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. 2020 Jan;26(1):98-109.
doi: 10.1038/s41591-019-0705-y. Epub 2020 Jan 13.

Phenome-based approach identifies RIC1-linked Mendelian syndrome through zebrafish models, biobank associations and clinical studies

Gokhan Unlu  1   2   3   4 , Xinzi Qi  1   2 , Eric R Gamazon #  1   2   5 , David B Melville #  1   6 , Nisha Patel #  7 , Amy R Rushing  1   2 , Mais Hashem  7 , Abdullah Al-Faifi  8 , Rui Chen  2   9 , Bingshan Li  2   9 , Nancy J Cox  1   2 , Fowzan S Alkuraya  7 , Ela W Knapik  10   11   12
Affiliations

Phenome-based approach identifies RIC1-linked Mendelian syndrome through zebrafish models, biobank associations and clinical studies

Gokhan Unlu et al. Nat Med. 2020 Jan.

Abstract

Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.

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Figures

Extended Data Fig. 1
Extended Data Fig. 1. Genetic analysis of ric1 mutations and dental phenotypes
a, Genetic linkage analysis and positional cloning mapped the rnd mutation to zebrafish chromosome 21. Microsatellite markers and number of recombination events (recs, blue) in critical region that contains 4 protein-coding genes (orange). b, Sequence conservation analysis (using Clustal Omega, EMBL-EBI) of human and zebrafish Ric1 proteins exhibit 81% similarity, and 71% identity. Positions of mutations detected with direct sequencing of rnd alleles (m641, m713, m715) are shown with arrows. Multiple sequence alignment shows highly conserved R882 residue (red) of the Rab6-interacting region across vertebrate species analyzed, i.e. zebrafish, rat, mouse and human. c, Electropherograms of direct sequencing results from genomic DNA of homozygous WT (+/+), heterozygous (+/−) and mutant (−/−) embryos for all three alleles, shading highlights mutation sites. 10 independent embryos of each genotype were analyzed and all revealed identical results as presented here. d, Ventral view of the teeth (arrows) stained by Alcian blue (cartilage) and Alizarin red (ossification), n=6 animals per group, e, lateral view of the pharyngeal teeth (white arrows) on 7th pharyngeal arch, n=6 animals per group. Orange arrows point to lack of cleithrum bone elongation in ric1−/− mutant embryo, 7 dpf; a: anterior, p: posterior.
Extended Data Fig. 2
Extended Data Fig. 2. Genetic analysis of RIC1 in biobanks and additional CATIFA patients
a, Correlation of imputation (R2) values for RIC1 expression across tissues. Sample sizes are indicated as ‘n’ next to each corresponding tissue on the graph. b, Q-Q plot showing the distribution of all association p-values with asthma in UK biobank. RIC1 association to asthma is statistically significant with p=7×10−9. Significance threshold is shown with black line set to p=1.5×10−5. c, Association of genes 4Mb around RIC1 locus with asthma. Note that association of RIC1 reduced expression with asthma is the most significant within this region. d, Pedigree of the additional family contributing to this study (modified from Patel et al., 201730). Standard pedigree symbols used; affected individuals are shaded, arrow points to patient 15DG2428 whose skin biopsy fibroblasts were sequenced (e), and analyzed by cellular and molecular methods. The mutation site is boxed, and results in Arg to Pro substitution in the protein sequence.
