1. Discovery of a new disease causing gene in the iron-sulfur cluster biosynthesis pathway: IBA57 1 Arnaud Vanlander, M.D., PhD student at the Ghent mitochondrial investigatins laboratory. 1 Mutation of the iron-sulfur cluster assembly gene IBA57 causes severe myopathy and encephalopathy. Ajit Bolar N, Vanlander AV, Wilbrecht C, Van der Aa N, Smet J, De Paepe B, Vandeweyer G, Kooy F, Eyskens F, De Latter E, Delanghe G, Govaert P, Leroy JG, Loeys B, Lill R, Van Laer L, Van Coster R. Hum Mol Genet. 2013 Jul 1;22(13):2590-602

2. Case presentation Two siblings presented with: –Intra uterine growth retardation –Muscular weakness –Severe brain malformations –Encephalopathy –Neonatal death The elevated serum lactate, was an argument to investigate the mitochondrial oxidative phosphorylation (OXPHOS).

3. Mitochondrial respiratory chain activity in skeletal muscle In accordance with our in gel activity staining of OXPHOS complexes after BN-PAGE, … … we could confirm lowered complex I, II and IV amount by western blot with the total OXPHOS human antibody cocktail. This unusual combination of complex deficiency was an argument in favour of a possible iron sulfur cluster biogenesis defect.

4. NFU1 BOLA3 cysteine Frataxin sulfur ISCU Fe ++ ABCB7 Mitoferrin =MFRN2/1 transferrin receptor 1 =TFR1 DMT1 TRPML1 Plasma membrane Outer mitochondrial membrane Inner mitochondrial membrane nucleus NUBPL, =Ind1 Nfs1/ LYRM4 Iron regulatory Protein (IRP) tran- scription FRD-R Mortalin HSC20 GRPEL1/2 Apo Holo Aconitase Lipoic acid synthase Complex II Complex III Complex I NFU1 BOLA3 GLRX5 ISCA1, ISCA2, IBA57 I IIIII The iron-sulfur cluster (ISC) biosynthesis pathway The intramitochondrial ISC biosynthetic pathway can be devided into three major steps. At first, iron (Fe 2+ ) enters the mitochondria. Together with sulfur it is assembled on a scaffold protein, ISCU, to form a small ISC (2Fe-2S). For the second step, assisting proteins stand in for maturation of the ISC on the scaffold protein and eventually for dissociation of the ISC and incorporation into apo-proteins. Finally for the third step, ISC can be enlarged to 4Fe-4S clusters for incorporation into complex I, complex II of the respiratory chain or into the enzym lipoic acid synthase. Small (2Fe-2S) clusters can also be exported out of the mitochondria. The genes encoding proteins of the ISC biosyntesis pathway for which mutations have already been described are highlighted by circles.

5. Confirmation of ISC pathway deficiency To confirm the faulty ISC pathway, presence of lipoylated residues was evaluated by WB on cultured skin fibroblasts and skeletal muscle… … and were found deficient in both patients, for both tissues.

6. NFU1 BOLA3 cysteine Frataxin sulfur ISCU Fe ++ ABCB7 Mitoferrin =MFRN2/1 transferrin receptor 1 =TFR1 DMT1 TRPML1 Plasma membrane Outer mitochondrial membrane Inner mitochondrial membrane nucleus NUBPL, =Ind1 Nfs1/ LYRM4 Iron regulatory Protein (IRP) tran- scription FRD-R Mortalin HSC20 GRPEL1/2 Apo Holo Aconitase Lipoic acid synthase Complex II Complex III Complex I NFU1 BOLA3 GLRX5 ISCA1, ISCA2, IBA57 Identification of the culprit gene Our molecular investigations together with our biochemical data… … showing combined complex I and II deficiency and absent lipoylation …. …brought us to the identification of the culprit gene: IBA57

7. Conclusions IBA57 was identified as a new disease causing gene in the human ISC pathway. Evaluation of lipoylation status in patients with combined OXPHOS complex deficiencies can help in targeting the molecular investigations.