Lansdon LA et al. (2023), Genome-wide analysis of copy-number variation i...

XB-ART-59509

Am J Hum Genet 2023 Jan 05;1101:71-91. doi: 10.1016/j.ajhg.2022.11.012.

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Genome-wide analysis of copy-number variation in humans with cleft lip and/or cleft palate identifies COBLL1, RIC1, and ARHGEF38 as clefting genes.

Lansdon LA , Dickinson A , Arlis S , Liu H , Hlas A , Hahn A , Bonde G , Long A , Standley J , Tyryshkina A , Wehby G , Lee NR , Daack-Hirsch S , Mohlke K , Girirajan S , Darbro BW , Cornell RA , Houston DW , Murray JC , Manak JR .

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Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders.

???displayArticle.pubmedLink??? 36493769
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Species referenced: Xenopus laevis
Genes referenced: arhgef38 cobll1 lpl nhlh1 pkp2 psmd6 ric1
GO keywords: palate development
???displayArticle.morpholinos??? arhgef38 MO1 arhgef38 MO2 cobll1 MO1 cobll1 MO2 ric1 MO1 ric1 MO2
gRNAs referenced: arhgef38 gRNA1 arhgef38 gRNA2 cobll1 gRNA1 cobll1 gRNA2 ric1 gRNA1 ric1 gRNA2

???displayArticle.disOnts??? orofacial cleft [+]
???displayArticle.omims??? VAN DER WOUDE SYNDROME 1; VWS1 [+]