Extended Data Fig. 3
Extended Data Fig. 3. Analysis of matrix components in sheath cells and notochord basement membrane (BM)
a, Representative images of Tg(Col2a1a:caax-EGFP) transgenic zebrafish stained with Col2 antibody in WT and ric1−/− notochord sections (Collagen-2, magenta; EGFP, green). Note accumulation of intracellular Col2 in mutant sheath cells (arrowheads). 3 animals per group were analyzed and similar results were confirmed. b, TEM images of notochord tissue at 3 dpf. Magnified views of sheath cells and extracellular sheath / basement membrane (BM) are displayed on the right panels. Arrowheads point to intracellular inclusions. ER: endoplasmic reticulum, N: nucleus. Size bars = 500 nm. 3 cells per genotype were imaged and similar observations were confirmed. Collagen II fails to be transported out of Ric1-deficient chondrocytes, while other ECM cargos are secreted normally. c, Experimental design for immunohistochemistry (IHC) analysis with antibodies against Col2, matrilin, β-catenin and fibronectin epitopes on cryosections. d, Col2 (arrows) and Matrilin (arrowheads) co-immunostaining in 3 dpf chondrocytes. 3 animals per group and 5 cells per animal were analyzed and similar results were confirmed. e, β-catenin (arrows) and Fibronectin (arrowheads) co-immunostaining at 60 hours post fertilization (hpf) stage. DAPI (blue) marks nuclei. 3 animals per group and 5 cells per animal were analyzed and similar results were confirmed.
Extended Data Fig. 4
Extended Data Fig. 4. CRISPR/Cas9 genome editing-mediated depletion of ric1 recapitulates collagen secretion defects in a cell-autonomous manner
a, Co-immunostaining for collagen type-II (Col2) and WGA-labeled glycosylated matrix proteins at 3 dpf zebrafish cartilage. Arrowheads in ric1gRNA mosaic mutant (ric1−/− chondrocyte clones in the WT embryo injected with gRNA targeting ric1) point to collagen accumulations. Arrows indicate secreted, extracellular Col2. DAPI (blue) marks nuclei. This experiment was repeated with similar results using 4 animals per each genotype. b, Representative mutations detected in ric1gRNA mosaic mutants by direct sequencing. PAM: Protospacer adjacent motif.
Extended Data Fig. 5
Extended Data Fig. 5. Ric1 expression in zebrafish and RIC1R1265P/R1265P patient fibroblasts
a, Temporal expression profile of ric1 across zebrafish developmental stages by PCR. 3 independent experiments were run and presented here. Each experiment contained pooled RNA from 10 embryos of the indicated genotype and stage. Center line indicates the mean; bars show standard error of the mean (SEM). b, Temporal expression profile of ric1 by RNA-seq from whole embryos (data from Expression Atlas, White et al., 201731). c, Gene expression analysis of ric1 in sorted, live chondrocytes and non-chondrocytic cells by RNA-seq. R/FPKM: reads/ fragments per kilobase of transcript in million reads. d, QPCR analysis of ric1 in whole zebrafish embryos at 5 dpf stage, replicated with two primer sets. 3 independent experiments were run and presented here. Each experiment contained pooled RNA from 10 embryos of the indicated genotype and stage. Center line indicates the mean; bars show SD. e, Expression analysis of RIC1 in CATIFA patient’s fibroblasts (RIC1R1265P/R1265P) by qPCR. Two independent primer sets were used for each assay. QPCR data in d and e were normalized to β-actin or GAPDH. Mean expression levels relative to control (WT) are reported in center lines, SD bars are indicated. Two-tailed Mann-Whitney U-test, CI = 95%.
Extended Data Fig. 6
Extended Data Fig. 6. Constricted cell shapes of ric1−/− chondrocytes at 3 dpf and normal ultrastructure of WT cells by TEM at 3 and 4 dpf
a, TEM image of WT craniofacial cartilage showing normal chondrocyte shapes. b, A representative WT chondrocyte at 3 dpf b’, Higher magnification reveals vesicular structures associated with normal secretion (arrows). c, A representative TEM image of WT chondrocytes at 4 dpf. c’, Magnified view of boxed area is shown on the right, arrows point to normal vesicular compartments. Intracellular vesicles accumulate in ric1−/− chondrocytes. d, ric1−/− craniofacial cartilage chondrocytes contain large inclusions (arrowheads). e, Representative examples of chondrocytes constricted at the midline (arrows), green overlays on b & e are drawn after image acquisition to help with cell shape demarcation. f, TEM micrograph of a representative 3 dpf ric1−/− craniofacial chondrocyte displaying 3 types of vesicles, classified based on their electron densities: low density (arrows), medium density (double headed arrow) and vacuolar/high density (arrowheads). f’, Magnified view of the boxed region from f. g, Size distribution and average number of vesicles in 3 and 4 dpf ric1−/− chondrocytes. (20 cells at 3dpf; 10 cells at 4dpf stage). Vesicle diameters were measured both in parallel (W: width) and perpendicular (H: height) dimension to image plane; and plotted as length (μm) values. ECM: extracellular matrix, ER: endoplasmic reticulum, M: mitochondrion, N: nucleus. Mean and SD values are indicated with bars. Three ric1−/− embryos at 3 dpf and one at 4dpf were imaged with similar results. One representative WT of each stage was imaged with similar results across 10 cells imaged per embryo at each stage analyzed.
