Exploration of Human Xylosyltransferase for Chemoenzymatic Synthesis of Proteoglycan Linkage Region

Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature,...

b3galt6 Knock-Out Zebrafish Recapitulate β3GalT6-Deficiency Disorders in Human and Reveal a Trisaccharide Proteoglycan Linkage Region

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human β3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study, therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

Abstract 16997: Reaffirmation of GPD1L-A280V as a Brugada Syndrome Disease-Causing Variant

Introduction: Brugada Syndrome (BrS) is an inherited arrhythmia syndrome characterized by ST-segment elevation in the right precordial leads (V1-V3) and sudden cardiac death; ~20% of cases are caused by mutations in SCN5A . Over the past two decades, over 20 genes that may cause BrS have been identified. We reported that a Glycerol-3-Phosphate Dehydrogenase 1-Like mutation (GPD1L;p.A280V, chromosome 3p22.3) was linked to BrS in a large, multigenerational family with a LOD score ~4. Recent efforts in the field have called into question the pathogenicity of non- SCN5A genetic variants in BrS. Hypothesis: We sought to confirm the role of GPD1L in this family using a high density SNP array to narrow the linkage region and whole exome sequencing (WES) of the proband to rule out other possible mutations. We also sought to test whether BrS susceptibility SNPs in SCN5A and SCN10A linked to GPD1L may play a role in this family. Methods: SNPs were identified on an Illumina Global Screening Array and used for fine mapping. The exome of the proband was captured with the Agilent V6+UTR kit. SNPs were called using GATK3. BrS susceptibility SNPs were confirmed by PCR amplification and Sanger Sequencing. Results: The linkage region in this family resolved to ~3.07 MB (hg19; 3:29,899,567-32,970,637; Figure). This region contained 14 genes including GPD1L , and lacking SCN5A , SCN10A , or any other BrS-associated gene. WES identified GPD1L;p.A280V as the only coding variant in the linkage region with an allele frequency ≤ 1%. Previously identified BrS risk alleles in SCN5A and SCN10A were linked to the GPD1L;p.A280V allele in most family members (Figure). Conclusions: Our data suggests that GPD1L;p.A280V causes BrS in this family. The pathogenicity of GPD1L;p.A280V may be increased by linkage with SCN5A and SCN10A risk SNPs. The role of GPD1L mutations in the pathogenesis of BrS should be reaffirmed.

b3galt6 knock-out zebrafish recapitulate β3GalT6-deficiency disorders in human and reveal a trisaccharide proteoglycan linkage region

Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human β3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.

Synthesis of oligosaccharides of the linkage region of proteoglycans using regioselective glycosylation

A collection of various sulfoforms of di- and trisaccharides of the linkage region of proteoglycans were prepared using a regioselective glycosylation. Preliminary results of the impact of sulfation on CSGalNAcT-1 is also reported.

Multi-hit autism genomic architecture evidenced from consanguineous families with involvement of FEZF2 and mutations in high-risk genes

ABSTRACTAutism Spectrum Disorders (ASDs) are a heterogeneous collection of neurodevelopmental disorders with a strong genetic basis. Recent studies identified that a single hit of either a de novo or transmitted gene-disrupting, or likely gene-disrupting, mutation in a subset of 65 strongly associated genes can be sufficient to generate an ASD phenotype. We took advantage of consanguineous families with an ASD proband to evaluate this model. By a genome-wide homozygosity mapping of ten families with eleven children displaying ASD, we identified a linkage region of 133 kb in five families at the 3p14.2 locus that includes FEZF2 with a LOD score of 5.8 suggesting a founder effect. Sequencing FEZF2 revealed a common deletion of four codons. However, the damaging FEZF2 mutation did not appear to be sufficient to induce the disease as non-affected parents also carry the mutation and, similarly, Fezf2 knockout mouse embryos electroporated with the mutant human FEZF2 construct did not display any obvious defects in the corticospinal tract, a pathway whose development depends on FEZF2. We extended the genetic analysis of these five FEZF2-linked families versus five FEZF2 non-linked families by studying de novo and transmitted copy number variation (CNV) and performing Whole Exome Sequencing (WES). We identified damaging mutations in the subset of 65 genes strongly associated with ASD whose co-expression analysis suggests an impact on the prefrontal cortex during the mid-fetal periods. From these results, we propose that both FEZF2 deletion and multiple hits in the repertoire of these 65 genes are necessary to generate an ASD phenotype.Significance StatementThe human neocortex is a highly organized laminar structure with neuron positioning and identity of deep-layer cortical neurons that depend on key transcription factors, such as FEZF2, SATB2, TSHZ3 and TBR1. These genes have a specific spatio-temporal pattern of expression in human midfetal deep cortical projection neurons and display mutations in patients with Autism Spectrum Disorder (ASD). Here, we identified a linkage region involving FEZF2 gene in five consanguineous families with an ASD proband. For these FEZF2-allele linked probands, we identified a four-codon deletion in FEZF2 and damaging mutations in other high-risk ASD genes, that exhibit regional and cell type–specific convergence in neocortical deep-layer excitatory neurons, suggesting a multi-hit genomic architecture of ASD in these consanguineous families.