Glycosaminoglycans (GAGs) are a heterogeneous family of linear polysaccharides that constitute the carbohydrate moiety covalently attached to the protein core of proteoglycans, macromolecules present on the cell surface and in the extracellular matrix. Several genetic disorders of bone and connective tissue are caused by mutations in genes encoding for glycosyltransferases, sulfotransferases and transporters that are responsible for the synthesis of sulfated GAGs. Phenotypically, these disorders all reflect alterations in crucial biological functions of GAGs in the development, growth and homoeostasis of cartilage and bone. To date, up to 27 different skeletal phenotypes have been linked to mutations in 23 genes encoding for proteins involved in GAG biosynthesis. This review focuses on recent genetic, molecular and biochemical studies of bone and connective tissue disorders caused by GAG synthesis defects. These insights and future research in the field will provide a deeper understanding of the molecular pathogenesis of these disorders and will pave the way for developing common therapeutic strategies that might be targeted to a range of individual phenotypes.

Desbuquois dysplasia type 1 (DBQD1) is a chondrodysplasia caused by mutations in CANT1 gene encoding an ER/Golgi calcium activated nucleotidase 1 that hydrolyses UDP. Here, using Cant1 knock-in and knock-out mice recapitulating DBQD1 phenotype, we report that CANT1 plays a crucial role in cartilage proteoglycan synthesis and in endochondral ossification. Specifically, the glycosaminoglycan synthesis was decreased in chondrocytes from Cant1 knock-out mice and their hydrodynamic size was reduced, whilst the sulfation was increased and the overall proteoglycan secretion was delayed. Interestingly, knock-out chondrocytes had dilated ER cisternae suggesting delayed protein secretion and cellular stress; however, no canonical ER stress response was detected using microarray analysis, Xbp1 splicing and protein levels of BiP and ATF4. The observed proteoglycan defects caused deregulated chondrocyte proliferation and maturation in the growth plate resulting in the reduced skeletal growth. In conclusion, the pathogenic mechanism of DBQD1 comprises deregulated chondrocyte performance due to defective intracellular proteoglycan synthesis and altered proteoglycan properties in the extracellular matrix.

Mutations in genes encoding enzymes responsible for the biosynthesis and structural diversity of glycosaminoglycans (GAGs) cause a variety of disorders affecting bone and connective tissues, including Desbuquois dysplasia (DD). In an infant with prenatal-onset disproportionate short stature, joint laxity, and radiographic findings typical for DD compound-heterozygosity for a large intragenic deletion, and a p.Pro384Arg missense mutation in CSGALNACT1 was found. CSGALNACT1 encodes chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1, ChGn-1), which initiates chondroitin sulfate (CS) chain biosynthesis on the so-called GAG-protein linker region tetrasaccharide. Biochemical studies revealed a reduced GalNAc-transferase activity of the Arg-384 mutant protein, whereas no differences in proteoglycan synthesis in fibroblasts and the GAG content in the urine were found between patient and controls. This is the first description of bi-allelic loss-of-function mutations in CSGALNACT1 that produce a skeletal dysplasia reminiscent of the skeletal dysplasia of Csgalnact1 mice, and adds to the genetic heterogeneity of DD.

The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. In this work, we have tested this strategy in vivo with the aim to generate two conditional knock-in mice for missense mutations in the Impad1 and Clcn7 genes causing two different skeletal dysplasias. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* and the Clcn7 exon 7 and an inverted exon 7* containing the point mutations were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon.The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation. Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Similarly, the phenotype of homozygous Clcn7 floxed mice overlaps with Clcn7 knock-out mice. Expression studies by qPCR and RT-PCR demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. Thus, the skeletal phenotypes in both murine strains were not caused by the missense mutations, but by aberrant splicing. Our data demonstrate that the Cre mediated genetic switch strategy should be considered cautiously for the generation of conditional knock-in mice.

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