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Cranio-lenticulo-sutural dysplasia is
caused by a SEC23A mutation
leading to abnormal endoplasmicreticulumto-Golgi trafficking
SEC23A is an essential component of the COPIIcoated 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.
CLSD was
originally
described in five
males and one
female from a
large
consanguineous
Saudi Arabian
family of Bedouin
Descent.
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).
The F382L substitution involved a residue that is
invariably conserved in at least ten species (Fig. 1c).
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).
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).
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.
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).
In vitro studies of F382L SEC23A. (a) Liposomebinding 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.
(b) Vesicle formation assay shows that F382L
SEC23A has markedly lower activity for generating
argocontaining vesicles as compared with the wildtype 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.
Developmental expression of sec23a and loss-of-function
phenotype in zebrafish.
(a)RT-PCR analysis detected sec23a transcript at the onecell stage, suggesting that sec23a is present as a
maternal transcript.
(b) (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.
(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).
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.
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-reticulumto-Golgi trafficking.
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.