Inherited Proteoglycan Biosynthesis Defects-Current Laboratory Tools and Bikunin as a Promising Blood Biomarker.
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ABSTRACT:
SUBMITTER: Haouari W
PROVIDER: S-EPMC8625474 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.
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Project description:BACKGROUND:Pseudodiastrophic dysplasia (PDD) is a severe skeletal dysplasia associated with prenatal manifestation and early lethality. Clinically, PDD is classified as a 'dysplasia with multiple joint dislocations'; however, the molecular aetiology of the disorder is currently unknown. METHODS:Whole exome sequencing (WES) was performed on three patients from two unrelated families, clinically diagnosed with PDD, in order to identify the underlying genetic cause. The functional effects of the identified variants were characterised using primary cells and human cell-based overexpression assays. RESULTS:WES resulted in the identification of biallelic variants in the established skeletal dysplasia genes, B3GAT3 (family 1) and CANT1 (family 2). Mutations in these genes have previously been reported to cause 'multiple joint dislocations, short stature, and craniofacial dysmorphism with or without congenital heart defects' ('JDSCD'; B3GAT3) and Desbuquois dysplasia 1 (CANT1), disorders in the same nosological group as PDD. Follow-up of the B3GAT3 variants demonstrated significantly reduced B3GAT3/GlcAT-I expression. Downstream in vitro functional analysis revealed abolished biosynthesis of glycosaminoglycan side chains on proteoglycans. Functional evaluation of the CANT1 variant showed impaired nucleotidase activity, which results in inhibition of glycosaminoglycan synthesis through accumulation of uridine diphosphate. CONCLUSION:For the families described in this study, the PDD phenotype was caused by mutations in the known skeletal dysplasia genes B3GAT3 and CANT1, demonstrating the advantage of genomic analyses in delineating the molecular diagnosis of skeletal dysplasias. This finding expands the phenotypic spectrum of B3GAT3-related and CANT1-related skeletal dysplasias to include PDD and highlights the significant phenotypic overlap of conditions within the proteoglycan biosynthesis pathway.
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Project description:Pseudodiastrophic dysplasia (PDD) is a severe skeletal dysplasia associated with prenatal manifestation and early lethality. Clinically, PDD is classified as a ‘dysplasia with multiple joint dislocations’ however, the molecular aetiology of the disorder is currently unknown. In order to identify the genetic defects underlying PDD, we performed whole exome sequencing (WES) on 3 patients from 2 unrelated families, clinically diagnosed with PDD. WES resulted in the identification of bi-allelic variants in the established skeletal dysplasia genes, B3GAT3 (Family 1) and CANT1 (Family 2). Mutations in these genes have previously been reported to cause ‘multiple joint dislocations, short stature, and craniofacial dysmorphism with or without congenital heart defects’ (B3GAT3) and Desbuquois dysplasia 1 (CANT1); disorders in the same nosological group as PDD. Follow-up of the B3GAT3 variants demonstrated significantly reduced B3GAT3/GlcAT-I expression. Downstream in vitro functional analysis revealed abolished biosynthesis of glycosaminoglycan side chains on proteoglycans. Functional evaluation of the CANT1 variant showed impaired nucleotidase activity, which results in inhibition of glycosaminoglycan synthesis through accumulation of uridine diphosphate. For the families described in this study, the PDD phenotype was caused by mutations in the known skeletal dysplasia genes B3GAT3 and CANT1, demonstrating the advantage of genomic analyses in delineating the molecular diagnosis of skeletal dysplasias. This finding expands the phenotypic spectrum of B3GAT3- and CANT1- related skeletal dysplasias to include PDD, and highlights the significant phenotypic overlap of conditions within the proteoglycan biosynthesis pathway.
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Project description:Proteoglycans are a major component of extracellular matrix and contribute to normal embryonic and postnatal development by ensuring tissue stability and signaling functions. We studied five patients with recessive joint dislocations and congenital heart defects, including bicuspid aortic valve (BAV) and aortic root dilatation. We identified linkage to chromosome 11 and detected a mutation (c.830G>A, p.Arg277Gln) in B3GAT3, the gene coding for glucuronosyltransferase-I (GlcAT-I). The enzyme catalyzes an initial step in the synthesis of glycosaminoglycan side chains of proteoglycans. Patients' cells as well as recombinant mutant protein showed reduced glucuronyltransferase activity. Patient fibroblasts demonstrated decreased levels of dermatan sulfate, chondroitin sulfate, and heparan sulfate proteoglycans, indicating that the defect in linker synthesis affected all three lines of O-glycanated proteoglycans. Further studies demonstrated that GlcAT-I resides in the cis and cis-medial Golgi apparatus and is expressed in the affected tissues, i.e., heart, aorta, and bone. The study shows that reduced GlcAT-I activity impairs skeletal as well as heart development and results in variable combinations of heart malformations, including mitral valve prolapse, ventricular septal defect, and bicuspid aortic valve. The described family constitutes a syndrome characterized by heart defects and joint dislocations resulting from altered initiation of proteoglycan synthesis (Larsen-like syndrome, B3GAT3 type).
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Project description:Colorectal cancer (CRC) heritability has been estimated to be around 30%. However, mutations in the known CRC-susceptibility genes explain CRC risk in fewer than 10% of affected individuals. Germline mutations in DNA-repair genes (DRGs) have recently been reported in CRC, but their contribution to CRC risk is largely unknown. We evaluated the gene-level germline mutation enrichment of 40 DRGs in 680 unselected CRC individuals and 27,728 ancestry-matched cancer-free adults. Significant findings were then examined in independent cohorts of 1,661 unselected CRC individuals and 1,456 individuals with early-onset CRC. Of the 680 individuals in the discovery set, 31 (4.56%) individuals harbored germline pathogenic mutations in known CRC-susceptibility genes, and another 33 (4.85%) individuals had DRG mutations that have not been previously associated with CRC risk. Germline pathogenic mutations in ATM and PALB2 were enriched in both the discovery (OR = 2.81 and p = 0.035 for ATM and OR = 4.91 and p = 0.024 for PALB2) and validation (OR = 2.97 and adjusted p = 0.0013 for ATM and OR = 3.42 and adjusted p = 0.034 for PALB2) sets. Biallelic loss of ATM was evident in all individuals with matched tumor profiling. CRC individuals also had higher rates of actionable mutations in the HR pathway, which can substantially increase the risk of developing cancers other than CRC. Our analysis provides evidence for ATM and PALB2 as CRC-risk genes, underscoring the importance of the homologous recombination pathway in CRC. In addition, we identified frequent complete homologous recombination deficiency in CRC tumors, representing a unique opportunity to explore targeted therapeutic interventions such as poly-ADP ribose polymerase inhibitor (PARPi).
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Project description:Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13- and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4- and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5- and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells.
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