Extended Data Fig. 7
Extended Data Fig. 7. Muscle attachment defects in ric1−/− zebrafish
a, Whole-mount zebrafish embryos immunostained for myosin. Ventral head views are displayed. Arrows point to retracted fibers, in the form of bright punctate structures. ih: interhyoideus, imp: intermandibularis posterior, ima: intermandibularis anterior. b, Transmission electron micrographs of cranial muscles. Note non-continuous muscle fibers in ric1−/− (arrow). M: M-line, Z: Z-line. Right panels show magnified views of boxed regions in the left panels (2 independent mutant examples, one representative WT). c, Schematic of tendon matrix and consequent muscle fiber attachment defects. d, Quantification of swim behavior patterns, categorized as either burst (fast, sharp-angle turn) or slow (slower routine, wide-angle motion) swim pattern (Budick & O’Malley, 2000). Mean and SEM are shown as bars. Each data point represents a single embryo, n=16 independent embryos per genotype. Locomotion tracks of individual embryos, e, WT embryos, f, ric1−/− mutant embryos.
Extended Data Fig. 8
Extended Data Fig. 8. Analysis of brain and eye phenotypes in ric1−/− zebrafish
a, Whole mounted zebrafish embryos immunostained for HuC/D (pan-neuronal marker) and WGA (marking N-glycosylated proteins in neural ECM) at 5 dpf (Higher objective view of the same embryos in Fig. 4f). b, Digital zoom-in views showing sparser matrix in optic tectum and cerebellum regions of ric1−/−. c, Forebrain (n=8 independent animals per genotype) and d, Cerebellum area measurements (n=7 independent animals per genotype) performed by ImageJ, Measure Tool using whole mount immunostained images. Arbitrary units (a.u.) were plotted. Bars indicate the mean, lines are SD. Two-tailed Mann-Whitney U-test, CI = 95%. e, Acetylated tubulin whole-mount immunostaining that labels axonal projections. Right panel shows magnified views. Notable poor bundle structure of axonal projections in ric1−/− (arrows) and sparse axon tracks in cerebellum (arrowheads). f, Images of dissected whole zebrafish eyes under DIC illumination. g, Transmitted light illumination images of dissected zebrafish lenses. Note comparable smooth lens surface of WT and ric1−/− in both panels. n: number of embryos analyzed.
Extended Data Fig. 9
Extended Data Fig. 9. Rgp1-depletion recapitulates ric1−/− mutant phenotype
a, Schematic for genomic structure of rgp1 gene. Exons are shown as boxes, protein coding in blue and untranslated in gray, introns as red lines. Orange arrow marks guide RNA target site within exon 2. b, Deleterious mutations detected in mosaic rgp1CRISPR embryos by direct sequencing. c, Proposed model for Ric1-Rgp1 dependent activation of Rab6a on consequent collagen secretion, which modulates cartilage development. d, Live images of 5 dpf WT, ric1−/− (rndm713) and rgp1CRISPR (mosaic mutant embryo generated with CRISPR/Cas9 genome editing). Blue arrows on dorsal views point to body length differences. e, Alcian blue staining of craniofacial head skeletons at 5 dpf, black arrowheads point to Meckel’s cartilage protrusion and pink arrowheads to ceratohyal cartilage element. f, Graph for ceratohyal length measurements shows a range of lengths, likely due to mosaic nature of rgp1CRISPR mutants. Both left and right elements are plotted. N: number of embryos, n: number of cartilage elements analyzed (Student’s t-test, two-tailed, CI= 95%). Central line shows mean; bars indicate SD. g, Histological analysis of zebrafish cartilage at 3 dpf with Toluidine blue staining. Arrows point to the secreted ECM surrounding WT chondrocytes, arrowheads to diffused matrix staining around mutant cells. Clone-1 in rgp1CRISPR mosaic mutant is inferred to be composed of un-edited WT cells, while clone-2 is inferred to be of rgp1-mutant chondrocytes due to phenotypic resemblance to ric1−/− cells in the middle panel. This experiment was repeated with similar results from 3 independent animals from each indicated genotype.
