Developmental expression of sec23a and loss-of-function phenotype in zebrafish.

1. Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulumto-Golgi trafficking

2. SEC23A is an essential component of the COPII-coated vesicles that transport secretory proteins from the endoplasmic reticulum to the Golgi complex. Electron microscopy and immunofluorescence show that there is gross dilatation of the endoplasmic reticulum in fibroblasts from individuals affected with CLSD. The COPII coat is a polymer complex formed by at least five well-characterized proteins: SAR1, SEC23, SEC24, SEC13 and SEC31.

3. CLSD was originally described in five males and one female from a large consanguineous Saudi Arabian family of Bedouin Descent.

4. We identified 47 known and predicted genes in the candidate region. PSMA6, GARNL1, BRMS1L and MBIP were screened by direct sequencing of cDNA generated by RT-PCR, and no variations were identified. We observed a 1144T-C transition in SEC23A that segregated in a homozygous form in all affected individuals and was not present in 600 control chromosomes (Fig. 1b).

5. The F382L substitution involved a residue that is invariably conserved in at least ten species (Fig. 1c).

7. On the basis of the known biological function of SEC23A, we predicted excessive accumulation of secretory proteins in the rough endoplasmic reticulum of the mutant fibroblasts. Using an antibody to the intralumenal endoplasmic reticulum chaperone PDI18, we detected abundant vacuolar structures in homozygous mutant cells that we nterpreted as distended endoplasmic reticulum (Fig. 2a–f).

8. Immunofluorescence with an antibody to procollagen COL1A1 showed substantial accumulation of this protein in endoplasmic reticulum cisternae identical in morphology to those marked by PDI (Fig. 2d–f). Immunofluorescence with an antibody to SEC31 showed diffuse cytoplasmic mislocalization of this protein in the mutant fibroblasts, suggestive of abnormal formation of the COPII complex (Fig. 2g,h).

9. Wild-type and F382L SEC23A heterozygous and homozygous mutant fibroblasts were examined by thin-section electron microscopy. Most wild-type cells showed a typical organization of the rough endoplasmic reticulum with narrow cisternae (Fig. 3a), and about 10% of cells showed mild focal dilatation of the endoplasmic reticulum.

10. Roughly 35% of heterozygous fibroblast sections showed a moderate generalized dilatation of endoplasmic reticulum (Fig. 3b). More than 80% of the homozygous mutant cells had endoplasmic reticulum cisternae that were greatly distended by an accumulation of secretory material (Fig. 3c).

11. In vitro studies of F382L SEC23A. (a) Liposome-binding assay shows that, when SAR1B is activated (triphosphate bound), the mutant protein binds to synthetic membranes to an extent similar to that of the wild-type protein. D, GDPbS; T, GTPgS.

12. (b) Vesicle formation assay shows that F382L SEC23A has markedly lower activity for generating argocontaining vesicles as compared with the wild-type protein. The endoplasmic reticulum resident protein ribophorin-I was used as a negative control; p58 and Sec22b are COPII cargo proteins. ATPr, ATP regeneration system.

14. Developmental expression of sec23a and loss-of-function phenotype in zebrafish. • RT-PCR analysis detected sec23a transcript at the one-cell stage, suggesting that sec23a is present as a maternal transcript. • (b,c) Wholemount in situ hybridization analysis confirmed the presence of maternal transcript at the onecell stage (b) until the 1,000-cell mid-blastula transition stage (c). • (d) At the 12-somite stage, weak but distinct expression is detected in the developing notochord.

15. (e) Notochord expression is strongest in the 1-d.p.f. embryo, especially in the tail bud region and the ventral tail edge (inset:25-somite stage). In 2-d.p.f. embryos, expression is no longer detectable in the notochord, but begins to be observed in the developing head cartilages (not shown). (j–q) Loss-of-function 5-d.p.f. morphants show reduced body length and dorsal curvature (j) as compared with wild-type larvae (k), kinked pectoral fins (l,m) owing to a larger non-cartilaginous fin segment at the distal edge (n,o), and malformation or dysgenesis of the head cartilages (p,q).

16. In conclusion, we have delineated CLSD as a dysmorphic genetic syndrome with characteristics of a skeletal dysplasia and have identified its genetic cause. Our experiments show that CLSD occurs as a result of defective COPII-mediated endoplasmic reticulum export owing to loss of function of SEC23A. As a result, collagen and (probably) other secretory proteins accumulate and distend the endoplasmic reticulum, ultimately leading to the clinical manifestations of CLSD.

17. The relatively mild phenotype of affected individuals suggests that the 1144T-C SEC23A mutation is a hypomorph and that the mutant protein retains some residual functional activity. Further studies of the mutant cells and/or a SEC23A animal model will allow more precise identification of the cargo proteins retained in the endoplasmic reticulum as a result of mutations in SEC23A. The characteristic phenotype of the SEC23A mutant cells suggests that screening methods could be developed to facilitate the identification of other human disorders caused by defects in endoplasmic-reticulum- to-Golgi trafficking.

18. Analysis of the orthologous sec23a gene in zebrafish revealed an anatomical and morphological correlation with the human CLSD phenotype, providing further evidence that loss of SEC23A function is responsible for this genetic syndrome.