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	Figure 1. Bioinformatics copy-number-variant prioritization pipeline Of the original 1,218 arrays, 1,102 passed quality controls and were used for downstream analyses. Copy-number variants (CNVs) which were overlapping an exon of a gene which passed minimum quality-control metrics (probe coverage and shift value) but occurring in less than 70% of the cohort or sharing less than 70% overlap with common CNVs or segmental duplications, were visually inspected. Genes that were recurrently deleted but at a frequency of less than 1% of the cohort with CL/P and at a higher frequency in individuals with clefts versus control subjects were prioritized for functional analysis.
	Figure 2. Log2 plots of top candidate clefting genes Log2 plots of (A) ARHGEF38, (B) COBLL1, and (C) RIC1 were generated using BioDiscovery’s Nexus 7 software and the UCSC genome browser. NSCLP, non-syndromic cleft lip and palate; NSCL, non-syndromic cleft lip only; NSC, non-syndromic cleft; SCLP, syndromic cleft lip and palate.
	Figure 3. Knockdown of Ric1, Cobll1, and Arhgef38 cause craniofacial malformations in Xenopus laevis (A) Schematic showing injection of reagents at the one-cell stage followed by imaging at stage 42–43 (80–87 hpf). Xenopus illustrations © Natalya Zahn (2022).74 (B and C) Frontal view of the face of representative tadpoles injected with control morpholinos (MOs) or Cas9. (D) Stacked bar graphs showing that 100% of the tadpoles were normal with respect to their craniofacial morphology. (E–H) Frontal views of the faces of representative tadpoles injected with splice-blocking MOs or short guide RNA (sgRNA)/Cas9 targeting ric1.L and ric1.S, respectively (three biological replicates for each). (I) Stacked bar graphs showing the percentage of the tadpoles that had normal faces, had craniofacial malformations, or triangular mouths that appeared cleft-like. (J–M) Frontal views of the faces of representative tadpoles injected with MOs or sgRNA/Cas9 targeting cobll1.L and cobll1.S (two biological replicates for each). (N) Stacked bar graphs showing the percentage of the tadpoles that had normal faces, had craniofacial malformations, or triangular mouths that appeared cleft-like. (O–R) Frontal views of the faces of representative tadpoles injected with MOs or sgRNA/Cas9 targeting arhgef38.L and arhgef38.S (two biological replicates for each). (S) Stacked bar graphs showing the percentage of the tadpoles that had normal faces, had craniofacial malformations, or triangular mouths that appeared cleft-like. The tadpole mouth opening is outlined in pink dots. Numbers of tadpoles are reported in the bottom right corner. CMO, control morpholino; CR, CRISPR-Cas9; st, stage; cg, cement gland; L, Xenopus laevis L homeolog; S, Xenopus laevis S homeolog.
	Figure S1: Copy number variant profiles detected in individuals from the Philippines by cleft type. The total number of copy number variants (CNVs; A-D), total amount of genomic content overlapped by CNVs (“CNV load”; E-H), and total amount of coding genomic content overlapped by CNVs (“CNV burden”; I-L), detected within the cohort from the Philippines were compared between clefting category [All; NSCLP, non-syndromic cleft lip and/or cleft palate; SCLP, syndromic cleft lip and/or cleft palate] and stratified by all calls (A, B; E, F; I, J), gains (C, G, K), or losses (D, H, L), respectively. No significant differences between cleft type and CNV profile were observed using a 2-sided Wilcoxon Rank Sum test and after correcting for multiple comparisons (27; comparisons for CNVs occurring at a 1-5% frequency and <1% frequency not shown) using Bonferroni (pvalue threshold for significance: <0.00185). Calculated p-values for each comparison shown are as follows: Total Number of CNVs (B) = 0.5093; Total Number of Gains (C) = 0.3898; Total Number of Losses (D) = 0.1141; Total CNV Load (F) = 0.4473; CNV Load Gains (G) = 0.4902; CNV Load Losses (H) = 0.8966; Total CNV Burden (J) = 0.3080; CNV Burden Gains (K) = 0.3077; CNV Burden Losses (L) = 0.7188.
	Figure S2: Sub-stratification of copy number variants within individuals with NSCL/P from the Philippines. The total number of copy number variants (CNVs; A-D), total amount of genomic content overlapped by CNVs (“CNV load”; E-H), and total amount of coding genomic content overlapped by CNVs (“CNV burden”; I-L), detected within individuals with non-syndromic cleft lip and/or cleft palate (NSCL/P) from the Philippines were compared between each individual’s genotypic sex (male, female; A, E, I), cleft type (CL, cleft lip only; CLP, cleft lip and cleft palate; CPO, cleft palate only; B, F, J), unilateral cleft sidedness (left, right; C, G, K), and cleft lip laterality (bilateral, unilateral; D, H, L). No significant differences between clefting category and CNV profile were observed using a 2-sided Mann-Whitney test (sex, sidedness, and laterality) or Kruskal-Wallis test (cleft type) after correcting for multiple comparisons (36; comparisons for gains and losses not shown) using Bonferroni (p-value threshold for significance: <0.00139). Calculated p-values for each comparison shown are as follows: Number of CNVs by Sex (A) = 0.1499; Number of CNVs by Cleft Type (B) = 0.4296; Number of CNVs by Cleft Sidedness (C) = 0.1389; Number of CNVs by Cleft Laterality (D) = 0.2301; CNV Load by Sex (E) = 0.0316; CNV Load by Cleft Type (F) = 0.4382; CNV Load by Cleft Sidedness (G) = 0.0455; CNV Load by Cleft Laterality (H) = 0.4902; CNV Burden by Sex (I) = 0.5619; CNV Burden by Cleft Type (J) = 0.4449; CNV Burden by Cleft Sidedness (K) = 0.1052; CNV Burden by Cleft Laterality (L) = 0.4902.
