THE ROLE OF NUCLEAR-ENCODED SUBUNIT GENES IN

MITOCHONDRIAL COMPLEX I DEFICIENCY

LISA CATHERINE WORGAN

A thesis submitted in fulfillment of the requirements for the degree of Master of Science

School of Women and Children’s Health University of N.S.W.

February 2005 ABSTRACT

BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A>G). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency.

i

ACKNOWLEDGEMENTS

I would like to sincerely thank my supervisors Dr Edwin Kirk and Dr Michael Buckley for all their help, guidance and support.

Dr David Thorburn, our collaborator at the Murdoch Institute in Melbourne, supplied all the patient samples and much technical advice and guidance for this project. He also provided much inspiration and enthusiasm for the task. In addition, he and his laboratory staff, in particular Renato Salemi, followed up results, including the genomic sequencing of patient and parental samples as well as the sequencing of controls. Without Dr. Thorburn’s ongoing involvement and encouragement this project would not have been completed.

This project could not have proceeded without financial support. I would like to acknowledge Rhonda and Shane Reaiche for their tremendous fundraising efforts which provided essential financial support for this project. I would also like to thank the UMDF (United Mitochondrial Diseases Foundation) who provided a grant to support this project. In addition, salary support was provided by the Sydney Children’s Hospital Foundation which was greatly appreciated.

As I did not have molecular genetic laboratory experience, I could not have started this project without the training I received by many staff members at the Molecular and Cytogenetics Unit, SEALS, Prince of Wales Hospital. In particular, Peter Taylor’s advice and daily patience was invaluable. I would also like to acknowledge Glenda Mullen and Dr Ying Ge for extensive technical training and ongoing advice with dHPLC and DNA sequencing.

Finally, I would like to thank all the physicians that referred patient samples for investigation of mitochondrial disease. I would also like to sincerely thank the patients and families for providing the samples that allowed this project to proceed.

ii TABLE OF CONTENTS

Page Number

ABSTRACT i Acknowledgements ii Table of contents iii List of tables vi List of figures viii Abbreviations x

1. INRODUCTION 1.1 Overview 1 1.2 Background 2 1.3 Complex I deficiency- clinical aspects 10 1.4 Complex I deficiency- genetic aspects 15 1.5 Project outline 21

2. METHODS 2.1. Ethics approval 23 2.2. Patient and control samples 23 2.3. RNA extraction 27 2.4. Quantification of RNA 29 2.5. Formaldehyde Agarose Gel Electrophoresis 29 2.6. Reverse Transcription (RT) 31 2.7. PCR (polymerase chain reaction) 32 2.8. Denaturing Polyacrylamide gel electrophoresis 35 2.9. dHPLC 37 2.10. PCR product purification 45 2.11. DNA cycle sequencing 45

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Page Number

3. RESULTS 3.1 Summary of mutations identified 49 3.2 dHPLC results 50 3.2.1 NDUFV1 dHPLC results 50 3.2.2 NDUFS1 dHPLC results 52 3.2.3 NDUFS2 dHPLC results 55 3.2.4 NDUFS4 dHPLC results 56 3.2.5 NDUFS7 dHPLC results 57 3.2.6 NDUFS8 dHPLC results 58 3.3 Sequencing results 3.3.1 NDUFV1 sequencing results 60 3.3.2 NDUFS1 sequencing results 65 3.3.3 NDUFS2 sequencing results 66 3.3.4 NDUFS4 sequencing results 71 3.3.5 NDUFS7 sequencing results 75 3.3.6 NDUFS8 sequencing results 77

4. DISCUSSION 4.1 Overview 78 4.2 Discussion of Methods 79 4.2.1 complementary DNA 79 4.2.2 dHPLC 81 4.2.3 Sequencing 88 4.3 Discussion of Results 88 4.3.1 NDUFV1 88 4.3.2 NDUFS2 92 4.3.3 NDUFS4 96

iv TABLE OF CONTENTS

Page Number

4. DISCUSSION continued 4.3.4 NDUFS1, NDUFS8 and NDUFS7 102 4.3.5 Assessment of the pathogenicity of the mutations identified 103 4.4 Where are the mutations in the remaining patients? 105 4.4.1 Summary of aetiology in our 34 complex I deficient patients 105 4.4.2 Complex I assembly and activation 105 4.5 Clinical and laboratory implications of subunit mutation analysis 108 4.5.1 Approach to molecular genetic diagnosis of complex I deficiency 108 4.5.2 Blue native gel Electrophoresis and complex I assembly 108 4.6 Conclusions and Significance of findings 112

5. REFERENCES 116

APPENDICES 1 RNA concentrations 133 2 dHPLC melt curves (Figures 18-23) 134-149 3 Sequencing of mutations (Figures 24-30) 150-156 4 dHPLC chromatograms (Figures 31-36) 157-161

v LIST OF TABLES Page number Table 1: Nuclear and mitochondrial gene contribution to Complexes I-V 2 Table 2: E. coli Nuo genes and their complex I homologues 4 Table 3: mtDNA mutations and complex I deficiency 16 Table 4: Nuclear complex I subunit mutations in complex I deficiency 18-20 Table 5: Characteristics of the 6 genes studied 22 Table 6: Clinical and biochemical features of patients 25 Table 7: Reverse transcription master mix 31 Table 8: PCR master mix components 32 Table 9: Oligonucleotide primers used in PCR and sequencing 34 Table 10: Optimised PCR conditions and amplicon size 35 Table 11: dHPLC temperatures used for each amplicon 39 Table 12: Additional primers used for PCR and sequencing 46 Table 13: Sequencing reaction mix 47 Table 14: Thermocycler program for the sequencing reaction 47 Table 15: Complex I subunit gene mutations 49 Table 16: NDUFV1 dHPLC results 51 Table 17: NDUFS1 dHPLC results 54 Table 18: NDUFS2 dHPLC results 56 Table 19: NDUFS4 dHPLC results 57 Table 20: NDUFS7 dHPLC results 58 Table 21: NDUFS8 dHPLC results 59 Table 22: NDUFV1 sequencing results 60 Table 23: NDUFV1 polymorphisms 64 Table 24: NDUFS1 sequencing results 65 Table 25: NDUFS1 polymorphisms 66 Table 26: NDUFS2 sequencing results 67

vi LIST OF TABLES Page number

Table 27: NDUFS2 polymorphisms 70 Table 28: NDUFS4 sequencing results 71 Table 29: NDUFS4 polymorphisms 75 Table 30: NDUFS7 sequencing results 76 Table 31: NDUFS7 polymorphisms 76 Table 32: NDUFS8 Sequencing results 77 Table 33: NDUFS8 polymorphisms 77 Table 34: Amplicons not screened at the Stanford recommended dHPLC temperature 85 Table 35: Genetic defects identified in our patients with complex I deficiency 107

vii LIST OF FIGURES

Page number

Figure 1: A: Complex I structure (Ugalde et al. 2004) 7 B: Subcomplexes of complex I 7 Figure 2: Polyacrylamide gel electrophoresis with silver staining 37 Figure 3: Heterozygous mutations and dHPLC 40 Figure 4: dHPLC chromatogram of pUC HaeIII 43 Figure 5: The NDUFV1 amino acid sequence 62 Figure 6: Conservation of NDUFV1 amino acids 63 Figure 7: Conservation of amino acids in a segment of NDUFS2 69 Figure 8: Conservation of NDUFS2 and the P352A polymorphism 70 Figure 9: Patient 26 sequencing results for amplicon 1 of NDUFS4 72 Figure 10: The NDUFS4 exon 2 / exon 3 cDNA junction in Patient 26 72 Figure 11: Reverse sequencing of NDUFS4 amplicon 1 in Patient 26 73 Figure 12: Amino acid conservation in NDUFS4 74 Figure 13: Organization of NDUFV1 89 Figure 14: Organization of NDUFS2 93 Figure 15: The amino acid sequence of NDUFS2 94 Figure 16: NDUFS4 organization 97 Figure 17: Human NDUFS4 cDNA and amino acid sequences 98

viii

LIST OF FIGURES (APPENDICES)

Page number Appendix 2 dHPLC melt curves Figure 18: NDUFV1, dHPLC melt curves for each amplicon 134 Figure 19: NDUFS1, dHPLC melt curves for each amplicon 137 Figure 20: NDUFS2, dHPLC melt curves for each amplicon 140 Figure 21: NDUFS4, dHPLC melt curves for each amplicon 143 Figure 22: NDUFS7, dHPLC melt curves for each amplicon 145 Figure 23: NDUFS8, dHPLC melt curves for each amplicon 147

