As most subunits of COX are nuclear-encoded and many nuclear-encoded factors are essential for assembly of the holoenzyme complex, it is thought that most cases of COX deficiency are caused by nuclear gene mutations. However, in contrast to the exponentially expanding number of reported mtDNA mutations, very few nuclear gene defects have been described in OXPHOS proteins. Although mutations have been reported in nuclear-encoded subunits of complexes I and II (Bourgeron et al. 1995; Loeffen et al. 1998; van den Heuvel et al. 1998; Triepels et al. 1999;
Schuelke et al. 1999; Parfait et al. 2000; Baysal et al. 2000; Budde et al. 2000; Petruzzella et al. 2001; Bénit et al. 2001; Loeffen et al. 2001), mutations have yet to be described in the nuclear COX subunit genes, despite extensive sequence
analysis (Adams et al. 1997; Lee et al. 1998; Jaksch et al. 1998).
The nuclear-encoded factors required for correct assembly of COX are candidate genes for autosomally inherited COX deficiency syndromes. So far only a few of the human genes encoding these factors have been characterised (Table 1.3) and only 4 of these have been demonstrated to be mutated in human COX deficiency (Table 1.6).
1.11.3.1 SURF1
The first nuclear gene mutations reported to cause COX deficiency were in the
SURF1 gene (Zhu et al. 1998; Tiranti et al. 1998b). The SURF1 gene is part of the
surfeit locus, an extremely tight cluster of housekeeping genes that maps to chromosome 9q34 in humans. There are 6 genes in the surfeit locus, but these do not share any sequence or amino acid homology (Duhig et al. 1998). The structure of the gene cluster is thought to be functionally significant, since it has been
conserved during 250 million years of divergent evolution (Colombo et al. 1992).
SURFS is the only gene in the surfeit locus whose function is known. It encodes the
L7a ribosomal protein. The SURF4 product appears to be an integral membrane protein associated with the endoplasmic reticulum whilst SURFS encodes 2 cytoplasmic proteins (as a result of differential splicing). The SURF6 protein has been localised to the nucleolus.
SURF1 encodes a 300 amino acid protein, human surfeit locus protein 1, whose precise function is unknown. It is a homologue of yeast Shy1p which has been localised to the inner mitochondrial membrane (Mashkevich et al. 1997). SURF1 has an N-terminal mitochondrial targeting signal and has also been shown to be an integral inner mitochondrial membrane protein (Yao and Shoubridge, 1999; Tiranti et al. 1999b). It is thought to have an essential role in the assembly or maintenance of the functional COX holoenzyme.
Mutations in the SURF1 gene have now been reported in a significant proportion of patients with COX-deficient Leigh syndrome (Zhu et al. 1998; Tiranti et al. 1998b; Tiranti et al. 1999c). This proportion varies between centres, from 26% (Sue et al. 2000) to 75% (Tiranti et al. 1999c). The clinical phenotype associated with SURF1
mutations appears to be remarkably homogeneous, which is particularly surprising when one considers the enormous clinical variability associated with respiratory chain defects. It has been suggested that SURF1 mutations are exclusively associated with COX-deficient Leigh syndrome of infantile onset (Tiranti et al.
1999c), but the reason for this is not clear. It is possible that it may reflect
ascertainment bias since mutation analysis of a large number of patients with other COX-deficient phenotypes has not yet been described.
Most SURF1 mutations appear to be small-scale rearrangements, leading to a frameshift and predicting a truncated protein product (Table 1.7). In addition several nonsense and splice-junction mutations, which also result in a truncated protein product, have been reported. Relatively few missense mutations have been
identified, but these are also associated with typical Leigh syndrome (Teraoka et al. 1999; Poyau et al. 2000). Several mutations have occurred more than once in different ethnic groups and are thus likely to represent mutational ‘hotspots’. In particular the 312del10/insAT mutation in exon 4 appears to be the most common
SURF1 mutation in Caucasian patients, and has been estimated to account for 35-
50% of all disease-causing SURF1 alleles (Robinson, 2000).
