MITOCIRCLE

1. MITOCIRCLE • WP 3.2: Positional cloning of the gene defect Maastricht L. van den Heuvel

2. Gene hunting in human OXPHOS deficiency • Positional gene cloning (1) • Candidate gene approach (2) • Positional candidate gene approach (3)

3. Positional cloning (1): • Large families for total genome scan with linkage analysis are rarely available. Linkage analysis: cosegregation between disease phenotype and microsatellite markers

5. Candidate gene approach (2): • Candidate genes obtained from biochemical diagnostics

6. Respiratory chain enzyme activities in different tissues of patient N. Fibroblast (control range) Muscle Liver Complex I 0.067 (0.10-0.24) 8.83 (4.7-29) 7.15 Complex II + III 0.25 (0.21-0.33) 11.4 (4-28) 30.8 Complex IV 146 (128-250) 52.1 (52-186) 72.9 Citrate synthase 158 (140-257) 84.6 (48-162) 34.2 Enzyme activities are expressed in mU/mg protein.

7. mitochondrial donor denucleation rho 0 mitochondrial donor mix and fuse hybrid transmitochondrial recipient rho 0 Rho zero test

8. Case report • Leigh Syndrome • 4-months-old male Spanish infant with a negative family history • Normal enzymatic activities of respiratory chain complexes in muscle and liver • Low complex I activity in fibroblasts (67%) • No mutations found in nuclear CI subunits

9. Mutation in ND6 T14487C (M63V) ” CONTROL PATIENT

10. 156 CONTROL TspRI 23 133 PATIENT C1 C2 C3 ♀ M L F 175 102 80 24% 65% 65% 86% mutant mtDNA Heteroplasmy of ND6 (T14487C) mutation

11. Features of the mutation (ND6): M63V • Localized in the most • conserved region of ND6 • Transmembrane segment • ND6 mutations: • Hot spot gene for LHON mutations (A14484G → M64V) • Mutation in MELAS (G14453A) • Mutation in LHON/dystonia or Leigh Syndrome (G14459A)

12. Assembly in a ND6 patient • Role of ND6: • Essential for the assembly of CI • - affects assembly of membrane arm and respiratory function in a mouse cell line with a frameshift mutation in ND6 • (Bai & Attardi, EMBO J., 1998) • - absence of intact ND6 prevents assembly of CI in Chlamydomonas (Cardol et al., JMB, 2002)

13. Co 1 Pat Co 2 1D 880 kD 2D 440 kD Assembly in a ND6 patient CI V NDUFA9 (39 kD) CONTROL PATIENT Low levels of fully-assembled CI Detection of a lower MW subcomplex

14. Candidate gene approach (2): • Evolutionary conserved genes.

16. Complex IV vs Complex I

17. Evolutionary building blocks of Complex I T Friedrich J. Bioenerg. Biomembr. 2001

18. Human complex I NDUFA1 NDUFA2 NDUFA3 NDUFA4 NDUFA6 NDUFA7 NDUFA8 NDUFA9 NDUFA10 NDUFAB1 NDUFB1 NDUFB2 NDUFB3 NDUFB4 NDUFB5 NDUFB6 NDUFB7 NDUFB8 NDUFB9 NDUFB10 NDUFC1 NDUFC2 NDUFS7 NDUFS8 17.2 kDa ? ND1 ND2 ND3 ND4L ND4 ND5 ND6 ? NADH NAD+ +H+ e- NDUFV1 NDUFV2 NDUFV3 FMN FP Fe-S NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFA5 e- Fe-S Fe-S e- Matrix IP Fe-S H+ Ubiquinone e- e- Fe-S HP HP H+ Ims

20. Candidate gene approach (3): • Candidate genes obtained from recognized protein function (for example function in complex I assembly).

22. Nijmegen Center for Mitochondrial Disorders International Center for Clinical Care, Diagnostics and Research of Patients with disturbed Mitochondrial Energy Metabolism BIO INFORMATICS

23. Human CIA30 genomic structure • Chromosomal location: 15q13.3 • Promoter region: TATA box CG box NRF-2 site CpG elements • Gene length: ~ 15 kb • Gene composition: 5 exons (75 to 654 bp) 4 introns (0.8 to 6.3 kb) • mRNA sequence: 1436 bp encoding 327 amino acids • Predicted mitochondrial targeting sequence of the first 24 amino acids

24. Tissue distribution of CIA30 mRNA • CIA30 expression is ubiquitous • There is a slightly higher expression in the various heart tissues, kidney, lung, and liver

25. CIA30: Results and Further Studies • Mutational detection: 13 patients • Five polymorphisms: 4/5 resulting in amino acid substitutions (frequent in ESTs) - Probably other Complex I assembly factors are involved in complex I deficiency

27. Positional candidate geneapproach (3): • Chromosome transfer in combination with linkage analyses and/or candidate gene approach. * OXPHOS defect is expressed in fibroblasts. * Fibroblast are immortalized.

32. Chromosome transfer: • Cloning of the SURF1 gene in COX deficient Leigh patients. • Map the gene for combined OXPHOS deficiency.

33. Regulation of COX assembly • Identified assembly intermediates • Part of the assembly route could be identified. • In COX deficient patients (e.g. SURF1 mutants) give a disturbed assembly

34. SurfI affects this step in the assembly of COX

35. Nuclear gene defects in OXPHOS deficiency: • Complex I: NDUFS1, -S2, -S3, -S4, -S6,-S7, -S8 NDUFV1, -V2 • Complex II: SDHA, -B, -C, -D • Complex III: BCS1L and UQCRB • Complex IV: SURF1, SCO1, SCO2, COX10, COX15, LRPPC • MtDNA replication/maintenance: TP, ANT1, C10 ORF 2, DGUOK, TK2, DNC, THTR-1, SLC19A2, SPG7, FRDA, OPA1, TAZ.

36. MITOCIRCLE • WP 3.2: Positional cloning of the gene defect

37. Objectives: • 1.)The identification of the disease locus in cohorts of patients belonging to the same complementation group by linkage analysis or functional complementation. • 2.)The characterization of the causative gene defect.

38. Description of work: • 1.)The discovery of new genes involved in the defects of complex I, complex IV, or both. - The identification of the disease locus in cohorts of patients belonging to the same complementation group by: • * linkage analysis on informative families. • * functional complementation strategies based on chromosome transfer. • 2.)Screening of candidate genes, identified by bioinformatics tools, for mutations.

39. Deliverables: • 1.)A list of candidate loci for CI, CIV and CI+IV deficiency. • 2.)A list of new genes involved in these deficiencies and OXPHOS dysfunction. • 3.)A list of new pathogenic mutations causing these deficiencies. • 4.)Diagnostic protocols to screen the genes involved for mutations.

40. Discussion(1): • 1.)Who is going to deliver the bioinformatics support? • 2.)Which patients (+biochemical and genetic characteristics) are available for these studies? • - complex I • - complex IV • - complex I+IV • - individual cases/ informative families • - Known genetic defects have been screened for?

41. Discussion(2): • 4.)Are complementation groups available? • 5.)Which are the candidate genes for the different patient groups? • 6.)Which group is going to study specific patients groups?