Appendix 3 Sequencing of mutations Figure 24: Patients 13 and 27, NDUFV1 mutation R386H 150 Figure 25: Patient 13, NDUFV1 mutation K111E 151 Figure 26: Patient 27, NDUFV1 mutation P252R 152 Figure 27: Patient 17, NDUFS2 mutation M292T 153 Figure 28: Patient 25, NDUFS2 mutation R221X 154 Figure 29: Patient 25, NDUFS2 mutation R333Q 155 Figure 30: Patients 19 and 26, NDUFS4 mutation c.221delC 156

Appendix 4 dHPLC chromatograms Figure 31: dHPLC chromatograms NDUFV1 amplicon 4 157 Figure 32: dHPLC chromatograms for NDUFV1 amplicon 2 158 Figure 33: dHPLC chromatograms for NDUFV1 amplicon 3 159 Figure 34: dHPLC chromatograms for NDUFS2 amplicon 3 159 Figure 35: dHPLC chromatograms for NDUFS2 amplicon 4 160 Figure 36: dHPLC chromatograms for NDUFS4 amplicon 1 161

ix

ABBREVIATIONS

BN-PAGE Blue Native Polyacrylamide Gel Electrophoresis bp base pairs cDNA complementary DNA gDNA genomic DNA mtDNA mitochondrial DNA NDUFS1-8 NADH-ubiquinone oxidoreductase Fe-S Protein 1-8 NDUFV1-2 NADH-ubiquinone oxidoreductase flavoprotein 1-2 NMD nonsense mediated mRNA decay OXPHOS oxidative phosphorylation SDS-PAGE Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis SNP single nucleotide polymorphism

x 1. INTRODUCTION

1.1 OVERVIEW

Disorders of mitochondrial energy metabolism have an incidence of around 1 in 10,000 live births (Skladal et al. 2003). Isolated complex I deficiency (OMIM#252010) is the most common, accounting for one third of diagnosed respiratory chain disorders (Kleist- Retzow et al. 1998). Complex I deficiency is associated with many different clinical phenotypes; the spectrum extending from lethal neonatal disease to adult onset neurodegenerative disorders such Parkinson disease (Robinson 1998). The phenotype in infancy and childhood is also variable, but is commonly characterised by an encephalomyopathy (Munnich et al. 1992). Affected children usually die in infancy or childhood from a progressive multisystem disorder (Smeitink et al. 1999).

Complex I deficiency can be either “isolated” or associated with deficiency of other respiratory chain enzymes. This research focuses on infants and children with isolated complex I deficiency. For the majority of children with complex I deficiency, inheritance is autosomal recessive (Kirby et al. 1999). Mitochondrial DNA mutations are estimated to account for 5-20% of cases (Triepels et al. 2001, Lebon et al. 2003). Autosomal dominant and X-linked inheritance of complex I deficiency are also possible, though as yet undocumented in human disease.

There are at least 46 complex I subunit genes, including seven mitochondrially encoded subunits and at least 39 nuclear encoded subunits (Carroll et al. 2003). As there are likely to be other genes involved in complex I assembly and regulation, there are potentially more than 46 genes involved in complex I deficiency. This has made identification of the causative mutation in a particular patient with complex I deficiency very difficult. For the vast majority of families with a child with complex I deficiency, the underlying genetic basis of this devastating condition remains unknown.

1 1.2 BACKGROUND Mitochondria are double membrane bound, intracellular organelles containing their own DNA. Human mitochondrial DNA (mtDNA) consists of a circular double-stranded DNA molecule made up of 16, 569 nucleotide pairs (Anderson et al. 1981). The mitochondrial genome is not static due to a high sequence evolution rate, 10-20 times higher than nuclear DNA (Wallace 1992). Consequently mtDNA contains extensive sequence variations when different individuals and populations are compared (Wallace 1992).

Mitochondrial DNA encodes 37 genes; 24 of these genes are needed for translation of mitochondrial genes (2 ribosomal RNAs and 22 transfer RNAs) and the other 13 mitochondrial genes encode respiratory chain subunits (Wallace 1992, DiMauro 1999A). These 13 subunits are synthesized on mitochondrial ribosomes, while all the other respiratory chain proteins that make up the complexes I-IV, are encoded by nuclear genes and are translated on cytoplasmic ribosomes before transport into the mitochondria (Wallace 1992).

Oxidative phosphorylation is a crucial process in cellular energy production in which ATP is generated (Shoffner and Wallace 1995 p.1535). The oxidative phosphorylation system spans the inner mitochondrial membrane and consists of 5 multiprotein enzyme

complexes (Complexes I-V) and two additional electron carriers, coenzyme Q10 and cytochrome c. Oxidative phosphorylation in the mitochondria relies on the products of both nuclear-encoded and mitochondrial genes (Shoffner and Wallace 1995 p.1535). The contribution to each complex from mitochondrial and nuclear genes is summarised in Table 1.

Table 1: Nuclear and mitochondrial gene contribution to Complexes I-V

Enzyme Complex Enzyme1 Encoded by Encoded by mtDNA Nuclear DNA Complex I : NADH dehydrogenase (ubiquinone) EC 1.6.5.3 7 subunits ~39 subunits Complex II: succinate dehydrogenase (ubiquinone) EC1.3.5.1 0 subunits 4 subunits Complex III: ubiquinol--cytochrome-c reductase EC 1.10.2.2 1 subunits 10 subunits Complex IV: cytochrome c oxidase EC 1.9.3.1 3 subunits 10 subunits Complex V: ATPase synthase EC 3.6.3.14 2 subunits 14 subunits 1 Footnote: Enzyme EC reference numbers from http://www.expasy.org/cgi-bin/nicezyme

2 Evolutionary conservation of complex I genes

Many of the 46 complex I subunit genes – including mitochondrial and nuclear encoded genes - are highly conserved and of ancient evolutionary origin. Bacterial complex I is referred to as NDH-1 (NADH dehydrogenase 1) and is composed of 14 subunits, all of which have homologues in mammalian complex I (Finel 1998, Yagi et al. 1998) (Table

2). These 14 highly conserved subunits are referred to as the “core” complex I subunits

(Carroll et al. 2002). Like mammalian complex I, bacterial complex I is an L-shaped protein complex, that links electron transfer to proton translocation and contains FMN

(flavin mononucleotide) and iron-sulphur clusters (Weidner et al. 1993, Yagi and

Matsuno-Yagi 2003).

Weidner et al. (1993) studied the Escherichia coli NDH-1. The E. coli gene locus for NDH-1 is called nuo (NADH:ubiquinone oxidoreductase) and was reported to contain these 14 conserved subunits (Weidner et al. 1993) (Table 2). Seven of these highly conserved genes are the mitochondrially encoded subunits (ND1-6, ND4L) and seven are the nuclear encoded subunits of mammalian complex I (NDUFS1, NDUFS2, NDUFS3, NDUFS7, NDUFS8, NDUFV1 and NDUFV2) (Table 2). It was later noted that E. coli NDH-1 actually consists of 13 subunits as nuoC and nuoD are fused (Yagi et al. 1998).

As these 14 genes are conserved throughout evolution they are likely to be involved in essential aspects of complex I structure or function, and are considered to be the “minimal” form of the enzyme. Therefore, these core genes were considered to be strong candidates for mutation in human complex I deficiency (Smeitink et al. 1998). This proved to be a correct assumption, as is discussed later in this review.

3

Table 2: E. coli Nuo genes and their complex I homologues

E.coli NDH-1 Complex I homologue % amino acid sequence subunits identity 1 Nuo 1 (NuoA) ND3 46% Nuo 2 (NuoB) NDUFS7 47% Nuo 3 (NuoC)2 NDUFS3 39% Nuo 4 (NuoD)2 NDUFS2 40% Nuo 5 (NuoE) NDUFV2 35% Nuo 6 (NuoF) NDUFV1 44% Nuo 7 (NuoG) NDUFS1 25% Nuo 8 (NuoH) ND1 42% Nuo 9 (NuoI) NDUFS8 38% Nuo 10 (NuoJ) ND6 20% Nuo 11 (NuoK) ND4L * Nuo 12 (NuoL) ND5 33% Nuo 13 (NuoM) ND4 31% Nuo 14 (NuoN) ND2 21% Footnote: 1the human and E.coli amino acid sequences were obtained from Genbank. The NCBI web program (blast2 sequences and sequence alignment) was used to determine percentage sequence identity 2 (www.ncbi.nlm.nih.gov/blast/bl2seq). In E.coli NuoC and NuoD are fused, which is not the case for other prokaryotes. *This particular web program did not identify homology between the human and E. coli protein sequences for NDL4. However homology between the B. Taurus and E. coli genes has been reported by multiple groups (Weidner et al. 1993, Yagi et al. 1998), and failure to detect homology is possibly related to the web program used.

Complex I structure Complex I (nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase) is the largest of the five multiprotein complexes involved in mitochondrial oxidative phosphorylation. Complex I composition has been best characterised in studies of bovine heart mitochondria where 46 subunits have been identified (Carroll et al. 2003).

