Fig 1: Genetic analysis results. (A) Pedigree of the Family 1. Affected individuals are labeled with black-colored (filled) symbols. Carriers are represented with half-filled symbols; squares represent males, circles represent females, and double line indicates consanguinity. (B) Pedigree for the family 2. Brief Sanger sequencing results of the c.475 G>T variant under the pedigrees show the results for the Families. (C) Homozygosity results for all the affected individuals in both families. The patients are aligned with the normal individuals in both families. The shared ROH block is shown in the red bracket on chromosome 6 where RTN4IP1 is located. Numbers 1-6 indicate family members from Family 1, individuals 1 to Family 2, and individual 3, (F1-II-1, F1-II-2, F1-II-3, F2-II-1, F2-II-2, and F2-II-3), respectively. (D) Protein sequence alignment of RTN4IP1, the blue arrow shows the affected Valine amino acids at position 159 by the novel variant. The alignment showed that the amino acid in this region is highly conserved among different species.
Fig 2: Structural analysis and immunoblotting experiment results. (A) The location of the variant within the 3-D crystal structure of RTN4IP1 (PDB ID: 2VN8). RTN4IP1 forms a dimer (green and blue chains). Val159 is shown as a red stick model. The cofactor NADPH is shown as a stick model with carbon atoms in white. The zoom view shows the effect of the p.Val159Phe substitution. Clashes of the Phenylalanine side chain (yellow) are shown as red discs. Key side chains in the vicinity are highlighted as stick models. (B) Immunoblotting analysis results. The Western blotting analysis revealed significantly decreased RTN4IP1 protein levels in the patient’s extracts from cultured fibroblasts in comparison to controls. A polyclonal antibody was targeted against the protein, and beta-actin was used as a control-loading marker. (C) Schematic presentation of RTN4IP1. The top panel shows localization of all previously reported variants (black) and the newly identified novel variant (red) distributed among the nine exons (blue). The bottom panel shows both the alcohol dehydrogenase domain (ADH_N) and the zinc-binding motif (ADH_Zinc).
Supplier Page from Abcam for Anti-RTN4IP1 antibody