Klaric, T. | bioRxiv Science

bioRxiv2023

Total cell N-glycosylation is altered during neuronal differentiation of iPSC to NSC and is disturbed by trisomy 21

Down syndrome (DS) is a genetic condition caused by trisomy 21 (T21) and characterized by a range of neurological symptoms including intellectual disability, early onset of neurodegeneration and dementia, some of which can be attributed to accelerated aging. N-glycosylation is a protein modification that plays a critical role in numerous biological processes and its dysregulation is associated with a wide range of diseases, in some even before the onset of symptoms. N-glycosylation of total plasma proteins, as well as specific plasma proteins, such as immunoglobulin G, has been shown to change in DS, displaying an accelerated aging phenotype consistent with the various symptoms of premature aging that occur in DS. However, little is known about how T21 affects the N-glycosylation of other cellular proteins. To better understand how T21 affects N-glycosylation during neural differentiation, we characterized and compared the total released N-glycans of induced pluripotent stem cells (iPSCs) and their neural stem cell (NSC) derivatives. We analyzed six different isogenic clones all derived from a single individual with mosaic DS and thus all sharing the same genetic background; however, three had a normal disomic karyotype (D21), while the other three had an additional copy of chromosome 21 (T21). We characterized the total cell N-glycosylation profiles using ultra high performance liquid chromatography (UHPLC) and subsequent tandem mass spectrometry analysis to determine proposed glycan structures. Our results revealed both qualitative and quantitative differences in the composition of N-glycomes between iPSCs and NSCs, with NSCs showing a higher amount of complex N-glycans and a lower amount of mannosidic N-glycans when compared to iPSCs. Moreover, we found differences in N-glycosylation patterns between D21 and T21 cells. Notably, T21 cells exhibited a significant increase in the amount of pseudohybrid N-glycans. Trisomy 21 also caused a significant decrease in the abundance of a hybrid monoantennary fucosylated glycan (H6N3F1). Our findings define the released N-glycan profile of total cells for both D21 and T21 iPSCs and NSCs and suggest that the presence of a third copy of chromosome 21 impacts N-glycosylation patterns already in the stem cell state.

molecular biology↗

bioRxiv2019

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

Autism 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.\n\nSignificance 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.

neuroscience↗