The three dimensional structure of complex I has been studied by electron microscopy in B. Taurus (Grigorieff 1998), E. coli and N. crassa (Guénebaut et al. 1998) and found to be similar in all three organisms. Bovine complex I has high homology with human complex I. It has a total molecular mass of about 980kDa (Carroll et al. 2003) and an L- shaped configuration (Figure 1A) with two domains of approximately equal length (Sasanov et al. 2000); a peripheral arm and a membrane arm. The water soluble, peripheral arm protrudes into the mitochondrial matrix and is involved in the catalytic function of the enzyme. It contains all the redox cofactors such as the flavin mononucleotide and the iron-sulphur clusters (Carroll et al. 2003, Petruzzella and Papa 2002). The hydrophobic, membrane arm is embedded in the inner mitochondrial

4 membrane (Triepels et al. 2001) and is involved in proton translocation. The location of some of the complex I subunits within complex I can be seen on Figure 1A.

Complex I can be fragmented into three fractions using chaotropic agents (Triepels et al. 2001): 1. The flavoprotein (FP) fraction (water soluble arm) contains the flavin mononucleotide (FMN) and the NADH binding site. It is composed of NDUFV1, NDUFV2 and NDUFV3 subunits (Petruzzella and Papa 2002). 2. Iron-sulphur protein (IP) fraction (water soluble arm) contains several iron-sulphur clusters. It contains the subunits NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS5, NDUFS6 and NDUFA5 (Petruzzella and Papa 2002). 3. The hydrophobic (HP) protein (membrane bound arm) binds quinone in the inner membrane (Petruzzella and Papa 2002) and is involved in proton translocation into the mitochondrial intermembrane space. All seven mitochondrially encoded subunits belong to this fraction, as well as the remaining nuclear encoded subunits (Bénit et al. 2004).

Alternatively, treatment with the non-denaturing detergent N,N-dimethyl- dodecyclamine N-oxide (LDAO) resolves bovine complex I into two subcomplexes, Iα and Iβ (Finel et al.1992) (Figure 1B). Subcomplex Iα is composed of the peripheral matrix arm in combination with a small part of the membrane arm. Subcomplex Iβ contains mainly hydrophobic subunits and represents a substantial part of the membrane arm (Figure 1B). Under slightly different conditions of dissociation, subcomplex Iλ is identified, which represents the peripheral arm part of subcomplex Iα. In addition, a further subunit Iγ has been identified, which represents a part of the membrane arm that is separate from Iβ (Sazanov et al.2000) (Figure 1B).

Division of complex I into the different subcomplexes or fractions has allowed analysis of subunit positions within complex I (Sazanov et al. 2000, Carroll et al. 2003), and as such provided information on the general structural organization of complex I. The 14 core subunits of bovine complex I are surrounded by many additional subunits. The function of these supernumerary subunits is still largely unknown, though they may

5 structurally stabilize complex I and keep the redox centres in the correct position (Guénebaut et al. 1998). These subunits envelope the core subunits and this may prevent electrons from escaping and reacting with oxygen to form toxic radicals (Guénebaut et al. 1998). Other supernumerary subunits may be involved in complex I assembly (Guénebaut et al. 1998) or complex I regulation (eg. NDUFS4, as discussed below).

Complex I assembly The process of complex I assembly remains poorly understood. A proposed model for mammalian complex I assembly has been described by Antonicka et al. (2003). By using two dimensional Blue Native / Sodium Dodecyl Sulphate (SDS) polyacrylamide gel electrophoresis and a panel of 11 antibodies against structural subunits, they identified a common constellation of subcomplexes in all complex I patients and controls. These subcomplexes are likely to be intermediates in complex I assembly. In this model of complex I assembly, the peripheral and membrane arms are not assembled separately, unlike the situation for complex I assembly in N. crassa (Schulte 2001).

Schägger et al. (2004) has shown that respiratory chain complexes are further assembled into supercomplexes, called the respirasomes. Complex I and complex III combine to form a stable core respirasome in the inner mitochondrial membrane and Complex IV can also bind to this. The respirasomes were studied in patients with single respiratory chain defects and the formation of respirasomes was essential for normal complex I assembly or stability. Loss of complex III led to secondary complex I deficiency. Combined complex I and III deficiency has been reported in patients with NDUFS4 mutations (Budde et al. 2000) and it is hypothesized that the truncated NDUFS4 protein interferes with the assembly of the complex I / III respirasome.

6 Figure 1: 1A: Complex I structure (Ugalde et al. 2004)

1B: Subcomplexes of complex I (modified from Sazanov et al. 2000)

Footnotes: The location of some nuclear encoded subunits is indicated in Figure 1A. Subcomplexes Iα, (Iλ), Iγ and Iβ, are best indicated in Figure 1B. All mitochondrial encoded subunits are located in the membrane bound arm.

7 Complex I function Complex I plays a crucial role in cellular ATP production, the primary source of energy for most cell functions. Complex I is the first complex to mediate electron transfer through the electron transfer chain. Complex I transports electrons from NADH and passes them via a series of different protein-coupled redox centres, to the electron acceptor ubiquinone (van den Heuvel et al. 1998). There is simultaneous shunting of protons across the inner mitochondrial membrane to the inter-membrane space (Loeffen et al. 2000, Procaccio et al. 2000). This in turn provides the driving force for ATP production by complex V (ATP synthase) (Schuelke et al. 1998). The transfer of electrons through complex I is possible through the presence of several redox centres; a flavin mononucleotide and at least six iron-sulphur clusters (Loeffen et al. 1998A, Triepels et al. 1999).

The specific function of each of the individual complex I subunits is largely unknown. Of the six subunits chosen for this study, the NDUFV1 subunit forms the NADH binding site (Triepels et al. 2001). It also contains the flavin mononucleotide which is involved in entry of the electrons escaping from the oxidation of NADH. NDUFV1 contains a tetra-nuclear iron-sulphur cluster (Triepels et al. 2001). NDUFS1, NDUFV2, NDUFS8 and NDUFS7 also contain iron- sulphur clusters, suggesting that these subunits are involved in electron transfer (Procaccio et al.1997, Triepels et al. 2001, Papa et al. 2002). NDUFS7 couples electron transfer from an iron sulphur cluster to quinone, the final step in electron transfer through complex I (Triepels et al. 2001). NDUFS2 is thought to be involved in proton pumping and ubiquinone binding (Triepels et al. 2001). NDUFS4, the only gene studied that is not one of the 14 core complex I subunits, contains a conserved phosphorylation site and is involved in regulation of complex I activity (Papa et al. 2001).

Pathophysiology In patients with complex I deficiency there is reduced ATP synthesis and this is a major factor in pathophysiology, as it is the tissues with the highest energy demands that are most severely affected. However, insufficient ATP may not be the only factor contributing to the changes seen in complex I deficiency. Symptoms may also be due to free radical damage (Robinson 1998, Kirby et al. 1999). There is increasing evidence

8 that oxidative stress is a major factor in age-related mitochondrial damage (Murray et al. 2003). This mitochondrial damage, particularly to complex I, is thought to be involved in the neuronal cell death that causes Parkinson disease and other late onset neurodegenerative disease (Murray et al. 2003).

– Superoxide (O2 ) is normally produced as a byproduct of oxidative phosphorylation, as a small percentage of the electrons passing down the electron transport chain are diverted into superoxide radical production (Robinson 1998, Murray et al. 2003). However, there are cellular defenses against free radical accumulation such as the radical scavengers, superoxide dimutase and glutathione (Murray et al. 2003). When – – – free radicals do accumulate, O2 can react with nitric oxide (NO ) to form ONOO – – (peroxynitrite) (Murray et al. 2003). The free radicals (O2 and ONOO ) if present in excess, inhibit complex I activity (Murray et al. 2003). This oxidative stress is the proposed mechanism for complex I deficiency in Parkinson disease and other age- related neurodegenerative disorders (Murray et al. 2003).

Oxidative stress also plays a role in the pathophysiology of complex I deficiency in childhood. Oxygen radical production is increased in some patients with complex I deficiency, even though this is offset in part by induction of radical scavengers such as superoxide dimutase (Robinson 1998). As a consequence, excess hydroxyl radicals are produced which are thought to lead to high rates of morbidity and mortality in complex I deficiency (Robinson 1998). Superoxide overproduction has also been shown to trigger cardiomyocyte hypertrophy (Siwik et al. 1999), and this may be a factor in hypertrophic cardiomyopathy which is seen in complex I deficiency (Bénit et al. 2004). Complex I deficiency is also associated with defects in the assembly or stabilization of other mitochondrial OXPHOS complexes (Ugalde et al. 2004). This can lead to decreased OXPHOS activity and to structural changes to the inner mitochondrial membrane that could induce proton leakage and accumulation of reactive oxygen species (Ugalde et al. 2004).

