The rapid secretion of large cargoes such as collagens requires specialized transport machinery (Melville et al., 2011; Saito et al., 2009a). The four genes are highly divergent in sequence between the two groups (20% similarity) and approximately 50% similar between each pair, but each paralog is highly conserved within vertebrates (up to 90% sequence similarity between fish and human) (Sarmah et al ., 2010). This phenotype is largely recapitulated by the vbi/sec24d medaka mutant that carries a nonsense mutation predicted to delete C-terminal portion of Sec24d protein ( Ohisa et al., 2010 ).
Sec24B Vangl2 Failure of localization to plasma membrane Neural tube mouse Merte et al., 2010 Wansleeben et al., 2010. Enterocytes & chondrocytes Zebrafish Levic et al., 2015 Sec13 Collagen I Defective secretion and deposition Fibroblasts, Human Towns et al. in these processes results in craniorachischisis and neural tube closure defects in Sec24b mouse mutants (Merte et al., 2010; Wansleeben et al., 2010).
Vertebrate genomes contain two paralogs of the ancestral Sec23 gene, Sec23a and Sec23b ( Paccaud et al., 1996 ). No nonsense mutation homozygotes have been identified in approximately 370 reported cases (Iolascon et al., 2012).
Wild-type 2-day zebrafish embryos stained with o-dianisidine, which is oxidized by heme in the presence of peroxide and colored brown, display abundant hemoglobin within
Additionally, type I procollagen was retained in the ER and was not sorted to the ERES ( Cutrona et al., 2013 ). The concurrent well-sense zebrafish mutation study identified a missense variant in the creb3L2 coding region resulting in a hypomorphic allele (Melville et al., 2011). Proteoglycans produced and secreted by chondrocytes include heparan sulfate (HSPG) and chondroitin sulfate (CSPG) modifications ( Holmborn et al., 2012 ).
Embryos were collected after natural mating and cultured until 4 days post fertilization (dpf) (Kimmel et al., 1995). Next, reads were aligned to the zebrafish transcriptome (based on GRCz10 release 80) and genome (genome assembly: GRCz10) using Tophat2 (Kim et al., 2013) with default parameters. Next, I crossed the Tg[Col2a1a:caax-EGFP] line with a nuclear mCherry reporter (H2a-mCherry) line ( Luderman et al., 2017 ) to obtain double-labeled chondrocytes.
Several PG processing enzymes act in the post-translational modification of cartilage PGs (Luderman et al., 2017). These guide RNA (gRNA) target sites were cloned into the pT7cas9gRNA2 vector ( Jao et al., 2013 ). To detect potential heterozygous mutants, the heteroduplex formation assay was performed as previously described ( Yin et al., 2015 ).
Zebrafish: Quantitative real-time PCR (qRT-PCR) was performed as previously described (Sarmah et al., 2010).
Alcian blue staining of 5 dpf zebrafish larvae displaying craniofacial cartilage elements
This finding suggested that C-terminal portion may well play a role in collagen secretion activity of Creb3L2. Rescue experiments revealed that C-terminus' effect on collagen secretion is cell-autonomous as collagen accumulation in C-SP-deficient creb3L2g12/g12 mutant chondrocytes could be cell-autonomously rescued with overexpression of full-length Creb3L2 (GFP+ cells), whereas their immediate neighbors do have noticeably large collagen deposits (Fig. 3.5B, lower panel). Overall, these data suggest that only full-length Creb3L2 is sufficient to modulate physiological levels of collagen secretion in mature chondrocytes.
However, it has not been investigated in vivo whether Sec23a is the sole and sufficient target of Creb3L2 to activate collagen secretion. Overexpression of FLAG-Creb3L2 cell autonomously rescued collagen transport defects in creb3L2-/- chondrocytes in this independent experimental setup (Fig. 3.6B) as previously shown in Fig. 3.5. In contrast, overexpression of sec23a failed to restore collagen secretion in creb3L2-/- cells (Fig. 3.6B, lower panel).
These findings suggest that Creb3L2 targets multiple factors other than sec23a to regulate collagen secretion. Our laboratory has identified two paralogs of the COPII inner coat components, sec23a and sec24d, that are essential for efficient collagen secretion in zebrafish chondrocytes in vivo ( Lang et al., 2006 ; Sarmah et al., 2010 ). Experimental design to overexpress FLAG-Creb3L2 or Sec23a mosaic with V2a-EGFP tag and analyze collagen secretion by chondrocytes via IF on cryosections.
The WT panel shows plain WT cartilage with collagen secretion from the chondrocyte at both the outer edges of the cartilage (orange arrows) and intracellular regions (yellow arrow). In particular, we hypothesize that Creb3L2 may activate sec24d expression, like its binding partner sec23a, in chondrocytes to promote physiological levels of collagen secretion. However, it remains unclear whether their function is required in a cell-autonomous manner for physiological levels of collagen secretion.
These results indicate that both inner coat components, Sec23a and Sec24d, are cell-autonomously required for efficient collagen secretion during chondrocyte maturation. Here, we present a comprehensive in vivo study investigating the functions of Creb3L2 in collagen secretion during chondrocyte maturation. Experimental design to mosaically overexpress Sec23a with V2a-EGFP tag under ubiquitously active β-actin promoter and analyze collagen secretion by chondrocytes via.
Experimental design to mosaically overexpress Sec23a with V2a-EGFP tag under ubiquitously active β-actin promoter and analyze collagen secretion by chondrocytes via
Our previous exome sequencing (WES) of the pediatric cataract cohort identified a missense mutation in the RIC1 gene ( Patel et al., 2017 ). Genetic analysis of additional CATIFA patients. a) Pedigree of additional family contributing to this study (modified from Patel et al., 2016, Human Genetics). To assess the role that RIC1 may play in the etiology of a disease trait, we used the PrediXcan approach (Gamazon et al., 2015).
