Cranio-lenticulo-sutural-dysplasia (CLSD) is a recessive syndrome with craniofacial, skeletal and neural defects (9). CLSD was originally associated with a homozygous F382L mutation in SEC23A (8). SEC23A is an essential component of COPII vesicles, which are responsible for export of cargo molecules out of the endoplasmic reticulum (ER) (77). The SEC23A F382L mutation disrupts COPII vesicle assembly (20). Recently, we have identified a novel CLSD case with a heterozygous M702V SEC23A mutation (10, 27). Surprisingly, fibroblasts derived from this patient display a collagen-specific secretion defect. Depletion of Sec23a results in a reduction in the extracellular matrix/collagen and craniofacial/skeletal abnormalities in fish (8, 31). Combined, these findings define a critical link between COPII vesicle biogenesis, secretion of collagen and craniofacial development. In this study we investigate this link. Procollagen forms about a 300nm-long rigid fibril in the ER and requires COPII proteins for export (77). However, because of its size and rigidity, procollagen cannot be packaged into the standard COPII vesicles which have a 60nm diameter. Recently, it was shown that CUL3-KLHL12 drives the assembly of large COPII-coated structures and expedites secretion of collagen (23). However, how COPII proteins generate these megavesicles remains unclear. Our studies have demonstrated that the sizes of these unusually large COPII vesicles, but not the sizes of the standard COPII vesicles, are reduced in M702V SEC23A patient's fibroblasts, suggesting a pronounced lesion in the assembly of COPII megavesicles but not standard COPII vesicles. Additionally, we have recently identified a novel CLSD case in which the patient carries novel compound heterozygous mutations in SEC24D, a cargo- loading subunit of COPII. This is the first reported human disease with a SEC24D mutation. To test the in vivo function of Sec24d, we have generated Sec24d null mice. Sec24d null embryos presented with holoprosencephaly and craniofacial anomaly, features that are known to result from a defect of type II collagen (32). Therefore, the secretion block of collagens and/or other critical factors may account for the skeletal, craniofacial and neural defects of CLSD. Our long term goal is to understand how the COPII vesicle assembly is integrated into the blueprint of vertebrate development. We hypothesize that CLSD is a disease of aberrant COPII megavesicle assembly. We will test this hypothesis by pursuing following questions: 1) what are the molecular features of COPII paralogs that contribute to megavesicle assembly?; 2) what are the mechanisms that regulate the size of a COPII megavesicle?; and 3) what are the roles of COPII megavesicles during embryonic development?

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We have identified SEC23A and SEC24D mutations linked to cranio-lenticulo-sutural dysplasia. We have acquired patients' fibroblasts and have generated mice lines deficient for Sec23a, Sec31a and Sec24d. Analyses of the mutant proteins, patients' fibroblasts, and COPII-deficient cell lines will allow us to decipher the fundamental mechanism for the assembly of COPII megavesicles, which will deepen our understanding on craniofacial diseases as well as disorders of forebrain development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing Study Section (MBPP)
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Flicker, Paula F
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Iowa State University
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Justice, Cristina M; Kim, Jinoh; Kim, Sun-Don et al. (2017) A variant associated with sagittal nonsyndromic craniosynostosis alters the regulatory function of a non-coding element. Am J Med Genet A 173:2893-2897
Garbes, Lutz; Kim, Kyungho; RieƟ, Angelika et al. (2015) Mutations in SEC24D, encoding a component of the COPII machinery, cause a syndromic form of osteogenesis imperfecta. Am J Hum Genet 96:432-9