9 1.3 COMPLEX I DEFICIENCY - CLINICAL ASPECTS

Clinical features Inheritance in complex I deficiency is usually autosomal recessive (Kleist-Retzow et al. 1998). Mitochondrial DNA mutations have been estimated to account for 5-20% cases, while nuclear gene mutations account for the remaining cases (Triepels et al. 2001, Lebon et al 2003). A high incidence of consanguineous parents, lack of family history to suggest maternal inheritance, affected siblings with similar phenotypes, an empiric recurrence risk of 20-25% and the recent identification of autosomal recessive mutations in complex I subunit genes all support autosomal recessive inheritance in the majority of patients (Kirby et al. 1999, Loeffen et al. 2000).

The clinical features of complex I deficiency are extremely variable, ranging from lethal neonatal disorders to organ specific adult-onset neurodegenerative disorders such as Leber Hereditary Optic Neuropathy (LHON). This variability often makes recognition and diagnosis of mitochondrial complex I disease difficult. The most common clinical feature in childhood is severe brain dysfunction leading to global neurodevelopmental handicap. Leigh syndrome and Leigh-like phenotypes, as described below, are common. Most children with complex I deficiency have progressive multisystem involvement and the condition is usually lethal in early childhood (Smeitink et al. 1999). Tissues with the highest energy demands are the most frequently affected and these include the brain, heart, skeletal muscle, kidney and retina (Triepels et al. 2001).

There have been several clinical series of complex I deficient patients published, as well as a number of case reports. Robinson and colleagues (Robinson 1993, Pitkänen et al 1996A) have reported the clinical findings in 44 complex I patients. Kirby et al. (1999) reviewed 51 cases (47 pedigrees) of isolated complex I deficiency; seven families had mtDNA mutations and in 40 families the genetic defect was unknown at that time. Loeffen et al. (2000) reviewed the clinical, biochemical and genetic aspects of 27 patients with isolated complex I deficiency. A summary of the clinical features and syndromes reported in complex I deficiency is provided below. However, ascertainment bias is likely to influence the available information on clinical features in complex I deficiency.

10 Only those patients with a constellation of symptoms and signs that we recognise as associated with respiratory chain defects are investigated. There may be an even broader spectrum of presentations in complex I deficiency that currently goes unrecognised.

Leigh syndrome and Leigh-like syndrome are the most common clinical presentations of complex I deficiency in childhood, occurring in up to 50% cases (Loeffen et al. 2000). Leigh syndrome is a subacute necrotizing encephalopathy that is characterised by a specific pattern of neuropathological findings in the brain (Rahman et al. 1996). These include spongiform lesions, demyelination, gliosis and capillary proliferation occurring bilaterally and symmetrically, especially in the brain stem and basal ganglia (Rahman et al. 1996). Clinically, Leigh syndrome is characterised by progressive neurological disease, motor and intellectual developmental regression, brainstem disease (respiratory abnormalities, nystagmus, ophthalmoparesis) and basal ganglia disease (ataxia, dystonia) (Rahman et al. 1996). Optic atrophy may occur and lactate levels in serum and CSF are often elevated (Rahman et al. 1996, Loeffen et al. 2000). The condition is progressive and fatal (Rahman et al. 1996). Although the diagnosis relies on characteristic neuropathology, brain imaging can be highly suggestive (Rahman et al. 1996).

There are a number of further clinical features seen either alone or in combination, in complex I deficiency of childhood. These include cardiomyopathy (hypertrophic, dilated), developmental regression with neurological abnormalities, unspecified encephalomyopathy, myopathy, renal tubular dysfunction, liver disease and ophthalmological features, such as cataracts, optic atrophy and strabismus (Kirby et al. 1999, Loeffen et al. 2000). Lactic acidosis is commonly seen in complex I deficiency but fluctuates over time and is not present in all patients (Kirby et al. 1999). Rare presentations of complex I deficiency include arthrogryposis multiplex congenita (Laubscher et al. 1997, Vielhaber et al. 2000) and infantile spasms (Verdu et al. 1996).

A number of further specific clinical phenotypes have been described in complex I deficiency. Lethal infantile lactic acidosis has been described by several groups (Robinson 1993, Kirby et al. 1999, Loeffen et al 2000). Macrocephaly with progressive leukodystrophy is described in two patients (Dionisi-Vici et al 1997), one of whom has

11 subsequently had a mutation in NDUFV1 identified (Schuelke et al. 1999). In one further case, progressive macrocephaly was associated with a necrotizing encephalopathy (Feillet et al. 1999). Neonatal hypertrophic cardiomyopathy with lactic acidosis is reported in three cases (Loeffen et al. 2000). Cardiomyopathy with cataract and lactic acidosis is also described (Pitkänen et al. 1996B).

In addition there are a number of well defined clinical syndromes associated with specific mtDNA mutations and complex I deficiency. These include Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS), Myoclonic Epilepsy and Ragged Red Fibres (MERRF) and Leber Hereditary Optic Neuropathy (LHON) (Kirby et al. 1999). Complex I deficiency has also been implicated as a pathogenic factor in Parkinson disease and other late onset neurodegenerative disorders (Hattori et al. 1998, Murray et al. 2003).

Male predominance There has been an unexplained male predominance reported in complex I deficiency of childhood. Kirby et al. (1999) report a male to female ratio of 30:21 in their series of 51 complex I deficient patients. Loeffen et al. (2000) report a male to female ratio of 3.5:1 in their group of 27 complex I patients. Von Kleist-Retzow et al. (1998) also report a male predominance in 51 patients with complex I deficiency. Only one complex I subunit gene is located on the X chromosome (NDUFA1) and no mutations in this gene have been reported to date (Loeffen et al. 2000). As there is a male predominance in complex I deficiency, it seems likely that other X linked genes will be identified that are involved in regulation or assembly of complex I.

Treatment of complex I deficiency At present, there is no proven effective treatment for children with complex I deficiency. Treatment options have focussed on correction of the two main factors implicated in pathogenicity. Firstly, reduced ATP production can be potentially improved by provision of cofactors such as co-enzyme Q10, niacin, thiamine, and riboflavin (FMN contains riboflavin) or electron donors that circumvent complex I such as succinate and menadione (Bar-Meir et al. 2001). Secondly, the overproduction of

12 reactive oxygen species may be mitigated by administration of free-radical scavengers such as tocoferol or ascorbate (Bar-Meir et al 2001).

Riboflavin is reported to be effective in complex I patients with isolated skeletal myopathy. In four patients treated with riboflavin, who had complex I deficiency characterised primarily by myopathy and exercise intolerance, there was improvement in muscle endurance (Scholte et al. 1995). Riboflavin was also effective in treatment in a child with isolated myopathy associated with a mtDNA mutation and complex I deficiency (Ogle et al. 1997). Riboflavin was also shown to improve ATP production in a cell line with NDUFS2 mutations (Bar-Meir et al 2001). It is therefore recommended that patients with pure skeletal myopathies and complex I deficiency undergo a trial of treatment with riboflavin (Ogle et al. 1997). In a mouse model of complex I deficiency the optic nerve degeneration associated with LHON was rescued by administration of the gene encoding manganese superoxide dimutase (SOD2), offering hope for treatment options in the future. At this stage however, no treatments for complex I deficiency have been studied in controlled trials and reports of successes are mostly anecdotal. Treatment failures are often not reported and it is likely that treatment failure is common (Triepels et al 2001).

Investigation of suspected complex I deficiency Diagnosis of complex I deficiency is difficult. It relies on tissue biopsies and biochemical assays of complex I function in multiple tissues. However, a number of simpler investigations can suggest the diagnosis, prior to invasive testing. Complex I deficiency leads to an excess of NADH and a lack of NAD+ in the cells because of its effect on the NADH oxidation rate (Munnich et al 1992). This leads to impairment of the Krebs cycle and a subsequent elevation in blood lactate, an increase in ketone bodies and an elevated lactate/pyruvate ratio (Munnich et al 1992). However, some patients with complex I deficiency have normal lactate levels in serum and CSF, and in others the levels are only elevated intermittently (Kirby et al. 1999). A normal lactate level does not exclude complex I deficiency.

Cerebral imaging is often abnormal and can be very informative, although not specific to complex I deficiency (Loeffen et al. 2000). In Leigh disease there are typically

13 symmetrical lesions in the basal ganglia, particularly the putamen but also the globus pallidus and caudate nuclei. Other areas of involvement are the brain stem and peri- ventricular white matter (Medina et al.1990).

Ragged red fibres on muscle histology, a pathognomonic feature of adult mitochondrial disease, is not a common feature of complex I deficiency (Loeffen et al. 2000). In the study by Kirby et al. (1999), ragged red fibres were identified in only 5 of 37 muscle biopsies, although it was noted that ragged red fibres may not develop until later in life. A further 9 of 37 patients had subsarcolemmal mitochondrial aggregates or morphologically abnormal mitochondria. The remaining patients had either normal muscle histology or non-specific changes such as increased lipid, fiber atrophy, fiber size variation or necrosis.