Extended Data Fig. 10
Extended Data Fig. 10. Collagen-I accumulates in TGN-associated compartments in RIC1R1265P/R1265P patient’s fibroblasts
a, Representative images (of 3 experiments of independently grown cell cultures) for co-IF of BJ fibroblasts (control) and 15DG2428 (RIC1R1265P/R1265P patient’s dermal fibroblasts) using antibodies against collagen-I and p230 (TGN marker). Confocal images at low magnification (20x/0.80 Plan-Apochromat, WD=0.55mm) are presented. b, Representative low-magnification images (of 3 experiments of independently grown cell cultures) for co-IF with Col1 and alternative TGN marker Golgin-97 taken under the same conditions. Arrows point to TGN-associated Col1 signal. Overexpression of human RIC1 restores collagen secretion in ric1-deficient zebrafish chondrocytes. c, Experimental design to mosaically overexpress human RIC1 (hRIC1) gene in zebrafish. EGFP is linked to hRIC1 via self-cleavable viral 2A (v2A) peptide. d, Co-immunostaining of Col2 and EGFP in cartilage. Green cells express the construct and neighboring cells act as endogenous WT or mutant controls. Arrows point to collagen accumulation; arrowheads show secreted, extracellular collagen. Representative of 3 cells per group, with similar results.
Figure 1.
Figure 1.. Ric1 is required for craniofacial skeleton development and shape.
a, Zebrafish Ric1 protein is highly conserved with 81% similarity to human RIC1 (Clustal Omega, EMBL-EBI). Positional cloning identified mutations in round alleles (rnd m641, rnd m713, rnd m715; see arrows), Rab6 interacting region (blue bar, also Extended Data Fig. 1). b, Live images of ric1−/− mutant larvae show micrognathia (arrowhead), small head and shortened trunk (dashed line to arrow). c, Box-whisker plot for quantification of head size index (head to total body length ratio) as a measure of microcephaly. Mann-Whitney U-test (two-tailed) was used for statistical comparison; 95% confidence interval (CI). Boxes show 25th to 75th percentiles and medians. Whiskers are min to max values. d, Alcian blue and Alizarin red staining of the ric1−/− mutant shows shorter, malformed Meckel’s cartilage (arrowheads) and shorter, kinked pectoral fins (arrows). All three alleles exhibit indistinguishable phenotypes and fail to complement one another in genetic complementation assays. e, Live images of ric1−/− mutant embryos (5 dpf, days post-fertilization) overexpressing human RIC1 mRNA, show rescue of jaw protrusion (arrowheads) and elongation of the body length (arrows). Quantification of the rescue experiments, head in f, and body length in g. Statistical comparison by two-tailed Mann-Whitney U-test, CI = 95%, n=3 independent animals for ric1−/− and WT; n=7 animals for hRIC1 rescue group. h, Hyosymplectic cartilage (HC) dysmorphology shown in double transgenic Tg(Col2a1a:caax-EGFP; Col2a1a:H2A-mCherry) zebrafish that marks plasma membrane (green) and nucleus (red), white bar marks the symplectic arm (SA); maximum intensity projections of live z-stack images by confocal microscopy in WT and ric1−/− embryos (n=3 independent animals per group). i, 3D rendered structures of HC shapes highlight the malformed and short cartilage shape in ric1−/−. j, Magnified views of SA chondrocytes. 3D rendered volumes are overlaid onto maximum intensity projection views. k, 3D rendering underscores the dysmorphic shape and reduced volume of ric1−/− chondrocytes. l, Volume measurements of 5 cells per genotype by Imaris 8, color-coded to match the cells in k. Lines indicate mean and standard deviations in f, g and l.
