The membranes were probed with antibodies raised against the following: BCS1L (HPA037701, Atlas Antibodies Ltd.), RISP (see above), CORE1 (see above), NDUFA9 (MS111, Mitosciences), SDHB (ab14714, Abcam, Cambridge, Great Britain), COXI (MS404, Mitosciences), VDAC1/porin (ab154856, Abcam). from the patient but not from the control fibroblast cDNA. (PDF 2123?kb) 13023_2017_624_MOESM1_ESM.pdf (2.0M) GUID:?B68176A9-C866-4779-9A81-F190D9F84C60 Additional file 2: Figure S1: Muscle histology and electron microscopy. (A) NADH staining showing scattered fibers with enhanced reactivity. (B) Electron microscopy showing a fiber with increased amount of lipid droplets (L) and many mitochondria, some with structural abnormalities (arrow). Control muscle with normal mitochondria. Bars 2?m. (PDF 4485?kb) 13023_2017_624_MOESM2_ESM.pdf (4.3M) GUID:?0D0657C8-3469-43AA-916C-3B21298A111C Additional file 3: Figure S2: Coronal section at the level of the left amygdala. The bulk of the white matter is reduced and shows discoloration in the temporal lobe. The corpus callosum is thin and there is moderate lateral and third ventricular dilation. Cortical laminar necrosis is seen in the cingulate gyrus, the superior frontal gyrus, the precentral gyrus, the inferior temporal gyrus and the lateral occipitotemporal gyrus (arrows). (PDF 5698?kb) 13023_2017_624_MOESM3_ESM.pdf (5.5M) GUID:?FAAF952F-62FF-423D-A95E-A2CE438CEC74 Additional file 4: Figure S3: BNGE with immunblotting. The samples were run on two gels in quadruplicate. (A). The gel was stained with commassie blue after blotting to PVDF membrane to show that the loading was similar; the first (lanes 2-5) and second (lanes 7-10) loading of the samples with the ladder (lanes 1 and 6) are shown. The molecular weights of the ladder markers are indicated. (B). For the upper blot the CORE1 and RISP antibodies were used, for the second blot the BCS1L antibody and the combination of CI NDUFVI (to detect the subunit assembled at the final stage), CIV Va, and CII 30kD were used, respectively. The first blot was stripped and thereafter the antibodies against CIV COX and CI NDUFA9 were probed (remnants of the CORE1 and RISP bands can be seen). Despite weaker bands in the patient (lanes 1 and 6) the decrease in BCS1L and RISP is recognizable. (PDF 2082?kb) 13023_2017_624_MOESM4_ESM.pdf (2.0M) GUID:?D475DDCF-B9DE-41BD-8A45-8B1CE119EE2A Data Availability StatementThe datasets used and/or analyzed during the current study is available from the corresponding author on reasonable request. Abstract Background Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the gene constitute the most common diagnosed cause of CIII Integrin Antagonists 27 deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Integrin Antagonists 27 Exome sequencing was performed to search for the causative mutation(s). The patients platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by mutations. Electronic supplementary material The online version of this article (doi:10.1186/s13023-017-0624-2) contains supplementary material, which is available to authorized users. and and [6]and [5]. The BCS1L protein is required for the insertion of the RISP into the CIII pre-complex dimer (pre-CIII2). This step completes the structure of the mature, catalytically active complex. The corresponding protein in yeast, bcs1, is well characterized and has been shown to transport the RISP from the matrix of the mitochondria, where it has acquired its 2Fe-2S cluster, to the intermembrane space, where it assembles with the pre-CIII [7]. BCS1L is phylogenetically conserved and homologs are found in all eukaryotic genomes. Diseases caused by mutations range from the mild Bj?rnstad syndrome, with brittle hair (mutations, encephalopathy, together with IB1 tubulopathy and liver disease are common features. In total, less than 100 patients have been described worldwide with conditions attributed to mutations in this gene. Knock-in mice, carrying the same missense mutation as the GRACILE syndrome patients, develop a phenotype Integrin Antagonists 27 that is similar to that seen in neonates and thorough analysis of the renal and hepatic pathologies have been published [15, 16]. We here describe two novel mutations in the gene in a patient with a severe phenotype involving minimal psychomotor development, pronounced muscular hypotonia, aminoaciduria, growth restriction and premature death. The necropsy revealed specific changes in.