	Figure S3: Assessment of the largest copy number variants occurring within each individual by cleft type. The largest CNV within each individual in the study was binned by size and compared as a percent of the total cohort (All CNVs), or NSCLP and SCLP subgroups harboring a similarly sized CNV across any CNV type (A), losses (B), or gains (C). NSCLP, non-syndromic cleft lip and/or cleft palate; SCLP, syndromic cleft lip and/or cleft palate; kb, kilobase; Mb, megabase.
	Figure S4: Log2 plots of four candidate clefting genes with strong likelihood of involvement in craniofacial development which were not prioritized for functional analysis. Log2 plots of (A) LPL, (B) VWDE, (C) PKP2, and (D) EXOC4 were generated using BioDiscovery’s Nexus 7 software and the UCSC genome browser. NSCLP, non-syndromic cleft lip and palate; NSCL, non-syndromic cleft lip only; SCPO, syndromic cleft palate only.
	Figure S5: In situ hybridization of arhgef38, cobll1, and ric1 in Xenopus laevis. (A-C) Stages 25-29 lateral view, showing expression of arhgef38 throughout the head, with expression overlapping the region that includes the branchial arches shown by the red dotted lines in panels B and C. Sense shown as an inset in panel B. (D-F) Stages 25-29 lateral view, showing expression of cobll1 in craniofacial tissue, hatching gland, and cement gland. Expression overlapping the region that includes the branchial arches is indicated by the red dotted lines in panels E and F. Sense shown as an inset in panel E. (G-I) Stages 25-29 lateral view, showing expression of ric1 throughout the head, with expression overlapping the region that includes the branchial arches shown in red dotted lines in panels H and I. Sense shown as an inset in panel H. (A’-I’) Anterior view at stages 25-29 for each gene’s probe. Red arrows indicate the location of future mouth development. The brightness and contrast of these images have been adjusted to optimize the visualization of each probe. br, brain; cg, cement gland; hg, hatching gland; ov, otic vesicle; sc spinal cord; st, stage.
	Figure S6: Knockdown of arhgef38 in Xenopus laevis. (A, B) Verification of arhgef38 splice-blocking morpholinos. Shows schematic of the gene structure showing where the morpholino (MO) targets (orange) and the predicted exon deletion. Blue arrows represent the primers used to detect changes in the resulting mRNA. RT-PCR results shows alternative size product consistent with the predicted deletion (pink arrow). (C,D) Verification of arhgef38 CRISPR/Cas9 mutagenesis. Shows schematic of the gene structure and sgRNA target (red). Blue arrows represent the primers used to sequence the gene and detect mutations. Sequencing is shown for two controls (“C1”, “C2”) and five candidate mutants (“E1-E5”) with craniofacial malformations. The red shaded region is the sequence targeted by sgRNA. (E-W) Frontal and lateral views of representative tadpoles injected with MOs or sgRNA/Cas9 targeting arhgef38.L and arhgef38.S demonstrates the similar range of malformations as well the similarity of morphants and mutants. The mouth opening is outlined in pink dots. Pink arrows indicate a triangular shaped mouth. bp, base pair; cg, cement gland; cont, control; Cr, CRISPR mutant; MO, morpholino; sgRNA, short guide RNA; WT, wildtype.
	Figure S7: Knockdown of cobll1 in Xenopus laevis. (A, B) Verification of cobll1 splice-blocking morpholinos. Shows schematic of the gene structure showing where the morpholino (MO) targets (orange) and the predicted exon deletion. Blue arrows represent the primers used to detect changes in the resulting mRNA. RT-PCR results shows alternative size product consistent with the predicted deletion (pink arrow). (C, D) Verification of cobll1 CRISPR/Cas9 mutagenesis. Shows schematic of the gene structure and sgRNA target (red). Blue arrows represent the primers used to sequence the gene and detect mutations. Sequencing is shown for two controls (“C1”, “C2”) and five candidate mutants (“E1-E5”) with craniofacial malformations. The red shaded region is the sequence targeted by the sgRNA. (E-W) Frontal and lateral views of representative tadpoles injected with MOs or sgRNA/Cas9 targeting cobll1.L and cobll1.S demonstrates the similar range of malformations as well the similarity of morphants and mutants. The mouth opening is outlined in pink dots. Pink arrows indicate a triangular shaped mouth. bp, base pair; cg, cement gland; cont, control; MO, morpholino; sgRNA, short guide RNA; WT, wildtype.
