Mitochondrial complex III deficiency

associated with a homozygous mutation in

UQCRQ

Ortal BarelOrtal Barel

Laboratory of Dr. Ohad BirkLaboratory of Dr. Ohad Birk

Ben Gurion UniversityBen Gurion University

Beer-Sheva, IsraelBeer-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

B

Pedigree of the Affected Israeli-Bedouin Kindred

Severe neurological syndrome.

Autosomal recessive pattern of inheritance

62 DNA samples

More than 25 affected individuals 

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)

The hallmarks of the phenotype:

MRI studies

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.

MRI Studies of Affected Individual at Age 2 Years

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

AssayP1P2P3

Complex I45%45%52%

Complex I+III43%70%74%

Complex II+III86%89%114%

Complex II79%94%92%

Complex III62%66%44%

Complex IV95%94%80%

Complex V135%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)

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)

Mitochondrial complex III, background:

Genome-wide linkage analysis results

D5S471

D5S2115

15.84 Mb

Founder effect

Ruled out: BCSIL and UQCRB

Genome-wide linkage analysis

(11 affected individuals; 5 healthy individuals)

A single locus; 9 cM; chromosome 5q31

5q31 Fine mapping

DNA samples of the 62 available family members (20 affected)

Family B: Many crossing over events in the maternal haplotype

A B

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

Locus defined between markers D5S2057 and D5S2497

2.14 cM interval of homozygosity common to all affected individuals

Contains 30 known or predicted genes

The disease locus

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)

Affected Carrier unaffected

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).

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

242bp

190bp

147bp

111bp

203bp173bp

Healthy

HinfI

Affected

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.

Ser45: Phe45:

PDB: Bovine complex III with chain 7 (UQCRQ) in blue

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 LeviFoundation that supports research for the diagnosis, treatment and cure of mitochondrial disorder

(www.orifund.org)

Acknowledgements: