Stream	Late-breaking Abstracts
Topic	Basic and Clinical Science
Abstract title	A genetic defect of the mitochondrial energy supply: characterization of a novel NDUFAF5 mutation
Co-authors	I. Raz1, A. Kogot-Levin1, A. Saada2, S. Weksler-Zangen1.
1Hadassah University Hospital, Diabetes unit, Jerusalem, Israel.
2Hadassah University Hospital, Monique and Jaques Roboh Department of Genetic Research and The Department of Genetic and Metabolic diseases, Jerusalem, Israel.
Abstract body	Background
Background: Mitochondrial dysfunction is a contributor to β-cell failure in type-2-diabetes. The Cohen-diabetic-sensitive rat (CDs) is a unique model of mitochondrial-respiratory, complex-IV deficiency, developing hyperglycemia when fed a diabetogenic-high-sucrose, copper-deficient diet (DD). Copper is a key-element for the catalytic activity of complex-IV. NDUFAF5 (NADH:Ubiquinone Oxidoreductase-Complex-Assembly-Factor-5) gene, encodes a mitochondrial complex-I assembly-factor which was associated with ComplexI & IV deficiencies in Leigh-syndrome patients. 
Aim: We examined Complex-I and complex-IV activity in islets and fibroblasts in relation to progression to diabetes in CDs-rats attempting to elucidate the genetic-basis of their β-cell dysfunction.
Methods: Blood-glucose and insulin-concentrations were measured before and during OGTT performed at different periods on DD or copper-supplemented-DD. Islets and fibroblasts complex-I and complex-IV activity were measured spectrophotometrically. Whole-genome-sequencing was performed in blood-DNA using Illumina HiSeq2500 followed by in-silico variant-analysis. Protein-levels were determined by Western-blot and supercomplex assembly by BN-PAGE. Rescue experiments were conducted by lentiviral-vector overexpression of a wild-type NDUFAF5-gene, in CDs primary-fibroblasts.
Results: A highly significant positive correlation between complex-IV activity and glucose-stimulated-insulin-secretion and an inverse correlation with blood-glucose-levels (R2=0.984 and R2=-0.915, P<0.0001 respectively) were found in islets of CDs fed DD or copper-supplemented-DD. Complex-I activity was 30% reduced in CDs-islets relative to the control-islets (P<0.01). Whole-genome-sequencing identified a novel homozygous missense variant, p.P318L (c.C1002T), in the NDUFAF5 gene predicted to be highly pathogenic by in-silico tools. NDUFAF5 protein level was significantly decreased in CDs-islets compared to control-islets. BN-PAGE demonstrated reduced complex-IV supercomplex-assembly in CDs-islets (P<0.01). Overexpression of wild-type NDUFAF5-gene in CDs-fibroblasts increased both complex-I and complex-IV activity (P<0.01).
Discussion
Conclusion: Our results demonstrated a tight correlation between impaired mitochondrial function and β-cell dysfunction in CDs, a model of mitochondrial-disorder related diabetes. The identified novel homozygous missense variant, p.P318L in the NDUFAF5-gene may underlie the CDs-mitochondria defect and susceptibility to develop diabetes.