Figure Legends
Figure 1. Pedigrees for NMD02 and ID01 and segregation of
identified variants.
- Pedigree of family NMD02 from Pakistan. Black symbols denote
individuals affected with the disorder while open symbols represent
the unaffected individuals. Strikethrough denotes deceased individual.
Genotypes for the TMEM251 variant c.133C>T;
p.(Arg45Trp) of all participants are shown under the respective
symbols. Asterisks mark the individuals whose samples were subjected
to exome sequencing.
- Pedigree of family ID01 from Iran. Black symbols denote individuals
affected with the disorder while open symbols represent the unaffected
individuals. Strikethrough denotes deceased individual. Genotypes for
the TMEM251 variant c.215dupA; p.(Tyr72Ter) of all participants
are shown under the respective symbols. Asterisks mark the individuals
whose samples were subjected to exome sequencing.
- Individuals III:11 and III:9 at ages of 9 and 13 respectively.
Photographs show progressive coarsening of facial features, short
stature and skeletal deformities. The younger child does not have
facial dysmorphism but the older brother has mild coarsening of
features, a short stature and prominent wrists.
- Protruding abdomen of individual III:9.
- Bed ridden individual III:5, who is now deceased, at the age of 22
years. Note the coarse facial features with broad nose and prominent
lips. Arms and hands are short, wrist joints are prominent.
- Photographs of the affected child from Iranian family ID01. Coarse
facial features and deformity are evident.
Figure 2. Clinical features of family NMD02 and ID01.
- X-rays of individual III:9 of NMD02 show widened ribs with normal
shaped thorax. Thoracic and cervical vertebrae are of reduced height.
Femur, tibia and fibula are irregularly shaped with abnormal
epiphyses. Feet show poor mineralization, curving of the metatarsal
bones with osteolytic appearance of the joints. Epiphyses are
irregular and abnormal in shape. The first toe has a short distal
phalange and osteolytic appearance on both sides.
- X-rays of individual III:11 of NMD02. Thoracic and lower limb. X-rays
indicate similar features as in individual III:9 but with milder
changes. Additionally, irregularly shaped iliac wings and abnormal
prominent hips are also obvious. Forearms are clearly abnormal with
curved and abnormally shaped radius and ulna with distal osteolysis.
Overall, radiographs indicate dysostosis multiplex.
- X-rays of an affected child from Iranian family. Spine X-ray indicates
scoliosis due to multiple hemivertebrae and paddle shape ribs. Pelvic
X-ray shows hypoplasia and flattening of acetabular roof and bilateral
hip dislocation with short iliac wings.
- Variant trace of TMEM251 c.133C>T; p.(Arg45Trp) at
site of variation. The changed base is shown by an arrow, while the
affected codon is underlined.
- Variant trace of TMEM251 c.215dupA; p.(Tyr72Ter) at the site of
variation. The inserted base is shown by an arrow, while the affected
codon is underlined.Figure 3. Multiple Sequence alignment and graphical
representation of TMEM251.
- CLUSTAL Omega multiple sequence alignment of TMEM251 with orthologues
from diverse vertebrate species. The gaps in alignment are denoted by
dashes. Identical amino acids in all orthologues are indicated by
asterisks below the alignment. The colons and periods depict amino
acids which are highly conserved or less conserved, respectively.
The p.Arg45 residue affected by the variant is boxed. The first amino
acid of the short isoform of TMEM251 is depicted by a small horizontal
bar on top of the Methionine residue.
- Schematic representation of human TMEM251 . Both the long and
short isoforms are depicted. Gray boxes show the coding exons while
the untranslated regions are indicated by black bars. Intron is
represented by a horizontal line. The short isoform of the gene
encodes a protein which lacks 38 amino acids from the N-terminal which
are present in the long isoform.
- Illustration of the processed pseudogene of TMEM251 long
isoform which is located on chromosome 5 within intron 85 ofADGRV1 (- or antisense strand). The gray vertical lines denote
mismatches to TMEM251 sequence.
- Diagrammatic representation of TMEM251 as encoded by the long isoform,
inserted in the lipid bilayer of plasma membrane. The two
transmembrane regions are shown by shaded rectangles (amino acids
47-66 and 105-127, respectively). N and C denote the amino and
carboxyl terminal of the protein, respectively. The positions of the
wild-type protein with p.Arg45 (left panel) and the variant p. Trp45
(right panel) are depicted by arrows. Note that the variant is
predicted to result in inclusion of amino acid 45 into the first
transmembrane segment which will be inserted in the membrane (right
panel).
Figure 4. Localization of wild-type and mutant TMEM251 proteins
in Human Osteosarcoma cell line and knockdown of Tmem25 in rat
primary chondrocytes.
- Analysis of wild-type TMEM251 localization in cells transfected with
GFP-fused TMEM251 and immunostaining of Golgi complex with antibody
against Giantin (red) nuclei with DAPI (blue). Merged image (yellow)
indicates co-localization of wild-type TMEM251 with Golgi marker.
- Analysis of mutant TMEM251 localization in cells transfected with
GFP-fused mutant TMEM251 and immunostaining of Golgi complex with
antibody against Giantin (red) nuclei with DAPI (blue), merged image
(yellow) indicates co-localization of mutant TMEM251 with Golgi
marker.
- Immunostaining of Endoplasmic Reticulum with anti-PDI antibody after
transfection with wild-type TMEM251 merged image indicates separate
localization of wild-type TMEM251 and ER marker.
- Immunostaining of Endoplasmic Reticulum with anti-PDI antibody after
transfection with mutant TMEM251, merged image demonstrate mutant
TMEM251 does not localize to ER. Note the reduced and punctate
expression of mutant TMEM251 as compared to wild-type TEM251. (Images
were taken in green, red and blue channel, separately and then
merged).
- Transfection of rat chondrocytes with two siRNAs (siR1 and siR2)
against Tmem251 reduced mRNA expression of Tmem251 , as
determined by quantitative PCR.
- Alpl expression and
- Col2a1 expression measured by quantitative PCR in transfected
cells compared to scrambled control. Alpl expression was not
significantly changed in siR2 transfected cells, whereas Col2a1expression was reduced in cells transfected with either siR1 or siR2.
- Apoptosis in rat chondrocytes is not altered by Tmem251knock-down, as measured by detection of cytoplasmic
histone-associated-DNA-fragments.
- Proliferation of chondrocytes measured by BrdU incorporation indicates
no effect on proliferation upon Tmem251 knockdown.
All measurements were performed 48h post- transfection. Bars indicate
average ± SEM of two independent experiments (*, p<0.05;
***, p<0.001 relative to the scrambled-siRNA treated cells).