Figure Legends
Figure 1. Pedigrees for NMD02 and ID01 and segregation of identified variants.
  1. 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.
  2. 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.
  3. 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.
  4. Protruding abdomen of individual III:9.
  5. 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.
  6. 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.
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  1. 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.
  2. 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.
  3. 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.
  4. 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).
  1. Transfection of rat chondrocytes with two siRNAs (siR1 and siR2) against Tmem251 reduced mRNA expression of Tmem251 , as determined by quantitative PCR.
  2. Alpl expression and
  3. 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.
  4. Apoptosis in rat chondrocytes is not altered by Tmem251knock-down, as measured by detection of cytoplasmic histone-associated-DNA-fragments.
  5. 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).