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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Summary statistics from SNP- and gene-level (PrediXcan) associations are available in Supplementary Tables 1–8. The protected data for the GTEx project (for example, genotype and RNA-seq data) are available via access request to dbGaP accession no. phs000424.v6.p1. All requests for raw (for example, genotype and phenotype) data and materials are reviewed by Vanderbilt University Medical Center to determine whether the request is subject to any intellectual property or confidentiality obligations. For example, patient-related data not included in the paper may be subject to patient confidentiality. Any such data and materials that can be shared will be released via a material transfer agreement. All gene, transcript and protein sequences can be found at the NCBI and Ensembl databases (accession numbers are provided in Methods).
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We are grateful to the families and participating individuals for their contribution. We thank the Sequencing and Genotyping Core Facilities at KFSHRC for their technical help. We thank C. Guthrie and A. Goodrich of ZCORE at VUMC for excellent zebrafish care. We thank the Vanderbilt University Nikon Center of Excellence and Vanderbilt University Cell Imaging Shared Resource (CISR) for technical support with confocal imaging and data analysis, and M. Vinogradova (CISR) for expertise and technical assistance with TEM. This manuscript is dedicated to the memory of K. Anderson Faulds. We acknowledge the NIH (no. R01 MH113362 to N.J.C. and E.W.K., no. U01 HG009086 to N.J.C. and B.L., no. R35HG010718 to E.R.G. and no. T32 GM008554 to D.B.M.) for financial support. We acknowledge the support of the Saudi Human Genome Program and KSCDR (to F.S.A.). G.U. was supported by the Vanderbilt International Scholar Program, and AHA predoctoral fellowship (no. 15PRE22940041). E.R.G. is grateful to the President and Fellows of Clare Hall, University of Cambridge, UK for a stimulating academic home during his fellowship in the college. We acknowledge VANTAGE and VICTR for construction and maintenance of the BioVU biorepository and the funding sources that support them—nos. S10RR025141, UL1TR002243, UL1TR000445 and UL1RR024975. BioVU genomic data are also supported by investigator-led projects that include nos. U01HG004798, R01NS032830, RC2GM092618, P50GM115305, U01HG006378, U19HL065962 and R01HD074711.
E.R.G. receives an honorarium from the journal Circulation Research of the American Heart Association, as a member of the editorial board. He performs consulting on pharmacogenetic analysis with the City of Hope/Beckman Research Institute. The remaining authors declare no competing interests.
Peer review information Kate Gao and Brett Benedetti were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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=7x10-9. Significance threshold is shown with black line set to p=1.5x10-5. 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., 30). 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 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 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 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.
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., 31). 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 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.
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.
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.
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 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.
3D-rendered structures of HC of 3-dpf WT embryo.
3D-rendered structures of HC of 3-dpf ric1–/– embryo.
3D-rendered structures of symplectic arm chondrocytes of 3-dpf WT embryo.
3D-rendered structures of symplectic arm chondrocytes of 3-dpf ric1–/– embryo.
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Unlu, G., Qi, X., Gamazon, E.R. et al. Phenome-based approach identifies RIC1-linked Mendelian syndrome through zebrafish models, biobank associations and clinical studies. Nat Med 26, 98–109 (2020) doi:10.1038/s41591-019-0705-y