Figure 2.
Figure 2.. Human common-disease phenome significantly associated with genetically determined reduced RIC1 expression in BioVU biobank and CATIFA patient phenome.
a, Design of gene-based PheWAS study conducted in BioVU biobank. b, Summary graph of traits (phecodes) significantly associated with predicted reduced expression of RIC1 in patients from BioVU biobank, as analyzed by PrediXcan algorithm; complete dataset and sample numbers are in the Supplementary Table 2. Traits are categorized into systems (y-axis), and significance is displayed on x-axis. Significance is tested by logistic regression analysis (two-sided). Multiple testing adjustment is done using Bonferroni correction. c, Individual skeletal phecodes from panel b are listed on y-axis. ‘Number of cases / total cases analyzed’ is indicated as insets within bars. x-axis shows significance levels (logistic regression, two-sided, Bonferroni-corrected). d, SNPs in the RIC1 gene are highly significantly associated, in the UK Biobank, with RIC1 phenome observed in the BioVU biobank. Representative traits and the p-value for the most significant SNP in RIC1 plotted; complete list of SNP-level and gene-level associations can be found in Supplementary Table 4. e, Pedigree of a large multiplex, consanguineous family with affected individuals shaded. Standard pedigree symbols used, genotypes are listed for tested individuals. Het: Heterozygous, Homo: Homozygous. f, Photographs of patients with CATIFA syndrome. Written consent for the use of photographs was obtained from the parents of affected individuals. g, Human Phenotype Ontology heat map of patients’ common clinical features. Table of complete clinical description for each subject is presented in Supplementary Table 5. h, Linkage analysis and LOD score value of CATIFA syndrome-linked RIC1 c3794G>C variant on chromosome 9 (arrow). i, Schematic of RIC1 protein marking the R1265 variant site within the Rab6 binding region. j, Translated RIC1 protein sequence is highly conserved among vertebrates; human, mouse and zebrafish, including R1265 residue (arrowhead).
Figure 3.
Figure 3.. Ric1 modulates procollagen transport.
a, Experimental design for immunohistochemistry (IHC) analysis of chondrocytes in the head. b, Representative images of Tg(Col2a1a:caax-EGFP) transgenic zebrafish stained with Col2 antibody (14 μm-thick cryosections) in WT and ric1−/− cartilage, (Collagen 2, magenta; EGFP, green). In WT Col2 signal is in extracellular space, outside caax-EGFP boundaries (dashed line), whereas in ric1−/− collagen accumulates in chondrocytes (inside caax-EGFP boundaries, arrows). c, d, Quantification of the percentage of cytoplasmic area occupied by Col2 signal in chondrocytes (c), and notochord sheath cells (d). For chondrocytes: N=3 embryos per group, n=30 cells per group. Notochord sheath cells: N=4 embryos per group, n=21 cells for WT, n=35 cells for ric1−/−. Data in c, d were analyzed with Mann-Whitney U test, two-tailed, CI= 95%. Mean and SD values are indicated with bars. e, Gene expression levels by RNA-seq from live, FACS-sorted chondrocytes, columns indicate mean values, bars show SD. RPKM: reads per kilobase of transcript per million mapped reads. 2 independent samples analyzed per genotype (WT and ric1−/−); 100 animals were included in each sample. f, TEM images of 4 dpf ric1−/− craniofacial chondrocytes show large vacuolar structures (arrowhead), f’ higher magnification of boxed area, and f” further zoom in of the blue box showing striated ultrastructure of intracellular collagen fibrils. Control WT images in Extended Data Fig. 6. g, Progression of matrix crosslinking in WT cartilage ECM from 3 dpf to 4 dpf. Note the ECM paucity and deficit in crosslinking at 4 dpf in ric1−/− ECM. TEM imaging was repeated with three independent ric1−/− animals at 3 dpf, one at 4 dpf, one WT sibling at 3 dpf and one at 4 dpf. 5 cells were imaged per animal. ECM: extracellular matrix, N: nucleus.