We estimated the genetic component of gene expression in the ten thousand BioVU subjects (Denny et al., 2013) to identify associated phenotypes (despite the lack of directly measured gene expression data). The Q72L mutation (Martinez et al., 1994) (constitutively active Rab6a mutant) was generated on the destination vector via Q5® site-directed mutagenesis kit (New England Biolabs). CRISPR/Cas9 target sites within the zebrafish rgp1 gene (GRCz10 assembly) were identified using the CHOPCHOP (Montague et al., 2014) web tool.
A cloning-free method to generate sgRNA templates was performed as previously described (Varshney et al., 2015). This chapter was published under this title in The American Journal of Human Genetics (Unlu G., Gamazon E.R. et al., 2019). We refer to the imputed gene expression level as the genetically regulated expression (GReX) (Gamazon et al., 2015).
The Synthetic Derivative (SD) is a de-identified and continuously updated image of the EHR, including data on > 2.6 million subjects ( Roden et al., 2008 ). To evaluate the role that a gene may play in the etiology of a disease trait, we used the PrediXcan approach (Gamazon et al., 2015). We estimated the genetic component of gene expression in the BioVU samples ( Denny et al., 2013 ) to identify genes associated with a phenotype (despite the lack of directly measured gene expression data).
The clinical characteristics used in this analysis are represented by fecodes (Denny et al., 2013), which are algorithmically defined using ICD-9 codes in the EHR database. Quantitative real-time PCR (qRT-PCR) was performed as previously described (Melville et al., 2011; Sarmah et al., 2010). An antisense morpholino oligonucleotide (MO) (Gene Tools) was designed to target the 5'UTR of grik5 (5'-GAGATGCCTTCTGCTGCCTATAGCA-3') as previously described ( Levic et al., 2015 ).
A cloning-free method was used to generate gRNA templates as previously described ( Varshney et al., 2015 ). The small eye phenotype observed in zebrafish is likely related to the expression of grik5 in photoreceptor cells and the outer plexiform layer of the retina, as shown in mouse embryos ( Haumann et al., 2017 ).
Examples of pigment blocker (PTU) treated (C) and untreated (E) embryos
Reduced eGFP signal is noted in the eye and CNS of grik5-MO-injected group (arrows). Quantitative PCR (qPCR) analysis of whole embryos indicated that grik5 expression peaked at 3 dpf and was localized to the brain and retina (Fig. 5.6B), as confirmed by an independent set of primers and riboprobes (data not shown). These results were confirmed by independent RNA-seq data from FACS-sorted endothelial cells in zebrafish Tg(flk1:GFP+ cells) at the same developmental stages (Fig. 5.6C) (Kasper et al., 2017).
Taken together, grik5 is expressed at the time and place that may explain the bleeding (Fig. 5.3C and 5.3E) and smaller eye phenotypes (Fig. 5.6F and 5.8) in grik5 morphants and mutants. To test this hypothesis, we calculated the relative risk (RR) for eye diseases initially associated with reduced GReX of GRIK5 in individuals with a set of vascular disease phenotypes most strongly associated with reduced GReX of GRIK5 in studies of larger number of BioVU subjects using Vanderbilt's Synthetic Derivative, the de-identified image of the EHR in > 2.6 million subjects. There are substantial increases in the risk for eye disease across the spectrum of vascular phenotypes (2- to 15-fold; Fig. 5.9A).
We similarly estimated RRs (Kleinbaum et al., 1998) for these phenotypes in > 150 million subjects from the MarketScan insurance claims dataset (Blair et al., 2013) (Figs. 5.9B, 5.9C) by age and sex, confirming the magnitude of comorbidity for eye and vascular diseases (see Methods). Age was not significantly associated with the extent of comorbidity in men and women (Kruskal-Wallis test p=0.38 and p=0.70, respectively) and no significant difference was observed between men and women (Mann-Whitney U test p=0.43) . demonstrating the robustness of the observation to potential confounds from these demographic variables. Comorbidity estimates were calculated using the synthetic derivative of Vanderbilt, the de-identified image of electronic health records in > 2.6 million participants.
This extends similar results in the most recent BioVU sample of 60,000, in which we observed comorbidity of the vascular trait, cerebrovascular disease, with “retinal macular degeneration (senile) NOS” (p=9.73x10-4) among participants with the lowest GRIK5 expression (bottom 2 .5%; Figure 5.10). We developed a genetic risk score using effect alleles in the AMD dataset and found a significant association with disease status for each vascular trait in BioVU, including primarily peripheral vascular disease (p<2.2x10-16 for each phenotype). Although AMD-associated variants were not enriched for the most significant associations with peripheral vascular disease, a significant (Spearman's) correlation (p=7.1x10-9) in heritability per SNP (which is proportional to β2*p*(1- p ) where β is the estimated effect size and p the frequency of the minor allele in controls) among traits for the highest AMD variants contributes to the predictive performance of genetic risk assessment for a vascular trait.
Furthermore, the significant comorbidity in the BioVU participants with the lowest genetically determined GRIK5 expression (in the bottom 2.5%) of “macular degeneration (senile) of the retina is NOS” with vascular diseases, including cerebrovascular diseases (Fig. 5.10 ), as noted above and peripheral vascular disease (Fig. 5.2C) suggests that genetic regulation of GRIK5 contributes to the overall effect of the genetic risk score on these vascular traits. The availability of the large-scale AMD dataset also allowed us to evaluate the relevance of the imputation models in non-ocular tissues for ocular features. Comorbidity analysis between cerebrovascular diseases and eye phenotypes (traits) in the bottom 2.5% of the population, shown in Fig.