Respiratory chain enzyme activity measurement enables definitive diagnosis of complex I deficiency in many cases. Respiratory chain enzyme analysis is recommended in at least two different tissues, as tissue specificity of complex I deficiency has been well documented (Kirby et al. 1999). Skeletal muscle, liver and cultured fibroblasts are the tissues often used. Muscle is preferred, as it is frequently clinically affected, demands a high oxidative metabolism and so is likely to express any respiratory chain enzyme deficiency (diMauro et al. 1999B). Possible explanations for tissue specific expression of complex I deficiency include tissue specific isoenzymes of complex I subunits, a threshold effect with mtDNA heteroplasmy and tissue differences in RNA processing (Kirby et al. 1999).

Genetic Counseling and Prenatal Diagnosis Genetic counseling should be offered when a child is diagnosed with complex I deficiency. Unfortunately, for most families the genetic basis is unknown making accurate counseling impossible. The empiric recurrence risk in complex I deficiency is around 20-25% indicating that the mode of inheritance in most families is autosomal recessive (Kirby et al. 1999).

When the genetic basis of the complex I deficiency is unknown, there are limited options available to a family to avoid having further children with the same disorder.

14 Prenatal diagnosis with measurement of complex I enzyme activity in chorionic villus sample has limitations. It is only offered in three centers world-wide (Melbourne, Nijmegen, Toronto) (Thorburn and Dahl 2001) and some groups do not perform prenatal testing for complex I deficiency by enzyme activity, as they do not believe they can accurately assess complex I activity in fetal cells (Faivre et al. 2000).

Therefore, mutation identification in complex I deficient patients will provide many real benefits for families affected by this severe disease. Importantly it will mean that the family will have the option of accurate and timely prenatal DNA diagnosis in a situation where the recurrence risk can be high. DNA diagnostic testing as well as carrier testing will be available and the family can also be offered accurate genetic counseling.

1.4 COMPLEX I DEFICIENCY - GENETIC ASPECTS

Mitochondrial DNA (mtDNA) mutations and complex I deficiency It has been estimated that about 5-10% of complex I deficiency in childhood is caused by mtDNA mutations (Triepels et al. 2001). However, this may be an underestimate as in one recent study of fifty complex I deficient patients, mtDNA mutations were identified in 20% of patients (Lebon et al. 2003). The mitochondrial DNA mutations can involve either a complex I subunit (ND1, ND2, ND3, ND4, ND4L, ND5 or ND6) or a non- structural mitochondrial gene, such as a tRNA (Triepels et al. 2001).

Mitochondrial DNA mutations have been identified in all seven mitochondrially encoded subunits as well as a number of mitochondrial tRNAs. Examples are listed in Table 3. The eighteen mtDNA allelic variants associated with LHON are not all included. As is demonstrated in Table 3, complex I deficiency caused by mtDNA mutation can result in a variety of different phenotypes.

15

Table 3: mtDNA mutations and complex I deficiency (modified from Triepels et al. 2001) Mitochondrial Mutation Phenotype Reference Gene ND1 G3460A LHON Early et al. 1987 T3394C Long Q-T syndrome Matsuoka et al. 1999 ND2 G5244A LHON Brown et al. 1992 ND3 T10158C Infantile encephalopathy McFarland et al. 2004 T10191C Infantile encephalopathy McFarland et al. 2004 ND4L 10663C LHON Brown et al. 2002 ND4 C11777A Late-onset encephalopathy Deschauer et al. 2003 ND5 T12706C Leigh disease Taylor et al. 2002 G13513A Leigh disease Kirby et al. 2003 ND6 T14484C LHON Oostra et al. 1995 G14459A Leigh disease Kirby et al. 1999 tRNAleu A3243G MELAS Goto 1993 tRNAleu T3250C Isolated myopathy Ogle et al. 1997 tRNAlys G8344A MERFF Shoffner et al. 1990 tRNAleu T3271C MELAS Kirby et al. 1999 tRNAleu C3303T Isolated myopathy Kirby et al. 1999 Footnote: MELAS- Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes, MERRF- Myoclonic Epilepsy and Ragged Red Fibres, LHON- Leber Hereditary Optic Neuropathy

Nuclear genes and complex I deficiency Overall, there have been reports of mutations in nine different nuclear-encoded complex I subunit genes in a total of 25 families with isolated complex I deficiency (Table 4). All mutations have been novel, except for the NDUFS4 mutation 316C>T, R106X, which has been identified in two apparently unrelated patients ( Budde et al. 2000, Budde et al. 2003).

Several groups have been involved in screening for nuclear gene mutations in complex I deficient patients. Twenty four patients with isolated complex I deficiency were reported by Smeitink et al. (1999) and colleagues (Triepels et al. 2001, Ugalde 2004). All known nuclear encoded complex I subunit genes were sequenced and mutations were found in10/24 families (42% cases). Patients with mtDNA mutations were excluded from this group, so this percentage would overestimate the proportion of complex I deficiency associated with nuclear subunit mutations. The genes with identified mutations were NDUFV1 (Schuelke et al. 1999), NDUFS2 (Loeffen et al. 2001),

16 NDUFS4 (van den Heuvel et al. 1998, Budde et al. 2000), NDUFS7 (Triepels et al. 1999) and NDUFS8 (Loeffen et al. 1998A).

Further mutations have been identified in the genes NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS, NDUFS6 and NDUFS8 (Table 4). Bénit et al. 2001 screened six conserved complex I subunit genes (NDUFV1, NDUFS8, NDUFS7, NDUFS1, NDUFA8, NDUFB6) and found mutations in 6/36 (17%) families. The genes in which mutations were found were NDUFV1 and NDUFS1. Subsequently, mutations in NDUFV2 (Bénit et al. 2003A), NDUFS4 (Bénit et al. 2003B) and NDUFS3 (Bénit et al. 2004) were reported. The number of complex I subunit genes screened in these patients is not reported, so an accurate assessment of the involvement of nuclear genes in complex I deficiency is not possible, though is probably around 20-25%.

The nuclear genes in which mutations have been identified in complex I deficiency include all seven of the highly evolutionary conserved genes NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS7 and NDUFS8. As mutations have also been identified in all seven mitochondrially encoded subunits, all 14 core complex I genes have now been implicated in human complex I deficiency.

Mutations in two nuclear encoded supernumerary complex I subunits, NDUFS4 and NDUFS6 have also been identified. Although NDUFS4 does not have a prokaryotic homologue, it is involved in the regulation of complex I activity. Cyclic AMP- dependent phosphorylation of the NDUFS4 subunit has been shown to activate complex I (Papa et al. 1996, Papa et al. 2001), indicating that this gene is of functional importance in mammalian complex I. NDUFS6 is the most recently identified subunit involved in human disease and its role in complex I is unknown (Kirby et al. 2004). It may be important for complex I assembly or stability (Kirby et al. 2004).

The phenotypes seen with nuclear encoded complex I mutations are summarised in Table 4. Overall, mutations in all seven core genes and NDUFS4 commonly result in neurological disease, and Leigh or Leigh like syndromes are the most common phenotypes. NDUFS4, NDUFS2 and NDUFV2 have been associated with hypertrophic cardiomyopathy. The one patient with NDUFS3 mutations had late-onset Leigh

17 syndrome (Bénit et al. 2004). Both patients with NDUFS6 mutations had lethal neonatal mitochondrial disease (Kirby et al. 2004). At this stage, the numbers are too small to be certain of a clear association between subunit involvement and phenotype.

Table 4: Nuclear complex I subunit mutations in complex I deficiency

NDUFV1 PHENOTYPE REFER- MUTATIONS ENCE homozygous Presented at 6 months with myoclonic epilepsy and hypotonia. Schuelke et c.1,022C>T (A341V) Clinical features included spasticity, macrocephaly, al. 1999 psychomotor developmental arrest, macrocystic leukodystrophy and elevated CSF lactate. Surviving at 10 years with severe spasticity and blindness. c.1,268C>T Leigh-like syndrome in two brothers who both presented at 5 Schuelke et (T423M) / months with vomiting, strabismus and hypotonia. Other al. 1999 c.175C>T (R59X) features include myoclonic epilepsy, psychomotor regression and a progressive, unspecified encephalomyopathy. Death at 14 and 17months. c.640G>A (E214K) / Leigh like syndrome- presentation at 1 year, seizures, elevated Bénit et al. IVS8+4A>C lactate, ataxia, psychomotor regression, strabismus, ptosis. 2001 MRI imaging- brain atrophy and areas of hyperintensity in the brain stem. Death at 3 years c.1294G>C (A432P) Leigh like syndrome presenting at 6 months with vomiting Bénit et al. / and hypotonia. Progressive respiratory abnormality, lactic 2001 c.989-990delCT acidosis, areas of hyperintensity in the basal ganglia on MRI and death at 18 months. c.611A>G (Y204C) / Leigh like syndrome presenting at 5 months with ptosis, Bénit et al. c.616T>G (C206G) ophthalmoplegia, hypotonia, ataxia, lactic acidosis, 2001 hyperintensity of the locus niger on MRI homozygous Parents Caucasian, consanguineous. Presentation as neonate Schuelke et c.632C>T (A211V) with hypotonia, nystagmus, bitemporal retinal al. 2002 depigmentation, lactic acidosis and death at 4 weeks

NDUFV2 PHENOTYPE REFER- MUTATIONS ENCE homozygous Early onset hypertrophic cardiomyopathy and encephalopathy. Bénit et al. IVS2+5_+8del Truncal hypotonia, poor growth and persistent lactic acidemia 2003A GTAA resulting in death at 3 months.