Figure 4.
Figure 4.. Ric1 depletion leads to musculoskeletal, brain and locomotion defects.
a, Whole-mount immunostaining of WT and ric1−/− mutant larvae for myosin (MF20, muscle) and thrombospondin-4 (TSP4) for tendon area. Ventral view of head muscles, inset: magnified view of boxed tendon region; arrowheads point to TSP4+ domains in the Meckel’s (left) and palatoquadrate (right) elements. n=4 animals for each group. Abbreviations for muscles, ih: interhyoideus, imp: intermandibularis posterior, ima: intermandibularis anterior, sh: sternohyoideus. b, Cryo-section and immunostaining of tendon area along sh muscle for collagen-I (Col1, red), phalloidin (muscle, green) and DAPI nuclear counterstain (blue). Tenocyte nuclei are marked by DAPI, arrows to the Col1 punctae. n=3 animals for each group c, Locomotion tracking of zebrafish embryos at 4 dpf stage. 16 WT and 16 ric1−/− embryos were tracked and plotted. Each color line indicates a separate animal (individual tracks in Extended Data Fig. 7). d, e, Graphs showing mean velocity of total travel (d) and maximum velocity reached during recorded travel (e) for each embryo. Mean and SD bars are displayed. Two-tailed Mann-Whitney U-test, CI = 95%, 16 WT and 16 ric1−/− larvae were analyzed. f, Whole mounted zebrafish embryos immunostained for HuC/D (pan-neuronal marker) and WGA (wheat germ agglutinin) at 5 dpf, maximum intensity projection. Forebrain and cerebellum structures are demarcated by dashed lines. Quantification is in Extended Data Fig. 8. 8 independent larvae per each group were analyzed. Arrowheads point to eminentia granularis; arrows to lobus caudalis cerebelli.
Figure 5.
Figure 5.. Ric1-Rgp1 GEF complex regulates procollagen transport via Rab6a activation.
a, Model of Rab6a activation by the Ric1-Rgp1 GEF complex to regulate collagen transport, and constitutively active Rab6a bypassing GEF requirement for collagen transport. b, IF labeling with Col2 antibody and wheat germ agglutinin (WGA) in 3 dpf zebrafish cartilage shows disrupted cell shape and tissue organization in ric1-mutants and rgp1gRNA (rgp1-guide RNA for CRISPR-Cas9 genome editing, Extended Data Fig. 9) injected WT embryos. Yellow arrows point to intracellular collagen accumulation, orange arrowhead points to extracellular Col2. This experiment was repeated with similar results, 3 independent animals per each group. c, Experimental design for mosaic overexpression (OE) of EGFP-Rab6a (WT form or Q72L constitutively active mutant) fusion protein in zebrafish notochord sheath cells. d, Col2 and EGFP co-immunostaining of sheath cells over-expressing constitutively active Rab6a (Q72L) mutant and wild type forms (bottom panel). Yellow arrows point to intracellular accumulations; orange arrowheads mark intracellular collagen in WT and rescued cells. e, Quantification of intracellular collagen accumulation within the cytoplasm, represented as percentage of Col2-stained area over cell surface by measuring areas from maximum intensity projections using ImageJ ‘Measure’ tool. OE of constitutively active Rab6a (Q72L) rescues intracellular collagen accumulation to WT levels (CI=95%, two-tailed Mann-Whitney U-test); whereas WT Rab6a does not. Mean and SD values are indicated with bars.
Figure 6.
