- Title
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Biallelic loss-of-function variants in CACHD1 cause a novel neurodevelopmental syndrome with facial dysmorphism and multisystem congenital abnormalities
- Authors
- Scala, M., Khan, K., Beneteau, C., Fox, R.G., von Hardenberg, S., Khan, A., Joubert, M., Fievet, L., Musquer, M., Le Vaillant, C., Holsclaw, J.K., Lim, D., Berking, A.C., Accogli, A., Giacomini, T., Nobili, L., Striano, P., Zara, F., Torella, A., Nigro, V., Cogné, B., Salick, M.R., Kaykas, A., Eggan, K., Capra, V., Bézieau, S., Davis, E.E., Wells, M.F.
- Source
- Full text @ Genet. Med.
Genetic and clinical aspects of affected individuals harboring CACHD1 variants. A. Location of CACHD1 variants in relation to exonic location (top); and domain structure (bottom; GenBank: NM_020925.4, NP_065976.3). CACHD1 consists of an exofacial N terminus, a von Willebrand factor A (VWA) domain, 2 bacterial chemosensory-like cache domains, a short hydrophobic transmembrane domain, and an intracellular C terminus. Exonic variants affect exons 3, 4, and 6. Intronic variants localize in introns 2, 12, and 16. Most variants affect the early portion of the gene, predicted to lead to premature transcription termination and putative NMD. Numbers under the protein schematic indicate amino acid numbers. Cache, Ca2+ channel and chemotaxis receptor; CR, cysteine rich; HR, histidine rich; MIDAS, metal ion-dependent adhesion site; NLS, nuclear localization signal; TM, transmembrane domain. Locations of domains are approximate based on data from www.Uniprot.org. B. Pedigrees of the reported families with the segregation patterns of CACHD1 variants. Open shapes, unaffected individuals; filled shapes, affected individuals; square, male; circle, female, triangle, pregnancy not carried to term; wt, wild type. C. Clinical photographs. Individual #1 at 13 years shows sparse hair, medially sparse eyebrows, a pit on the left cheek, and small and posteriorly rotated ears with preauricular tags, underdeveloped crus of the helix, and uplifted earlobes. Individual #2 at 1 year shows bilateral preauricular skin tags associated with overfolding of the superior helices. Individuals #5 (31+5 weeks) and #6 (21+5 weeks) show long and thick eyebrows, periorbital rings, palpebral edema, low-set ears with dysplastic outer ear and bilateral preauricular skin tags, and macroglossia. D. Graph summarizing the distribution of the most common clinical features (present in at least two cases) in the reported cohort. Abbreviations: CMDTs, congenital malformations of the digestive tract; DD, developmental delay; ID, intellectual disability; NA, not applicable. |
CACHD1 depletion alters NPC proliferation and differentiation. A. Schematic describing the workflow of NPC cellular. B. Quantification of the percentage of HuCD+ post-mitotic neurons in Day 30 differentiation cultures from control and CACHD1-edited SNaP lines. Representative images depict nuclei in blue and HuCD in green. Scale bar, 50 μm. C. Quantification of the percentage of SOX2+ NPCs in Day 30 differentiation cultures from control and CACHD1-edited SNaP lines. Representative images depict nuclei in blue and SOX2 in green. Scale bar, 50 μm. D. Schematic describing the production of 3D neurospheres derived from 2D SNaP cultures. E. Lightsheet imaging of a day 7 SNaP-neurosphere stained with phospho-Vimentin (NPC marker) and MAP2 (neurite marker). F. Representative bright field images of control and CACHD1-edited neurospheres at day 15 post-plating. Scale bar, 1 mm. G. Quantification of (F). Data are represented as mean ± S.D. |
Genetic ablation of CACHD1 affects expression of key neurodevelopmental genes and signaling pathways. A. Venn diagram depicting the overlap of differentially expressed genes (DEGs) between the 2 CACHD1-depleted NPC lines. B, C. Volcano plots of DEGs, comparing each CACHD1-depleted line with NT control. Open circles reflect DEGs consistent in both CACHD1-depleted lines relative to NT control. Positive mean log2 fold change (FC) refers to genes that are upregulated in CACHD1-edited cells. Statistical significance is defined as Benjamini-Hochberg adjusted P values < .05. Vertical dashed lines represent log2FC = 0.6 (FC ∼ 1.5 fold), whereas horizontal dashed lines represent the adjusted P value threshold of P < .05. DEGs that pass both the significance and FC thresholds are colored in red (upregulated) or blue (downregulated). Genes in green are known Wnt pathway genes. D, E. Gene Ontology (GO) term analysis of 382 DEGs; (D) downregulated and (E) upregulated genes in CACHD1-depleted lines. |
Ablation of zebrafish cachd1 by CRISPR-Cas9 results in craniofacial abnormalities. A. Schematic of zebrafish cachd1 transcript (GRCz11, Ensembl transcript ID: ENSDART00000087964.7) generated by Exon-Intron Graphic Maker (http://wormweb.org/exonintron). The sgRNA target site on exon 9 is indicated by a red triangle. Scale bar, 10 kb. B. Representative sequence chromatograms of cachd1 +/+ (wild type), cachd1+/− (heterozygous mutant), and cachd1−/− (homozygous mutant) are shown. Protospacer adjacent motif (PAM) is indicated by a red box for each chromatogram. Mutants harbor a 16 bp deletion (19 bp deletion and 3 bp insertion) that results in a frameshift and putative protein truncation (p.Phe452LeufsTer3). C. Bar graph showing relative mRNA expression of cachd1 in genotype-matched 2 day post-fertilization (dpf) larvae generated from F1 in-crosses. n = 20 per batch, 3 technical replicates per experiment, 8 biological replicates (One way ANOVA; Tukey’s multiple comparisons test). Tails were used for genotyping, whereas RNA was extracted from the matched heads for quantitative PCR analysis. Relative expression was normalized to gapdh, and statistical differences were calculated using One way ANOVA (F [5, 42] = 50.39; P value < .0001]; Tukey’s multiple comparisons test’s P values for mutants versus +/+ are indicated above each bar. Error bars represent standard error of the mean. D. Schematic of 3 dpf zebrafish larva showing a ventral view of craniofacial cartilage structures. Abbreviations: Meckel’s cartilage (mk, blue), palatoquadrate (pq, yellow), ceratohyal (ch, orange), hyosymplectic (hs, gray), and ceratobranchial (cb, green). E. Representative images of −1.4col1a1:egfp;cachd1 larvae imaged live at 3 dpf. Top: bright field lateral images of wild-type and homozygous mutants. Scale bar, 200 μm. Bottom: fluorescent ventral images of wild-type and homozygous mutants. Region of interest (ROI) area, as outlined in red and bordered by ch, pq, and mk, was measured to detect statistical differences. Scale bar, 100 μm. F. Quantification of ROI indicated in (E); AU, arbitrary units. Statistical differences were calculated using an unpaired t test (n = 50-70 larvae per condition, repeated). Biological replicates (1 and 2 as indicated in x axis labels) were obtained from different parental pairs with the investigator masked to the experimental conditions. Regardless of their parental genotype, all embryos obtained were morphologically similar,and no animal was excluded from imaging and quantification. Error bars represent standard deviation of the mean. |