Supplementary Data (doc 42K)

Supplementary data
Supplementary methods
Transferrin- and Apolipoprotein-C isoelectric focusing
Transferrin isoelectric focusing (TIEF) was carried out as screening for glycosylation
disorders. Iron-saturated plasma or serum was separated on a hydrated immobiline gel
(pH 4–7) on Ultraphore system (Amersham Pharmacia Biotech). After IEF, the isoforms
were visualized by adding rabbit anti-human transferrin antibodies (Dako, Glostrup,
Denmark) and stained with Coomassie blue. Apolipoprotein-C isoelectrofocusing (apoCIII IEF) was carried out as described [2S]. Plasma was separated on a hydrated dry IEF gel
(pH 3.5–5.0 and 8 M urea) on a PhastSystem. After IEF, a Western blot was performed.
The isoforms were specified by adding rabbit anti-human antibody and visualized by
electrochemiluminescence and determined using densitometry.
Histology, electron microscopy, and immunohistochemistry and immuno-electron
3 mm punch biopsies of the skin were examined by light microscopy on 4 µm thick
paraffin slides stained with HE and a Verhoeff van-Gieson stain (EVG) which
specifically stains elastic material. Electron microscopy was performed in patients 1, 3, 7
and 9. In these cases half of the punch biopsies were fixed in 2% Glutaraldehyde
buffered with 0.1M Sodium cacodylate pH 7.4, postfixed in 1% Osmium teroxide in
Paladebuffer pH 7.4 with 1% Kaliumhexacyanoferrat(III)-Trihydrat and after dehydration
in ethanol and propyleenoxide, embedded in Epon. Semithin, 1µm thick transverse
sections were stained with 1% Toluidine Blue. Ultrathin sections were stained with
Uranyl acetate and Lead citrate. For each patient, images of representative electron
microscopic fields of view were recorded for papillary dermis and for reticular dermis
separately using a JEM-1200EX II (Jeol Europe B.V., The Netherlands) microscope at
low (1.5k – 3k) and medium (20-25k) magnification setting. At each level specific
attention was paid to the density and the organization of collagen and elastin fibers.
One-micron thick sections were incubated with rabbit polyclonal antibodies against
panspecific transforming growth factor-b (TGF-b) (R&D, Milano, Italy) and tumor
necrosis factor-a (TNF-a) (Sigma, St Louis, MO, USA) for 20 h at 4_C. To block
nonspecific reactions, sections were treated with 0.1% trypsin in phosphate-buffered
saline (PBS), pH7.7 for 30 min at room temperature before incubation with the
antibodies. Antibodies were assayed at different concentrations (from 1:20 to 1:100) and
were diluted with PBS containing 1% bovine serum albumin (Sigma), 0.1% sodium
azide, and 0.1% Triton X-100. After incubation with antibodies, sections were carefully
washed in PBS and stained by the streptavidin–biotin method by using diaminobenzidine
hydrochloride as the chromogen.
Ultrathin sections were collected on nickel grids and processed for immunogold labeling
as described by Polak and coworkers. Sections were incubated with polyclonal antibodies
against elastin (EPC) (1:300 diluted), and neutrophilic elastase (EPC) (1:200 diluted).
Incubation was performed for 20 h at 4oC. After exhaustive washing in PBS, sections
were incubated with secondary antibodies (goat anti-rabbit immunoglobulin G)
conjugated with colloidal gold particles of 15nm (EY Laboratories Inc., San Mateo, CA,
USA) for 1.5 h at room temperature [1S].
Fibroblast culture and Brefeldin A treatment
To analyze retrograde transport patient and control fibroblasts were cultured at 37oC and
5% CO2 in E199 medium (Gibco), supplemented with 10% fetal calf serum (Lonza) and
1% penicillin/ streptomycin (Gibco). At 70% confluence cells were subjected to 5 μg/ml
Brefeldin A (Sigma-Aldrich) as described (after which cells were fixed in 4% (w/v)
paraformaldehyde in PBS and stored overnight at 4oC. Permeabilization was performed
using 0.4% (v/v) Triton-X100 in 3% (w/v) BSA in PBS for 10 min at 4 oC. For
incubation with the primary antibody specific antibodies to GM130 (mouse anti-GM130,
BD Transduction Laboratories) and Giantin (rabbit anti-Giantin, Covance) were
dissolved in 3% BSA in PBS and cells were incubated for one and a half hour after which
cells were washed thrice in PBS.
For detection anti–rabbit IgG Alexa Fluor 488 conjugate (Invitrogen, 1:1000) and an
antimouse IgG Alexa Fluor 555 (Invitrogen, 1:1000) were applied in 3% BSA in PBS.
Cells were mounted in one step using ProLong Gold antifade reagent with DAPI
(Invitrogen). Images were recorded using an LSM 510 Meta (Carl Zeiss) with a 63 Plan
Apochromat oil immersion objective.