Figure 6.. Pathogenic RIC1 variant leads to collagen accumulation in CATIFA fibroblasts.
a, TEM images of control BJ fibroblasts and RIC1R1265P/R1265P patient’s dermal fibroblasts show Golgi complex stacks (arrowheads). Arrow points to enlarged Golgi lumen and post-Golgi structures in patient fibroblasts. 5 cells of each group were imaged by TEM. b, Co-localization of Collagen 1 (Col1) and the TGN marker p230 (arrow) by IF in RIC1R1265P/R1265P cells. c, d, Quantification of the intracellular Col1 content (c), and Col1 co-localization with p230 (d) based on images in b. (Student’s t-test, two-tailed, CI= 95%). Mean value (center line) and SD bars are indicated in ‘c’; center line indicates the median and whiskers show min to max values in ‘d’. e, Experimental design for mosaic OE of human RIC1 (hRIC1) gene in zebrafish. EGFP is linked to hRIC1 via self-dissociating viral 2A (v2A) peptide marking hRIC1 expressing cells. f, Antibody labeling for Col2 and EGFP in notochord sheath cells shows collagen deposits (arrows). hRIC1 overexpressing cell (demarcated by dashed line) has fewer deposits (arrowheads). g, Mosaic OE of CATIFA-linked R1265P variant of hRIC1 only partially clears collagen deposits (arrows) while neighboring cells continue to retain Col2 and serve as ric1−/− controls. Similar observations were made in 3 cells for each group in f, g. h, Experimental design for hRIC1 mRNA OE and analysis of craniofacial morphology rescue. i, Phenotypic scoring results of protruding jaw morphology in hRIC1 mRNA overexpressing larvae and controls. n: number of animals analyzed. j, Dissected Alcian blue preparations of ethmoid plate and trabeculae (dorsal head skeleton) of hRIC1 mRNA injected larvae and controls show mosaic rescue of ric1−/− cells (arrows) with characteristic clear cytoplasm as in the WT skeletal element. The R1265P variant rescue more closely resembles cell morphology in the round mutants; 3 independent animals of WT and ric1−/−. k, Phenome comparison table for SEC23A gene, associated with cranio-lenticulo-sutural dysplasia (CLSD) in animal models, gene-based PheWAS analysis in BioVU and Mendelian disease patients. l, Phenome comparison table for FBN1 (fibrillin-1) gene, associated with Marfan syndrome. AV: atrioventricular, EKG: electrocardiogram. m, Integrated approach for discovery of gene function and disease mechanism using the animal model, common-disease phenome in a biobank and monogenic, rare Mendelian disease phenome of RIC1/CATIFA syndrome.
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References

    1. Klein C & Gahl WA Patients with rare diseases: from therapeutic orphans to pioneers of personalized treatments. EMBO Mol Med 10, 1–3 (2018). - PMC - PubMed
    1. Maddirevula S et al. Expanding the phenome and variome of skeletal dysplasia. Genet Med, 12, 1609–1616 (2018). - PubMed
    1. Arnold WV & Fertala A Skeletal diseases caused by mutations that affect collagen structure and function. Int J Biochem Cell Biol 45, 1556–1567, (2013). - PubMed
    1. Luderman LN, Unlu G & Knapik EW Zebrafish Developmental Models of Skeletal Diseases. Curr Top Dev Biol 124, 81–124, (2017). - PubMed
    1. Unlu G, Levic DS, Melville DB & Knapik EW Trafficking mechanisms of extracellular matrix macromolecules: insights from vertebrate development and human diseases. Int J Biochem Cell Biol 47, 57–67, (2014). - PMC - PubMed

Methods-only References

    1. Muller II, Knapik EW & Hatzopoulos AK Expression of the protein related to Dan and Cerberus gene--prdc--During eye, pharyngeal arch, somite, and swim bladder development in zebrafish. Dev Dyn 235, 2881–2888, (2006). - PubMed
    1. Knapik EW et al. A microsatellite genetic linkage map for zebrafish (Danio rerio). Nat Genet 18, 338–343, (1998). - PubMed
    1. Hoffmann K & Lindner TH easyLINKAGE-Plus--automated linkage analyses using large-scale SNP data. Bioinformatics 21, 3565–3567, (2005). - PubMed
    1. Varshney GK et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res, (2015). - PMC - PubMed
    1. Li Y, Willer CJ, Ding J, Scheet P & Abecasis GR MaCH: Using Sequence and Genotype Data to Estimate Haplotypes and Unobserved Genotypes. Genet Epidemiol 34, 816–834, (2010). - PMC - PubMed

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