Northern blot analysis demonstrated ubiquitous expression of SURF1 (Yao and Shoubridge, 1999) although expression in brain appeared relatively low compared to heart, skeletal muscle and kidney. This is surprising since all patients with SURF1
mutations reported to date had Leigh syndrome, and had almost exclusively
Site of mutation Mutation Predicted Protein Sequence Clinical Phenotype R eference*
Exon 1 37 38ins17 Frameshift LS Tiranti et al. 1998b Exon 1-2 delCCCCGCA Frameshift (?ss) LS Hanson et al. 2001 Exon 2 74G>A W 25X LS Tiranti et al. 1999c Exon 3-4 240+1 G>T Frameshift LS Tiranti et al. 1999c
Exon 4 244C>T Q82X LS+periph
neuropathy
Santoro et al. 2000 Exon 4 312 321del10,
311 312insAT
Frameshift (ss) LS Tiranti et al. 1998b Exon 4 323+2T>C Frameshift (ss) LS Zhu et al. 1998 Exon 5 371 G>A G124E LS Poyau et al. 2000 Exon 5-6 515+2T>G Frameshift (ss) LS Tiranti et al. 1998b Exon 5-6 516-516 IdelAG Frameshift (ss) LS Pequignot et al. 2001a Exon 6 550 551delAG Frameshift LS Tiranti et al. 1998b Exon 6 552delG Frameshift LS Tiranti et al. 1999c Exon 6 539G>A G180E von Kleist-Retzow et
al. 2001
Exon 6 1 base deletion V187X LS von Kleist-Retzow et al. 1999
Exon 6 574 575insCTG C
Frameshift LS Tiranti et al. 1999c Exon 6 587_588insCAG
G
Frameshift LS Sue et al. 2000
Exon 6-7 588+1 deIG Frameshift (ss) LS Pequignot et al. 2001a Exon 6-7 589-1 G>C Deletion 1197 +
E l 98 (ss)
von Kleist-Retzow et al. 2001
Exon 7 688C>T R230X LS Coenen et al. 1999 Exon 7 737T>C I246T LS Poyau et al. 2000 Exon 7 751O T Q251X LS Tiranti et al. 1998b Exon 7-8 751+6T>G Frameshift (ss) LS Pequignot et al. 2001b Exon 7-8 752-3C>G Frameshift (ss) LS Poyau et al. 2000 Exon 8 758 759deiCA Frameshift LS Tiranti et al. 1999c Exon 8 771 774delACC
C
Frameshift LS Hanson et al. 2001 Exon 8 772 773delCC Frameshift LS Tiranti et al. 1998b Exon 8 790 IdelAG Frameshift LS Teraoka et al. 1999 Exon 8 808G>T E270X LS Tiranti et al. 1999c Exon 8 814 815delCT Frameshift LS Sue et al. 2000 Exon 8 820T>G Y274D LS Teraoka et al. 1999 Exon 8-9 821 835+3del18 Frameshift (ss) LS Williams et al. 2001 Exon 9 841 842delCT Frameshift LS Zhu et al. 1998 Exon 9 845 846delCT Frameshift LS Tiranti et al. 1998b Exon 9 868_869insT Frameshift LS Tiranti et al. 1998b
Mutation numbering is from the first ATG (start) codon. LS = Leigh syndrome
ss = splice-site mutation
* Refers to first report o f mutation.
**This patient had hypertrichosis, partial villous atrophy and neurological dysfunction.
Table 1.7
neurological symptoms. Imunohistochemical studies (Sue et al. 2000) and
immunoblot analysis (Yao and Shoubridge, 1999; Poyau et al. 2000) of patients with
SURF1 mutations demonstrated reduced immunoreactivity to both nDNA and
mtDNA-encoded subunits of COX. Two-dimensional blue-native gel electrophoresis of fibroblasts from 4 SURF1 deficient patients revealed reduced levels of both holo- COX (S4) and the final assembly intermediate S3, proportional to the decrease in COX activity, but relatively increased levels of the early assembly intermediates SI and S2 (Coenen et al. 1999). Taken together, these results indicate that SURF1 is not necessary for the biosynthesis of COX subunits but is required at a later stage in COX assembly or maintenance.