Supplementary results
Pathogeneicity prediction of novel mutations
The homozygous frameshift mutations c.754delT (p.Tyr252Ilefs*15), the heterozygous
frameshift mutations c.1101delC, c.1562_1563delins9 and c.600delC
(p.Thr368Leufs*43, p.Ile521Metfs*16 and p.Ile201Serfs*20 respectively) in ATP6V0A2
were predicted to result in truncated proteins and thus as pathogenic. The patients
(sisters), carrying the heterozygous c.600delC (p.Ile201Serfs*20) mutation is unique,
since she also carries the largest so far reported deletion, involving the whole ATP6V0A2
gene. Both parents were carrying either the frameshift mutation or the large deletion and
were confirmed to be healthy carriers. The splicing mutations c.1326+1G>A and
c.1605+1G>A in ATP6V0A2 were predicted to be truncating mutations and therefore
pathogenic alterations. They were not described in the dbSNP database and not present in
80 healthy controls. The missense mutation c.2287C>T (p.His763Tyr ) was predicted to
be pathogenic according to the SIFT and PolyPhen 2.0 algorithm database. Furthermore,
it alters an evolutionarily conserved amino acid, not listed in the dbSNP database and is
absent in 100 healthy controls. The missense mutations c.355C>T (p.Arg119Cys) and
c.772G>A (p.Val258Met) in PYCR1 involved conserved amino acid changes and were
predicted as pathogenic according to the SIFT and PolyPhen 2.0 algorithm database. All
parents were confirmed to be healthy carriers. The mutations were not described in
dbSNP database and not present in 80 healthy controls.
The mutations c.1273C>T (p.Ser742Ile) and c.2225G>T (p.Arg425Cys) in ALDH18A1
were predicted to be disease-causing by the SIFT and PolyPhen 2.0 algorithm and
recently reported as pathogenic [5S]. They alter evolutionarily conserved amino acids, are
not listed in the dbSNP database and were absent in 100 healthy controls. The mutation
c.524C>T (p.Ser175Phe) in GORAB involved a conserved amino acid change and
predicted as pathogenic according to the PolyPhen 2.0 algorithm database. The parents
were confirmed to be healthy carriers. The mutation was not described in dbSNP
database and not present in 100 healthy controls. The p. Glu123* mutations results in a
premature stop codon and is thus predicted to be pathogenic.
Histology/ Electron microscopy
Electron microscopy was performed in patients 1, 3, 7 and 9 and was characteristic for
the disease localization in patients 1,3,7 and 9. In the patients carrying PYCR1 mutations
(3 and 7) the epidermis was normal, whereas the dermis was thinner compared to
controls. It exhibited more scattered collagen bundles and very scarce and small elastic
fibers. The scarcity of elastic fibers was mostly pronounced in the reticular dermis
(Suppl. Fig. 1A and 1B). The ultrastructural organization of the reticular dermis the
elastic fibers were smaller and exhibited a criss-crossed structure. Collagen bundles and
collagen fibrils were normal in the patient’s dermis, although less compact than on
control. Fibroblasts of the dermis exhibited swollen mitochondria and a number of lipid
droplets in the cytoplasm higher than that in normal fibroblasts (data not shown).
Electron microscopy in fibroblasts of the patient carrying an ATP6V0A2 mutation (patient
9) show swollen, enlarged Golgi system (Figure 2F, left) comparable to the electron
microscopic results of abnormal Golgi structure in patient 1 with the common
homozygous RIN2 deletion (Figure 2F, right). In addition the elastic fibres in this patient
showed a "halo" of filamentous material and there is abnormal organisation of collagen,
which is in some areas densely packed in bundles, but in other intermittent areas
irregularly and loosely packed.
Brefeldin A assay
Brefeldin A assay was performed in fibroblast from patient 9 showing 80% of Golgi
remnants, indicating abnormal retrograde transport in fibroblasts.
Suppl. Figure 1
Flow chart representing diagnostic flow of 36 patients referred to our clinical suspected
for autosomal recessive cutis laxa.
Suppl. Figure 2
Suppl. Fig 2A: Electron microscopy images of the dermis of healthy control (left) and
patient with ARCL 2B due to a PYCR1 defect (right). Elastic fibers were immune-stained
with for elastin specific antibodies, and by secondary antibodies conjugated with
colloidal gold particles. In the patient with PYCR1 mutation the elastic fibers are scarce,
short and thin, and polymorphous. No halo-forming can be observed. Suppl. Fig 2B:
Light microscopy images of the dermis with elastin staining demonstrating short and
scarcely present elastin rods. Suppl. Fig 2C: Immunofluorescence in control fibroblasts
double stained with Giantin and GM130 demonstrating normal staining of Golgi stacks,
Suppl. Fig 2D: Immunofluorescence of control fibroblast after Brefeldin A treatment,
without Golgi remnants, indicating showing normal retrograde transport in control
fibroblasts Suppl. Fig 2E: Immunofluorescence of fibroblasts of a patient carrying an
ATP6V0A2 mutation after Brefeldin A treatment, showing Golgi remnants, indicating
abnormal retrograde transport. Suppl. Fig. 2F: Electronmicroscopy showing swollen
Golgi stacks in patient fibroblasts in patient carrying an ATP6V0A2 mutation. Suppl. Fig.
2G: Similar abnormalities with abnormal Golgi structure in fibroblasts of the patient with
the common homozygous RIN2 deletion.
Suppl. Figure 3
Suppl. Fig 3A, B and D: Facial features of patients [4S] with GORAB mutation showing
long face, bulbous nose, long philtrum, and prominent ear cups in patient D. No
significant facial skin laxity. Suppl. Fig 3C: Patient with PYCR1 mutation with triangular
face without significant facial skin wrinkling and no pergament-like skin at 1.5 years.
Note the large ear, beaked nose and short neck. Suppl. Fig 3E Patient with PYCR1
mutation with progerioid features, abnormal hair grow and prominent veins at the age of
1.5 years showing several facial feature overlap with Suppl. Fig 3F: SHOC2 mutation at
10 months of age showing progerioid features, sparse hair grow and visible venous
network. Note the discriminative nose form with beaked tip of the nose, characteristic for
PYCR1 mutations (see Suppl. Fig 3D and E). Strabismus was present in patients with
GORAB, PYCR1 and SHOC2 mutations.
Supplementary references
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