Shireen R. Lamandé

Genetic diseases of connective tissues: cellular and extracellular effects of ECM mutations.

Tissue-specific extracellular matrices (ECMs) are crucial for normal development and tissue function, and mutations in ECM genes result in a wide range of serious inherited connective tissue disorders. Mutations cause ECM dysfunction by combinations of two mechanisms. First, secretion of the mutated ECM components can be reduced by mutations affecting synthesis or by structural mutations causing cellular retention and/or degradation. Second, secretion of mutant protein can disturb crucial ECM interactions, structure and stability. Moreover, recent experiments suggest that endoplasmic reticulum (ER) stress, caused by mutant misfolded ECM proteins, contributes to the molecular pathology. Targeting ER stress might offer a new therapeutic strategy.

Autophagic elimination of misfolded procollagen aggregates in the endoplasmic reticulum as a means of cell protection.

Type I collagen is a major component of the extracellular matrix, and mutations in the collagen gene cause several matrix-associated diseases. These mutant procollagens are misfolded and often aggregated in the endoplasmic reticulum (ER). Although the misfolded procollagens are potentially toxic to the cell, little is known about how they are eliminated from the ER. Here, we show that procollagen that can initially trimerize but then aggregates in the ER are eliminated by an autophagy-lysosome pathway, but not by the ER-associated degradation (ERAD) pathway. Inhibition of autophagy by specific inhibitors or RNAi-mediated knockdown of an autophagy-related gene significantly stimulated accumulation of aggregated procollagen trimers in the ER, and activation of autophagy with rapamycin resulted in reduced amount of aggregates. In contrast, a mutant procollagen which has a compromised ability to form trimers was degraded by ERAD. Moreover, we found that autophagy plays an essential role in protecting cells against the toxicity of the ERAD-inefficient procollagen aggregates. The autophagic elimination of aggregated procollagen occurs independently of the ERAD system. These results indicate that autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that are not able to be removed by ERAD.

Mutations in TRPV4 cause an inherited arthropathy of hands and feet

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.

Diagnosis and etiology of congenital muscular dystrophy

Objective: We aimed to determine the frequency of all known forms of congenital muscular dystrophy (CMD) in a large Australasian cohort. Methods: We screened 101 patients with CMD with a combination of immunofluorescence, Western blotting, and DNA sequencing to identify disease-associated abnormalities in glycosylated α-dystroglycan, collagen VI, laminin α2, α7-integrin, and selenoprotein. Results: A total of 45% of the CMD cohort were assigned to an immunofluorescent subgroup based on their abnormal staining pattern. Abnormal staining for glycosylated α-dystroglycan was present in 25% of patients, and approximately half of these had reduced glycosylated α-dystroglycan by Western blot. Sequencing of the FKRP, fukutin, POMGnT1, and POMT1 genes in all patients with abnormal α-dystroglycan immunofluorescence identified mutations in one patient for each of these genes and two patients had mutations in POMT2. Twelve percent of patients had abnormalities in collagen VI immunofluorescence, and we identified disease-causing COL6 mutations in eight of nine patients in whom the genes were sequenced. Laminin α2 deficiency accounted for only 8% of CMD. α7-Integrin staining was absent in 12 of 45 patients studied, and ITGA7 gene mutations were excluded in all of these patients. Conclusions: We define the distribution of different forms of congenital muscular dystrophy in a large cohort of mixed ethnicity and demonstrate the utility and limitations of current diagnostic techniques.

Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability.

The protein truncation test (PTT) is a mutation‐detection method used to scan for premature termination (nonsense) mutations. PCR amplification of the DNA or mRNA source material is performed using forward primers containing a T7‐promoter sequence and translation initiation signals such that the resultant products can be transcribed and translated in vitro to identify the smaller truncated protein products. mRNA is commonly used as the source material, but success of the PTT and other RNA‐based mutation detection methods can be severely compromised by nonsense mutation‐induced mRNA decay, a well‐documented process that is often overlooked in mutation detection strategies. In this study, we develop an RNA‐based PTT that overcomes the problem of mRNA decay by preincubating cells with cycloheximide to stabilise the mutant mRNA. The effectiveness of this method for mutation detection in abundant mRNAs was demonstrated in osteogenesis imperfecta fibroblasts by the protection of type I collagen (COL1A1) mRNA containing nonsense mutations that normally resulted in mutant mRNA degradation. Stabilisation of mutant mismatch repair gene (MLH1) mRNA was also observed in transformed lymphocytes from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Importantly, our strategy also stabilised very low‐level (or illegitimate) nonsense‐containing transcripts in lymphoblasts from patients with Bethlem myopathy (COL6A1), familial adenomatous polyposis (APC), and breast cancer (BRCA1). The greatly increased sensitivity and reliability of this RT‐PCR/PTT protocol has broad applicability to the many genetic diseases in which only blood‐derived cells may be readily available for analysis. Hum Mutat 13:311–317, 1999.

Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and protein secretion.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, andCOL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the α1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant α1(VI) chains associated intracellularly with α2(VI) and α3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an α3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant α3(VI) chains associated with endogenous α1(VI) and α2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the α1(VI) and α3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy.

Proteasomal Degradation of Unassembled Mutant Type I Collagen Pro-α1(I) Chains

We have previously shown that type I procollagen pro-α1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995)J. Biol. Chem. 270, 8642–8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-α1(I), were stably transfected with a pro-α1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-α1(I) could be detected indicating that the majority of the mutant pro-α1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH4Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-α1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-α1(I) in OI26 is mediated by proteasomes.

Secretion and assembly of type IV and VI collagens depend on glycosylation of hydroxylysines.

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. These hydroxylysine-linked carbohydrates were shown recently to be indispensable for the formation of basement membranes (Ruotsalainen, H., Sipilä, L., Vapola, M., Sormunen, R., Salo, A. M., Uitto, L., Mercer, D. K., Robins, S. P., Risteli, M., Aszodi, A., Fässler, R., and Myllylä, R. (2006) J. Cell Sci. 119, 625–635). Analysis of LH3 knock-out embryos and cells in this work indicated that loss of glycosylated hydroxylysines prevents the intracellular tetramerization of type VI collagen and leads to impaired secretion of type IV and VI collagens. Mice lacking the LH activity of LH3 produced slightly underglycosylated type IV and VI collagens with abnormal distribution. The altered distribution and aggregation of type VI collagen led to similar ultrastructural alterations in muscle to those detected in collagen VI knockout and some Ullrich congenital muscular dystrophy patients. Our results provide new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion, assembly, and distribution of highly glycosylated collagen types.

Collagen VI glycine mutations: perturbed assembly and a spectrum of clinical severity

The collagen VI muscular dystrophies, Bethlem myopathy and Ullrich congenital muscular dystrophy, form a continuum of clinical phenotypes. Glycine mutations in the triple helix have been identified in both Bethlem and Ullrich congenital muscular dystrophy, but it is not known why they cause these different phenotypes.

Molecular consequences of dominant Bethlem myopathy collagen VI mutations

Dominant mutations in the three collagen VI genes cause Bethlem myopathy, a disorder characterized by proximal muscle weakness and commonly contractures of the fingers, wrists, and ankles. Although more than 20 different dominant mutations have been identified in Bethlem myopathy patients, the biosynthetic consequences of only a subset of these have been studied, and in many cases, the pathogenic mechanisms remain unknown.