M110.152520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963345PMC
October 2010

Reduction of lysyl hydroxylase 3 causes deleterious changes in the deposition and organization of extracellular matrix.

J Biol Chem 2009 Oct 20;284(41):28204-11. Epub 2009 Aug 20.

Department of Biochemistry, P.O. Box 3000, University of Oulu, FI-90014 Oulu, Finland.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF(+/-)) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF(+/-), may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.
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http://dx.doi.org/10.1074/jbc.M109.038190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788872PMC
October 2009