Mitochondrial complex III deficiency associated with a homozygous mutation in UQCRQ Ortal Barel

1. Mitochondrial complex III deficiency associated with a homozygous mutation in UQCRQ Ortal Barel Laboratory of Dr. Ohad Birk Ben Gurion University Beer-Sheva, Israel

2. The IsraeliBedouin • Isolated inbred population • High rate of consanguineous marriage • Negev region of southern Israel • High frequency of autosomal recessive diseases • Effective in mapping genes of rare Recessive diseases

3. Pedigree of the Affected Israeli-Bedouin Kindred • Severe neurological syndrome. • Autosomal recessive pattern of inheritance • 62 DNA samples • More than 25 affected individuals  B

4. The hallmarks of the phenotype: • Markedly uniform phenotype • Early onset in infancy • Severe psychomotor retardation • Extrapyramidal signs (age 2-3 years): Dystonic postures, athetoid movements and ataxia • Severe defects in verbal receptive communication • Near total absence of expressive communication skills (verbal) • Hypotonia (inability to walk unsupported)

5. MRI studies MRI Studies of Affected Individual at Age 2 Years • Bilateral symmetrical abnormal findings in the basal ganglia • Increased density in the putamen and decreased density and size of the caudate and globus pallidum nuclei. • No involvement of the brain-stem area. Normal section

6. Muscle biopsy • Muscle biopsies were done in three of the affected individuals (ages 3, 15 and 2 years). - Preserved architecture of the skeletal muscle • - Nonspecific histological findings • Metabolic work up: - All laboratories values were within the normal range except: Elevated serum lactate and pyruvate Elevated Cerbrospinal fluid (CSF) lactate

7. Enzymatic activities of the mitochondrial respiratory chain • Enzymatic activities of the five mitochondrial respiratory-chain complexes were measured in isolated muscle mitochondria. • 34%–56% decrease in the activity of mitochondrial complex III • Variable decreases complex III + I

8. Mitochondrial disease Summary of the phenotype and the clinical features: • Autosomal recessive • Elevated serum lactate and pyruvate • Decreased activity of mitochondrial complex III & I • Neurological abnormalities • Involvement of tissues characteristic of mitochondrial diseases (high energy consumption)

9. Mitochondrial complex III, background: • Complex III is located within the inner mitocondrial membrane • Transfers electrons from ubiquinol to cytochrome c • Complex III is made up of 11 subunits • Genes associated with complex III deficiency: Cytochrome b (mitochondrial DNA) BCSIL - CIII assembly essential factor (2q33) UQCRB - ubiquinone-binding protein (8q22)

10. D5S471 15.84 Mb D5S2115 Genome-wide linkage analysis results • Founder effect • Ruled out: BCSIL and UQCRB • Genome-wide linkage analysis (11 affected individuals; 5 healthy individuals) • A single locus; 9 cM; chromosome 5q31

11. 5q31 Fine mapping B A • DNA samples of the 62 available family members (20 affected) • Family B: Many crossing over events in the maternal haplotype

12. Region common to all affected individuals Fine mapping of the homozygosity regions in the haplotype, demonstrate a region around D5S2002 which is common to all affected individuals in the family

13. Mapping two more family branches resulted in setting the lower (telomeric) limit of the homozygosity interval at D5S2497

14. Compilation Table: Genotypes of All Affected Individuals • The shaded areas indicate the region of homozygosity markers. • Upper limit of the homozygosity interval: D5S2057 • Lower limit of the homozygosity interval: D5S2497

15. Results of Two-Point LOD Score Analysis • Analysis using SUPERLINK software • LOD score of 8.82 at θ= 0 for marker D5S_2

16. The disease locus • Locus defined between markers D5S2057 and D5S2497 • 2.14 cM interval of homozygosity common to all affected individuals • Contains 30 known or predicted genes

17. UQCRQ • Official Full Name: • Ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5kDa (QCR8; QP-C ) • Summary: • Encodes a ubiquinone-binding protein of low molecular mass. • The protein is a small core-associated protein and a subunit of mitochondrial complex III. • The bc1 complex (complex III ) contains 11 subunits: - 3 respiratory subunits (cytochrome b, cytochrome c1 and Rieske) - 2 core proteins (UQCRC1 and UQCRC2) - 6 low-molecular weight proteins (UQCRH, UQCRB, UQCRQ, UQCR10, UQCR11 and a cleavage product of Rieske)

18. Affected unaffected Carrier C208T Mutation in Exon 2 of UQCRQ • DNA: c.208 C to T mutation in exon 2 of UQCRQ • Protein: replacing serine at position 45 by phenylalanine (p.Ser45Phe) Barel et al. The American Journal of Human Genetics (2008).

19. NCBI database:rs11544803 242bp 203bp 190bp 173bp 147bp 111bp Healthy Affected HinfI Mutation or SNP ???  • Sequencing all other 29 genes at the locus revealed no other mutations • Predicted SNP only (without validation experiments). • Analysis of 62 DNA samples of the kindred: compatible with the mutation • Controls: 600 ethnically matched chromosomes 470 unrelated non-Bedouin chromosomes

20. ClustalW Results: • Mutation: Serine to Phenylalanine • There is no phenylalanine in any of the homologues of UQCRQ gene • Phenylalanine has an aromatic side chain, leading to a significantly different three-dimensional structure.

21. PDB: Bovine complex III with chain 7 (UQCRQ) in blue Ser45: Phe45:

22. Acknowledgements: Dr. Ohad Birk’s group Dr. Rivka Ofir Dr. Sara Sivan The Lab members Dr. Zamir Shorer Pediatric Neurology, Soroka Medical Center, Beer-Sheva, Israel     Dr. Ann Saada (Reisch)Metabolic Disease Unit, Hadassah Medical Center, Jerusalem Dr. Stavit Shalev Genetics Institute, HaEmek Medical Center, Afula Asi Cohen, Dr.Noam Zilberberg Lab, Department of Life Sciences, BGU The Kahn Family Foundation The Ori’s Foundation - In Memory of Ori Levi Foundation that supports research for the diagnosis, treatment and cure of mitochondrial disorder (www.orifund.org)