Transcript Mitochondrial complex III, background

Mitochondrial complex III deficiency
associated with a homozygous mutation in
UQCRQ
Ortal Barel
Laboratory of Dr. Ohad Birk
Ben Gurion University
Beer-Sheva, Israel
The Israeli Bedouin
 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
Pedigree of the Affected Israeli-Bedouin Kindred
 Severe neurological syndrome.
B
 Autosomal recessive pattern of inheritance
 62 DNA samples
 More than 25 affected individuals
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)
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
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
Enzymatic activities of the mitochondrial respiratory chain
Assay
P1
P2
P3
Complex I
45%
45%
52%
I+III Complex
43%
70%
74%
Complex II+III
86%
89%
114%
Complex II
79%
94%
92%
Complex III
62%
66%
44%
Complex IV
95%
94%
80%
Complex V
135%
94%
82%
 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
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)
Mitochondrial complex III,
background:
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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)
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
D5S471
15.84 Mb
D5S2115
5q31 Fine mapping
A
B
 DNA samples of the 62 available family members (20 affected)
 Family B: Many crossing over events in the maternal haplotype
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
Mapping two more family branches resulted in setting the lower
(telomeric) limit of the homozygosity interval at D5S2497
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
Results of Two-Point LOD Score Analysis
 Analysis using SUPERLINK software
 LOD score of 8.82 at θ= 0 for marker D5S_2
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
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)
C208T Mutation in Exon 2 of UQCRQ
Affected
Carrier
unaffected
 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).
Mutation or SNP ???
NCBI database: rs11544803
 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
Healthy
Affected
HinfI
203bp
173bp
242bp
190bp
147bp
111bp
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 threedimensional structure.
PDB: Bovine complex III with chain 7 (UQCRQ) in blue
Ser45:
Phe45:
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)