NDUFS1 PHENOTYPE REFER- MUTATIONS ENCE c.664_666del / Presented at 4 months, psychomotor retardation, hypotonia, Bénit et al. c.755A>G (D252G) nystagmus, optic atrophy, leukodystrophy, lactic acidosis. 2001 Death at 10 months c.721C>T (R241W) / Leigh like syndrome; presented at 2 months with growth Bénit et al. c.1669C>T (R557X) retardation, hypotonia, hepatomegaly, lactic acidosis, MRI 2001 showed hyperintensity of basal ganglia, macrocytic anaemia, dystonia. Death at 5 months. c.2119A>G Leigh like syndrome; neonatal presentation with failure to Bénit et al. (M707V) / de novo thrive, hypotonia, microcephaly, pyramidal signs, anaemia, 2001 deletion paternal lactic acidosis, MRI suggestive of Leigh syndrome allele

18 Table 4 continued NDUFS2 PHENOTYPE REFERENCE MUTATIONS homozygous Consanguineous parents. Two affected children with Loeffen et al. c.683G>A (R228Q) early onset of severe neurological regression, nystagmus, 2001 hypotonia, pyramidal signs, failure to thrive, optic atrophy, hypertrophic cardiomyopathy, basal ganglia hypodensities and severe lactic acidosis. Death at 18 and 24 months. homozygous Severe lactic acidosis presenting on first day of life and Loeffen et al. c.686C>A (P229Q) resulting in death at 4 days of age. Hypertrophic 2001 cardiomyopathy was confirmed on post mortem. homozygous Consanguineous parents with three affected children all Loeffen et al. c.1237T>C (S413P) presenting in the first year of life. Clinical features 2001 included vomiting, failure to thrive, nystagmus, hypotonia, optic atrophy, psychomotor retardation, lactic acidosis and hypodensity of basal ganglia and midbrain. Death was at 18 months, 3 years and 2 years.

NDUFS3 PHENOTYPE REFERENCE MUTATIONS c.434C>T (T145I) / Late onset Leigh syndrome (onset at 9 years of age), Bénit et al. 2004 c.595C>T (R199W) optic atrophy, death at 13 years

NDUFS4 PHENOTYPE REFERENCE MUTATIONS homozygous Leigh-like syndrome: term female infant presented as Petruzzella et al. c.44G>A, (W15X) neonate. Clinical features include vomiting, failure to 2001 thrive, psychomotor delay, seizures, hypotonia , lactic acidosis, hypertrophic cardiomyopathy, basal ganglia hyperechogenicity on ultrasound. Death at 7 months. homozygous Leigh syndrome: consanguineous parents with two Bénit et al. 2003B IVS1-1G>A affected female children presenting at 2 months and 3 months. Clinical features included poor sucking, drowsiness, hypotonia, psychomotor regression, squint, elevated lactate, bilateral hypodensity of periventricular white matter, brainstem involvement. **homozygous Leigh-like syndrome: male infant presented at 8 van den Heuvel et c.466_470dupAAGTC months. Features included vomiting, failure to thrive, al. 1998 hypotonia, severe psychomotor delay, seizures and bradypnea. Lactate normal. Brain MRI showed atrophy and basal ganglia abnormalities. Death at 16 months. **homozygous Leigh-like syndrome: consanguineous parents, female Budde et al. 2000 c.290delG (W96X) infant at 1 week developed hypotonia, lethargy and failure to thrive. At 3 months microcephaly, lactic acidemia and bilateral basal ganglia hypodensities on brain MRI. Normal cardiac echo. Death at 3 months. **homozygous Leigh-like syndrome: consanguineous parents, male Budde et al. 2000 c.316C>T (R106X) infant presented at 7 weeks. Clinical features included hypotonia, lack of responsiveness, cough, feeding problems, pneumonia, hypertrophy of the left ventricle. Lactate was elevated. Cranial MRI consistent with Leigh syndrome. Death at 3 months. homozygous Leigh-like syndrome, early lethality Budde et al. 2003 c.316C>T (R106X)

19 Table 4 continued

NDUFS6 PHENOTYPE REFERENCE MUTATIONS homozygous Lethal neonatal mitochondrial disease Kirby et al. 2004 c.186+2T>A 4.175 kb deletion Lethal neonatal mitochondrial disease Kirby et al. 2004 encompassing exons 3 and 4

NDUFS7 PHENOTYPE REFERENCE MUTATIONS homozygous Leigh syndrome Triepels et al.1999 c.364G>A (V122M)

NDUFS8 PHENOTYPE REFERENCE MUTATIONS c.236C>T (P79L ) Leigh syndrome Loeffen et al. c.305G>A (R102H) 1998A c.254C>T (P85L) Late onset Leigh syndrome Procaccio et al. c.413G>A (R138H) 2004 Footnote: The nomenclature used in this table to describe mutations was changed from the original reports. The system of nomenclature, described by Antonarakis et al. (1998) was implemented. ** these mutations were originally reported with combined complex I and III deficiency (Budde et al. 2000). However, the AAGTC duplication at 466-470 and the 290delG mutations were re-assessed by Scacco et al (2003), in combination with another NDUFS4 mutation G44A. All three patients had normal assembly and activity of complex III. In addition, 20 patients with combined complex I and III deficiency were studied and none had NDUFS4 mutations (Scacco et al 2003).

The aetiology of complex I deficiency when subunit mutations have been excluded Structural subunit gene mutations (mitochondrial and nuclear encoded) appear to account for only a proportion of complex I deficiency, somewhere less than 50% (Antonicka et al. 2003). Mutations in one or more non-structural nuclear encoded genes are likely to be the cause of complex I deficiency in the remaining patients (Triepels et al. 2001). These genes may be involved in movement of nuclear encoded complex I subunits into the mitochondria, the correct assembly of subunits into complex I, the implantation of complex I in the inner mitochondrial membrane, regulation of complex I function or perhaps nuclear and mitochondrial communication (Loeffen et al. 2000, Triepels et al. 2001). Two novel chaperones for complex I membrane arm assembly (CIA30 and CIA84) were identified in Neospora crassa (Küffner et al. 1998). The human homologue of CIA30 has been identified, proving that complex I assembly factors do exist (Ugalde et al. 2004), though mutations causing human disease have not yet been identified.

20

1.5 PROJECT OUTLINE This project aimed to identify the molecular basis of complex I deficiency, so that accurate genetic counseling and prenatal diagnosis could be offered to the families involved. Thirty four children with isolated complex I deficiency and three children with suspected complex I deficiency were studied. Six of the highly conserved nuclear encoded complex I subunit genes were screened for mutations. This patient group is a unique resource and one of the largest groups of complex I patients assembled and studied in this way. The patients are well defined biochemically and clinically and the patient group has been part of a published series (Kirby et al. 1999).

Mutation analysis was undertaken in six of the 39 nuclear genes that encode for subunits of complex I, and for which there is evidence of involvement in human mitochondrial disease. Five of these genes (NDUFV1, NDUFS1, NDUFS2, NDUFS7, NDUFS8) were selected because they have homology with the E. coli complex I subunit genes, indicating that these genes, conserved throughout evolution, carry out essential aspects of complex I function (Smeitink et al. 1999). The sixth gene NDUFS4 does not have a counterpart in E. coli though it is highly conserved in mammals. NDUFS4 has an important role in complex I regulation (Papa et al. 2001) and mutations causing complex I deficiency have been identified (van den Heuvel et al. 1998, Petruzzella et al. 2001). Since the completion of this project, three other complex I subunits genes (NDUFV2, NDUFS3 and NDUFS6) have been implicated in complex I deficiency, but these genes were not assessed in this project.

The chromosomal location, cDNA size and protein product size of the six genes studied are summarised in Table 5. Further information on the genomic DNA and cDNA organizational structures of these genes is presented in the Discussion. The cDNA sequence of all the genes is known. The six genes are all relatively small (Loeffen et al. 1998B) with their cDNA size ranging from 601-2503 base pairs (Table 5). The genes were initially screened by denaturing high performance liquid chromatography (dHPLC). Any patient samples with abnormal profiles by dHPLC then were directly sequenced.