1.11.3.2 S C 0 2
The gene encoding SC02, a mitochondrially-targeted protein thought to be required for insertion of copper into COX I and COX II (see section 1.8.2), has been found to be mutated in patients with a fatal infantile form of COX deficiency characterised by hypertrophic cardiomyopathy and encephalopathy (Papadopouiou et al. 1999). COX deficiency in these patients was expressed most severely in cardiac and skeletal muscle (0 to 18% residual COX activity), with relatively high residual COX activity in cultured skin fibroblasts (12 to 50%) compared to patients with SURF1 mutations (Sue et al. 2000; Jaksch et al. 2000). Immunohistochemical studies suggested that the enzyme deficiency is due to loss of mtDNA-encoded COX subunits
(Papadopouiou et al. 1999).
All patients reported to have SC02 mutations share a common missense mutation E140K near the putative CxxxC copper binding domain of SC02. This might be an ancient founder allele or a mutational hotspot. Most patients are compound
heterozygotes (Papadopouiou et al. 1999; Sue et al. 2000; Jaksch et al. 2000) but recently three patients were reported who were homozygous for this mutation (Jaksch et al. 2001). These patients had delayed onset of hypertrophic obstructive cardiomyopathy. The phenotype thus appears to be milder in E140K homozygotes and it has been postulated that two severe SC02 alleles might lead to embryonic lethality (Shoubridge, 2001).
1.11.3.3 S C 01
Mutations in the SC01 gene, whose product is also thought to be involved in copper delivery to COX, have been demonstrated in a French family with isolated COX deficiency associated with neonatal-onset hepatic failure and encephalopathy (Vainot et al. 2000a). Residual COX activity was 0.5% in skeletal muscle and 20% in liver of the index case. Affected infants were compound heterozygotes, with a 2 bp frameshift deletion in exon 2 resulting in a premature stop codon on the paternal allele, and a missense mutation P174L in exon 3 on the maternal allele. This
missense mutation alters a highly conserved proline residue adjacent to the copper- binding domain of SC01 and is thus analogous to the common E140K missense mutation in SC02.
The patients with SC01 mutations did not have the cardiac symptoms observed in patients with SC02 mutations. The observation of different clinical phenotypes associated with SC01 and SC02 mutations, with predominantly liver involvement in the former and cardiac involvement in the latter but neurological symptoms in both, has led to the suggestion that there might be tissue-specific pathways for
mitochondrial copper delivery in humans (Shoubridge, 2001).
A Canadian study that sequenced SC01 and C0X17 in 30 COX-deficient patients with heterogeneous clinical presentations suggested that mutations in SC01 are not a common cause of isolated COX deficiency (Horvath et al. 2000). Furthermore no mutations were identified in 00X77, whose product is also thought to be involved in the delivery of copper to the COX holoenzyme.
1.11.3.4 COX10
A genetic linkage study of an African consanguineous family with an isolated COX defect allowed identification of a homozygous missense mutation in the COX10
gene (Vainot et al. 2000b). The COX10 gene encodes haem A: farnesyltransferase, which catalyses the first step in the conversion of protohaem to the haem A
prosthetic group of COX subunit I. The clinical phenotype in this family was lactic acidosis and proximal renal tubulopathy associated with neurological features including hypotonia, myopathy, ataxia and seizures. Residual COX activities were 2%, 17% and 51% in skeletal muscle, kidney and liver respectively (Ogier et al.
1988). Immunoblot analysis of COX subunits in fibroblasts revealed severe
reduction of steady state levels of subunit II, moderate reduction of subunits III and Vic and only slight reduction of all other COX subunits studied including I and IV (Vainot et al. 2000b).