We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition. DOI: https://doi.org/10. 1038/ng.3578 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-130493 Accepted Version Originally published at: van Karnebeek, Clara D M; Bonafé, Luisa; Wen, Xiao-Yan; Tarailo-Graovac, Maja; Balzano, Sara; RoyerBertrand, Beryl; Ashikov, Angel; Garavelli, Livia; Mammi, Isabella; Turolla, Licia; Breen, Catherine; Donnai, Dian; Cormier, Valerie; Heron, Delphine; Nishimura, Gen; Uchikawa, Shinichi; Campos-Xavier, Belinda; Rossi, Antonio; Hennet, Thierry; Brand-Arzamendi, Koroboshka; Rozmus, Jacob; Harshman, Keith; Stevenson, Brian J; Girardi, Enrico; Superti-Furga, Giulio; Dewan, Tammie; Collingridge, Alissa; Halparin, Jessie; Ross, Colin J; Van Allen, Margot I;et al (2016). NANS-mediated synthesis of sialic acid is required for brain and skeletal development. Nature Genetics, 48 (7) insights into the molecular basis of neurocognitive impairment allows for the development and 89 application of targeted therapeutic strategies 5 . Although less frequent than IDD, genetic disorders 90 affecting skeletal development and growth (commonly called the "skeletal dysplasias") are a 91 group of over 500 distinct disorders 6 . Studying their molecular basis has provided precious 92 insights into the many factors necessary for skeletal development, ranging from minerals and 93 structural molecules to enzymes, to signaling molecules and transcription factors 6,7 . We report 94here a genetic disorder presenting with a combination of severe IDD with skeletal dysplasia and 95 short stature. Our data show that its pathogenic basis is an inborn error of metabolism that 96 affects the endogenous synthesis of N-acetyl neuraminic acid (NeuNAc; sialic acid). Exploration 97 of the biochemical and molecular features of this disorder provides new information on the role 98 of sialic acid in the development of brain and bone. 99 100 RESULTS 101 Clinical and radiographic phenotype of N...

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.

Mutations in the diastrophic dysplasia sulfate transporter (DTDST or SLC26A2) cause a family of recessively inherited chondrodysplasias including, in order of decreasing severity, achondrogenesis 1B, atelosteogenesis 2, diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia. The gene encodes a widely distributed sulfate/chloride antiporter of the cell membrane whose function is crucial for the uptake of inorganic sulfate, which is needed for proteoglycan sulfation. To provide new insights in the pathogenetic mechanisms leading to skeletal and connective tissue dysplasia and to obtain an in vivo model for therapeutic approaches to DTD, we generated a Dtdst knock-in mouse with a partial loss of function of the sulfate transporter. In addition, the intronic neomycine cassette in the mutant allele contributed to the hypomorphic phenotype by inducing abnormal splicing. Homozygous mutant mice were characterized by growth retardation, skeletal dysplasia and joint contractures, thereby recapitulating essential aspects of the DTD phenotype in man. The skeletal phenotype included reduced toluidine blue staining of cartilage, chondrocytes of irregular size, delay in the formation of the secondary ossification center and osteoporosis of long bones. Impaired sulfate uptake was demonstrated in chondrocytes, osteoblasts and fibroblasts. In spite of the generalized nature of the sulfate uptake defect, significant proteoglycan undersulfation was detected only in cartilage. Chondrocyte proliferation and apoptosis studies suggested that reduced proliferation and/or lack of terminal chondrocyte differentiation might contribute to reduced bone growth. The similarity with human DTD makes this mouse strain a useful model to explore pathogenetic and therapeutic aspects of DTDST-related disorders.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of ␤ 2 -microglobulin (␤ 2 -m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, ␤ 2 -m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of ␤ 2 -m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in ␤ 2 -m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role in ␤ 2 -m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-L-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-L-lysine is dissolved in solution with ␤ 2 -m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as ␤ 2 -m, that decay on moving away from the fiber surface.The deposition of ␤ 2 -microglobulin (␤ 2 -m) 2 into amyloid fibrils is the hallmark of dialysis-related amyloidosis (DRA), a disease arising as a complication of long-term hemodialysis. ␤ 2 -m is a 99-residue protein (molecular mass 11.7 kDa) that represents the light chain of the major histocompatibility complex class I (MHCI), an integral membrane protein involved in the immune response. As a result of normal MHCI catabolism, ␤ 2 -m is released in the serum from the cell surface and carried to the kidney for clearance. In the presence of kidney failure, the concentration of free circulating ␤ 2 -m can increase by up to 50-fold; the persistent increase in ␤ 2 -m concentration results in amyloid deposition, preferentially localized in the musculoskeletal system. The accumulation of ␤ 2 -m deposits has been shown to cause arthralgias, destructive osteoarthropathies, and carpal tunnel syndrome (1). Although a high concentration of ␤ 2 -m is a necessary condition for the onset of the disease, there is not a strict correlation between the disease severity and ␤ 2 -m levels (2), suggesting that other factors might be involved in ␤ 2 -m amyloid deposition.The aggregation process of ␤ 2 -m has been the object of extensive investigation for many years. Severa...

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Here we summarized what is known at the present about function, structure and effect of mutations in the human prolidase. Among the peptidases, prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement therapy for the prolidase deficiency (PD). Prolidase deficiency is a rare recessive disorder caused by mutations in the prolidase gene and characterized by severe skin lesions. Single amino acid substitutions, exon splicing, deletions and a duplication were described as causative for the disease and are mainly located at highly conserved amino acids in the sequence of prolidase from different species. The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized.

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of ␤ 2 -microglobulin (␤ 2 -m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of ␤ 2 -m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes ␤ 2 -m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of ␤ 2 -m (⌬N6␤ 2 -m) that is a ubiquitous constituent of the natural ␤ 2 -m fibrils. The formation of this ␤ 2 -m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition. Dialysis-related amyloidosis (DRA),2 a severe disease arising as a complication of long term hemodialysis, involves the deposition of ␤ 2 -microglobulin (␤ 2 -m) amyloid fibrils in bones and ligaments. ␤ 2 -m constitutes the light chain of the major histocompatibility complex class I and CD1 (1), and in normal catabolism, it is continuously released in the serum and cleared from the circulation by the kidney. The replacement of renal function by hemodialysis does not efficiently remove ␤ 2 -m. The persistent increase of its plasma concentration is associated with ␤ 2 -m deposition in the osteotendineous system, which is the specific target tissue of this type of amyloidosis. Among extra-cerebral amyloidoses, DRA represents the most striking case of tissue-specific targeting. Although other organs can be involved, bones and ligaments never escape amyloid deposition. Homma (2) first pointed out that collagen might be involved in determining this tissue specificity and demonstrated a collagen/␤ 2 -m interaction.We have recently determined the binding properties governing the collagen/␤ 2 -m interaction, and we found that the latter is quite weak but is enhanced when ␤ 2 -m is truncated at the N-terminal end, and the pH is reduced from 7.4 to 6.4 (3). We subsequently demonstrated that fibrillar collagen (type I), which is abundant in skeletal...

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.