21

In summary, novel mutations in NDUFV1, NDUFS2 and NDUFS4 were identified in 6/34 (18%) patients. Two patients were compound heterozygotes for NDUFV1 mutations and two patients were compound heterozygotes for NDUFS2 mutations. Two patients had mutations in NDUFS4 identified.

Table 5: Characteristics of the 6 genes studied

Gene Location cDNA size Amino Genbank References (base pairs) Acid Accession NDUFV1 11q13 1566 464 NM 007103 Ali et al. 1993, Schuelke et al. 1998, de Coo et al. 1999 NDUFS1 2q33 2503 727 NM 005006 OMIM 157655

NDUFS2 1q23 2061 463 NM 004550 Loeffen et al. 1998C

NDUFS4 5q11.1 601 175 NM 002495 van den Heuvel et al. 1998, Budde et al. 2000 NDUFS7 19p13 754 213 NM 024407 Hyslop et al. 1996

NDUFS8 11q13 779 210 NM 002496 Procaccio et al.1997, de Sury et al 1998

22 2. METHODS

A cohort of 34 patients with biochemically confirmed complex I deficiency was studied, looking for mutations in six of the nuclear encoded complex I subunit genes. Patient samples underwent RNA extraction followed by reverse transcription to form cDNA. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The PCR products were then screened for mutations using dHPLC (denaturing High Performance Liquid Chromatography). Samples with abnormal profiles by dHPLC were then purified and sequenced to enable mutation identification. The methods used are presented in detail in the following sections. The methods are presented in the format of laboratories, in sufficient detail to enable external review.

2.1 ETHIC COMMITTEE APPROVAL This study was approved by the Research Ethics Committee of the South Eastern Sydney Area Health Service (# 00/073).

2.2 PATIENT AND CONTROL SAMPLES Cultured fibroblasts from a cohort of 34 patients with biochemically proven complex I deficiency and 3 patients (Patients 11, 12 and 28) with suspected mitochondrial disease served as the patient samples for this study. The 34 patients with “isolated” complex I deficiency did not have deficiency of the other respiratory chain complexes on biochemical testing. Patients with known mitochondrial DNA complex I subunit gene mutations or known mitochondrial DNA tRNA mutations were excluded. Biochemical complex I deficiency had already been determined by measurement of respiratory chain enzymes activity and citrate synthase in tissue homogenates and mitochondrial extracts from cultured fibroblasts and Epstein Barr virus-transformed lymphocytes, as described previously (Rahman et al. 1996, Kirby et al. 1999). The cell pellets, derived from the cultured fibroblasts, were prepared at the Murdoch Institute, Melbourne. The cells were cultured in cycloheximide to minimize the risk of mRNA decay (Bateman et al. 1999). The fibroblasts were spun down, the supernatant removed and then the pellet was flash frozen on dry ice and stored at -70 degrees Celsius. Each patient cell pellet contained 2- 6 x107 cells.

23 The five control samples (C1-5) used in dHPLC were de-identified cell pellets derived from cultured chorionic villous samples that had been collected for the purpose of chromosome analysis. The chromosome analysis was normal. The pellets contained a similar number of cells to patient samples.

Clinical Information: The clinical and biochemical features of the patients studied are summarised in Table 6. Of the 34 patients with confirmed complex I deficiency, 21/34 had Leigh syndrome or a Leigh-like neurodegenerative syndrome. Four patients had primary lactic acidosis, three had Lethal Infantile Mitochondrial Disease (LIMD), three had non specific encephalomyopathy and three had cardiomyopathy as the major feature. In total there were six patients with documented cardiomyopathy.

24

Table 6: Clinical and biochemical features of patients

Con- Patient Age at sangui Complex No. Clinical Diagnosis presentation Ethnicity nity Deficiency 1 Leigh syndrome birth I 21 Primary Lactic acidosis neonatal Yes I 31 Primary Lactic acidosis neonatal Turkish No I 4 Primary Lactic acidosis I 5 ?Leigh like syndrome 2 years Tongan I 2 6 Leigh syndrome 9 months I

72 Leigh-like syndrome 6 weeks No I 8 affected pregnancy Egyptian Yes I 9 LIMD I (?III) 10 mitochondrial myopathy I 3 11 ?mitochondrial disease suspected 3 12 mitochondrial disease (I,IV) 13 Leigh syndrome 5 months No I

14 Leigh syndrome 4.5 months No I

4 Leigh syndrome 15 15 months I 16 Leigh syndrome 6 months No I 17 Lactic acidosis I 18 Leigh syndrome 1 day I 19 Leigh-like syndrome Lebanese Yes I 20 Leigh syndrome 4 months I 21 Leigh syndrome 3 months No I 22 Leigh syndrome birth No I

23 ?Leigh syndrome 5 years Thai I

24 Leigh syndrome Yes I 25 Leigh-like syndrome 2 months No I 26 Leigh syndrome 8 months Lebanese I 27 Leigh-like syndrome 5months No I 283 mt encephalomyopathy 6 weeks No I,IV (,III) 29 Leigh disease 10 months Lebanese Yes I 30 Cardiomyopathy birth I 31 Leigh syndrome 18 months I (IV?)

32 Primary Lactic acidosis neonatal I

33 Cardiomyopathy Chinese I 34 Cardiomyopathy 9 months Lebanese No I 35 mitochondrial disease I 36 Leigh-like syndrome 3 months I 37 Cardiomyopathy neonatal I

Footnote: 1 homozygous NDUFS6 mutation subsequently identified (Kirby et al. 2004) 2 mtDNA mutation in ND3, T10158C, subsequently identified (McFarland et al. 2004) 3These patients did not have proven isolated complex I deficiency 4mtDNA subunit gene ND5 mutation (12706T>C) subsequently identified (Dr David Thorburn)

25 Specific clinical details of the patients with identified mutations are summarized below. Patient 13: This female infant was born to nonconsanguineous parents. Her brother was deceased and had a post-mortem diagnosis of Leigh syndrome. She presented at 5 months of age. Clinical features included developmental regression, spasticity, involuntary movements, bilateral ptosis, ophthalmoplegia, dysarthria, dysphagia, increasing stridor, vomiting and failure to thrive. She died at 24 months of age and post mortem examination confirmed Leigh syndrome. Enzymology studies of fibroblasts identified isolated complex I deficiency Patient 17: There is no family history information available for this patient and clinical detail is limited. His clinical features included ataxia, nystagmus, optic atrophy, developmental delay and metabolic acidosis. CSF lactate was 2.7 mmol/l and serum lactates were 3.2 and 5.7 mmol/l (normal range 0.6-2.2 mmol/l). A CT scan (without contrast) at 6 years of age was normal. Enzymology showed isolated complex I deficiency. Patient 19: This boy had a diagnosis of Leigh-like syndrome. His parents are first cousins. He had a sister who died from Leigh syndrome. Two surviving siblings have mental retardation and two further siblings are well. Clinical features included failure to thrive, developmental delay, pryramidal tract signs, spastic quadraparesis and hyperkeratotic skin lesions. CSF lactate was 1.6 mmol/l and serum lactates 3.8 and 1.3 mmol/l. Clinical progression was consistent with Leigh syndrome. Enzymology revealed complex I deficiency. Patient 25: This male infant was the second child to nonconsanguineous parents. He presented at 2 months of age with poor feeding, poor weight gain and episodic hypoventilation. There was elevation of CSF lactate (4.7 and 10.9 mmol/l) and serum lactate (4.2 and 9.9 mmol/l). MRI scanning showed possible increased signal in the basal ganglia on T2 weighted image. This infant died at around 8 months of age with probable Leigh syndrome. Isolated complex I deficiency was identified by enzymology. Patient 26: Patient 26 was the second child to nonconsanguinous parents. His parents originated from villages about 100km apart in Lebanon. He presented at 8 months of age with vomiting and lethargy. At 15 months he was noted to have ptosis, bilateral convergent strabismus and intermittent upward ocular deviation. He suffered progressive neurological deterioration and died at 7 years of age. MRI brain scan

26 showed brain stem lesions consistent with Leigh syndrome. CSF lactate was 2.9 and serum lactate 2.1 mmol/l. Enzymology confirmed complex I deficiency. Patient 27: This male infant was the second child to nonconsanguineous parents and there was no relevant family history. He presented at five months of age with a history of listlessness, poor feeding and poor head control. He had progressive deterioration in conscious state over one week. He had nystagmus and a myopathic facies. He was hypotonic and developed apnea requiring mechanical ventilation. He subsequently died at 5 months of age. Investigations revealed a CSF lactate of 2.2 mmol/l and serum lactates of 1.3 and 2.5 mmol/l. MRI of brain showed symmetrical abnormal signal in cerebral peduncles, dorsal midbrain, medulla oblongata and cervical cord, consistent with Leigh syndrome. Enzymology of fibroblasts, muscle and liver identified complex I deficiency.