	Figure S8: Knockdown of ric1 in Xenopus laevis. (A, B) Verification of ric1 splice-blocking morpholinos. Shows schematic of the gene structure showing where the morpholino (MO) targets (orange) and the predicted exon deletion. Blue arrows represent the primers used to detect changes in the resulting mRNA. RT-PCR results shows alternative size product consistent with the predicted deletion (pink arrow). (C,D) Verification of ric1 CRISPR/Cas9 mutagenesis. Shows schematic of the gene structure and sgRNA target (red). Blue arrows represent the primers used to sequence the gene and detect mutations. Sequencing is shown for two controls (“C1”, “C2”) and five candidate mutants (“E1-E5”) with craniofacial malformations. The red shaded region is the sequence targeted by sgRNA. (E-W) Frontal and lateral views of representative tadpoles injected with MOs or sgRNA/Cas9 targeting ric1.L and ric1.S demonstrates the similar range of malformations as well the similarity of morphants and mutants. The mouth opening is outlined in pink dots. Pink arrows indicate a triangular shaped mouth. bm, buccopharyngeal membrane; bp, base pair; cg, cement gland; cont, control; MO, morpholino; sgRNA, short guide RNA; WT, wildtype.
	Figure S9: Alcian blue staining of craniofacial cartilage from arhgef38, cobll1, and ric1 splice-blocking morpholino knockdowns in Xenopus laevis tadpoles. (A) Schematic depicting the injection of splice-blocking morpholinos (MOs) at the one-cell stage followed by aging to stage 45 (98-106 hours post fertilization) and Alcian blue staining. Xenopus illustrations © Natalya Zahn (2022).150 (B, B’) Ventral and dorsal views of the craniofacial cartilage of a representative tadpole injected with a control morpholino (CMO). (C-D’) Ventral and dorsal views of the craniofacial cartilage of representative tadpoles injected with a splice-blocking MO targeting arhgef38.L that developed moderate (C, C’) or severe (D, D’) phenotypes. (E-F’) Ventral and dorsal views of the craniofacial cartilage of representative tadpoles injected with a splice-blocking MO targeting arhgef38.S that developed moderate (E, E’) or severe (F, F’) phenotypes. (G) Stacked bar graph showing percentage of tadpoles with normal faces, mild cartilage phenotypes (one cartilage structure affected), moderate cartilage phenotypes (multiple cartilage structures affected), or severe cartilage phenotypes (near absence or major disfiguration of the cartilage structures). (H-I’) Ventral and dorsal views of the craniofacial cartilage of representative tadpoles injected with a splice-blocking MO targeting cobll1.L that developed moderate (H, H’) or severe (I, I’) phenotypes. (J-K’) Ventral and dorsal views of the craniofacial cartilage of representative tadpoles injected with a splice-blocking MO targeting cobll1.S that developed moderate (J, J’) or severe (K, K’) phenotypes. (L) Stacked bar graph showing percentage of tadpoles with normal faces, mild cartilage phenotypes, moderate cartilage phenotypes, or severe cartilage phenotypes. (M-N’) Ventral and dorsal views of the craniofacial cartilage of representative tadpoles injected with a splice-blocking MO targeting ric1.L that developed moderate (M, M’) or severe (N, N’) phenotypes. (O-P’) Ventral and dorsal views of the craniofacial cartilage of representative tadpoles injected with a splice-blocking MO targeting ric1.S that developed moderate (O, O’) or severe (P, P’) phenotypes. (Q) Stacked bar graph showing percentage of tadpoles with normal faces, mild cartilage phenotypes, moderate cartilage phenotypes, or severe cartilage phenotypes. Red arrowhead indicates a defect in trabecular cartilage (tc), red arrow indicates a defect in ceratohyal cartilage (ch), black arrowhead indicates a defect in Meckel’s cartilage (mc), black arrow indicates a defect in ceratobranchial cartilage (cb). bhb, basihyobranchial cartilage; pq, palatoquadrate cartilage.
	Figure S10: Abnormal development of the craniofacial skeleton in F0 Danio rerio larvae after targeting ric1, arhgef38, and cobll1a with CRISPR. (A) Table showing genes of interest and summary of phenotypes in Danio rerio injected at single cell stage with CRISPR/Cas9 reagents targeting the indicated gene. Resulting abnormalities were quantified at 48 hours post fertilization (hpf) and four days post fertilization (dpf). All three candidate genes showed head deformities similar to a positive control (radil). (B-F) Ventral views, anterior to the left, of larvae fixed at four dpf and processed with alcian blue. Figure B was adapted from Albertson and Yelick, 2004 showing an uninjected larva (WT, wildtype). (C-F) larvae injected with Cas9 protein and sgRNAs targeting the indicated genes, with the two targeted regions shown to the right of each skeletal image and highlighted in blue (listed as sgRNA A and sgRNA B). Meckel’s cartilage (mc; red arrow) is hypoplastic in all the F0 mutants, and dysmorphogenesis of the ceratohyal arch (red asterisk) was commonly accompanied by the loss or reduction of the ceratobranchial arches. ch, ceratohyal arch; cb, ceratobranchial arches; gRNA, short guide RNA; pos cont, positive control.

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