Jos Jansen

70 Chapter 3 Figure 2. TMEM199 Mutations, TMEM199 Levels, and Liver Ultrastructure (A) Pedigrees and sequence profiles of TMEM199 mutations in families F1 to F3. (B) Sequencing of TMEM199 cDNA from individual F2-II2 showed homozygosity for the c.40G > C missense mutation. The insert shows the splice site prediction according to Alamut v.2.5.1 (Interactive Biosoftware), indicating a new splice site one nucleotide downstream. (C) Western blot analysis of TMEM199 levels was performed on fibroblasts from healthy control individuals and affected individuals with anti-TMEM199 antibody (1:250, Sigma, #HPA027051) (left). TMEM199-V5 was overexpressed in HeLa cells and the Western blot incubated with anti-TMEM199 antibody (right). As a protein loading control, the same blot was incubated with mouse anti-GAPDH antibody (1:2,500, Calbiochem, #CB1001). (D–F) Transmission electron microscopy of liver tissue from individual F2-II2. (D) Focally, lipid vacuoles were present in the paranuclear area (asterisk). (E) Enlarged hepatocytes show diffuse and severe vacuolization most likely derived from rough ER, smooth ER, and/or Golgi apparatus. (F) Mitochondria featured fragmented cristae and an altered inner matrix appearing as granulo-filamentous material. Sometimes electron-dense crystal-like structures were included in the mitochondria (arrow).