2.3 RNA EXTRACTION An RNA based mutation detection method was utilised. The reasons for this are presented in the discussion. QIAGEN RNeasy midipreps (QIAGEN Pty Ltd, cat. no. 75144) were used for RNA extraction of patient and control samples, allowing isolation of up to 1mg RNA and all RNA molecules greater than 200 nucleotides in size. RNA binds to a silica-gel based membrane, contaminants are washed away and RNA is eluted in water. The detailed protocol for RNA extraction is described in the handbook, RNeasy Midi/Maxi Handbook October 1999, and is summarised below. The exact chemical composition of all the supplied buffers is not provided by QIAGEN. The sample size recommended for use with the RNeasy midi preps is 5 x 106 to 1 x 108 cells, appropriate for the size of the patient samples.

In order to release all RNA contained in the sample, the plasma membranes of cells and organelles were disrupted. The frozen cell pellets were partially thawed and two ml of buffer RLT was added. Buffer RLT contains a highly denaturing guanidine isothiocyanate (GITC) which immediately inactivates RNases. 10µl 14.3 Molar β mercaptoethanol was added to each 1ml of Buffer RLT, using a fume hood and protective clothing and eye wear.

27 The cell lysates were then homogenized by vortexing for ten seconds and passing the lysate ten times through a 21 gauge needle attached to a sterile plastic syringe. Homogenisation reduces the viscosity of cell lysates by shearing high molecular weight genomic DNA and other high molecular weight cell components.

Two ml of 70% alcohol was added to the homogenized lysate and mixed thoroughly by shaking. The addition of alcohol promotes selective binding of RNA to the silica-gel membrane provided. The sample was then applied to the column which was placed in a 15 ml centrifuge tube. The tube was centrifuged for five minutes at 4000g so that the lysate passed through the column. RNA was now bound to the column membrane and the flow-through was discarded.

Four ml of Buffer RW1 was then added to the column, which was then centrifuged for five minutes at 4000g. This step removes contaminants left on the column. Flow through was discarded. Buffer RPE was supplied with the extraction kit as a concentrate to which four volumes of 96% ethanol was added. 2.5 ml of buffer RPE was applied to the column and the sample centrifuged for two minutes at 4000g, to wash away any remaining contaminants. The flow through was discarded. A further 2.5 ml of buffer RPE was then applied to the column which was then centrifuged again for five minutes at 4000g.

The column was then transferred to a new 15ml centrifuge tube for elution of RNA from the column. 150 µl of RNase free water was pipetted directly onto the column membrane and the tube was closed and allowed to stand for 1 minute. The tube containing the column was then centrifuged for three minutes at 4000g. The elution step was then repeated and the 300µl of eluted RNA stored at -80C.

Before the cells are disrupted and homogenised in the presence of RNase inhibiting or denaturing agents, the RNA in the cell samples can be degraded by ribonucleases (RNases). A number of precautions were taken to avoid RNase contamination during the process of RNA extraction. These included using an aseptic technique when handling patient samples, changing gloves frequently, keeping tubes closed when possible and using RNAse free pipette tips and plasticware.

28 2.4 QUANTIFICATION OF RNA RNA quantification in the patient samples was achieved by Ultra Violet (UV) spectrophotometry. The results of UV spectrophotometry are tabulated and recorded in Appendix 1. Measurements were taken at a wavelength of 260nm. At 260nm, an absorbance of one unit corresponds to an RNA concentration of 40 µg/ml RNA (for RNA diluted in water). The concentration of RNA in the patient samples was calculated according to the following formula;

RNA concentration (µg/ml) =

40 µg/ml x OD(optical densitometry)260 x dilution factor dilution factor = 50 (10 µl RNA diluted in 490 µl RNAse free water).

An estimate of the amount of protein contained in the RNA sample was also obtained using UV spectrophotometry. Proteins have a UV absorption maximum of 280 nm and so absorbance of the RNA sample at 280 nm gives an estimate of protein contamination.

The ratio of the absorbance at 260 nm/ absorbance at 280 nm (OD260/OD280) is a measure of the purity of a DNA or RNA sample. For DNA, a pure sample has an

OD260/OD280 ratio between 1.65 and 1.85. For RNA, it is recommended that the RNA be diluted in 10mM Tris.Cl, pH 7.5, rather than water, as the pH influences the

OD260/OD280 ratio. Lower pH results in a lower OD260/OD280 ratio. For RNA diluted in

10mM Tris.Cl, pH 7.5, pure RNA has a OD260/OD280 ratio of 1.9-2.1. Given the limited amounts of RNA available for analysis, the RNA was not diluted in Tris. Cl. The

OD260/OD280 measurements were done in water and so may underestimate protein contamination. The results are tabulated in Appendix 1.

2.5 FORMALDEHYDE AGAROSE (FA) GEL ELECTROPHORESIS The integrity and size distribution of total RNA was assessed using formaldehyde agarose (FA) gel electrophoresis with ethidium bromide staining.

1.2% FA gel was made by adding 1.2g agarose to 10 ml of 10x FA gel buffer (see below). RNase free water was added to make a total volume of 100 ml. This mixture was heated in a microwave oven until the agarose melted. The solution was cooled to 65oC in a water bath. Then 1.8 ml of 37% Formaldehyde was added and the solution

29 was mixed thoroughly and poured into a gel mould. After the gel solidified, it was equilibrated with 1 x FA gel running buffer (see below) for 30 minutes.

10x FA gel buffer consisting of 10 × MESA buffer (MOPS, EDTA, Sodium Acetate) was supplied by Sigma (catalogue no. M5755). When reconstituted it consisted of 0.4M MOPS(3-[N-morpholino] propanesulfonic acid), pH approx. 7.0, 0.1 M sodium acetate and 10 mM EDTA.

1x FA running buffer was made by combining 25 ml 10x FA gel buffer, 5 ml 37%Formaldehyde and 220 ml RNAse free water.

Electrophoresis: 10 µl of RNA loading buffer (Deionized formamide 62.5% (v/v), formaldehyde 1.14 M, bromphenol blue 200 µg/ml, xylene cyanole 200 µg/ml, MOPS- EDTA-sodium acetate at 1.25× working concentration, ethidium bromide 50 µg/ml) was mixed with 20- 40 µl RNA (~5µg RNA) and incubated for five minutes at 65oC then chilled on ice. The samples and a control water lane were then loaded onto the equilibrated 1.2% FA gel. The samples were electophoresed for 75 minutes at 100volts. The gel was then photographed with a UV transilluminator.

The quality of RNA extraction was considered adequate if two ribosomal RNA (rRNA) species were seen on the photograph, an 18S rRNA [1.9kb size] and a 28S rRNA [5.0kb size]. As expected, the fluorescence of the 28S ribosomal RNA bands was approximately twice the amount of the 18S band. There was no evidence of RNA degradation which would appear as smearing of the bands towards smaller sized RNA’s.

FA gel electrophoresis was performed on the first seven RNA samples extracted, to confirm the adequacy of RNA extraction. No samples showed evidence of RNA degradation. FA gel electrophoresis was not performed routinely on subsequent samples, to avoid RNA wastage. It was considered that reverse transcription and PCR would be unsuccessful if the RNA was degraded or of poor quality.

30 2.6 REVERSE TRANSCRIPTION Reverse Transcription (RT) uses the enzyme reverse transcriptase (RNA dependent DNA polymerase) to make a DNA copy that is complementary in base sequence to the mRNA (cDNA). Reverse Transcription was performed according to the Qiagen Omniscript Reverse Transcriptase protocol (April 1999 handbook) and is described below. The protocol is optimized for use with 50ng - 2µg RNA. All reactions were performed on ice and pipette tips with hydrophobic filters were used to minimize contamination.

RNA samples were thawed on ice. Oligo-(dT)12-18 primer (Life Technologies, product no. 18418-012), 10 x Buffer RT, dNTP mix, Ribonuclease inhibitor, cloned (Invitrogen cat. no. 15518-012) and RNase free water were thawed at room temperature then stored on ice. Each solution was mixed by vortexing briefly. A master mix was prepared on ice, according to Table 7 below. The mix was vortexed for 5 seconds and stored on ice.

Table 7: Reverse transcription master mix

Component Volume/Reaction Final Concentration Master Mix 10XBuffer RT 2.0 µl 1x dNTP Mix (5mM each dNTP) 2.0 µl 0.5 mM each dNTP Oligo-dT primer (10uM) 1.0 µl 0.5 µM RNase inhibitor (10 units/µl) 1.0 µl 10 units/20 µl reaction Omniscript reverse transcriptase 1.0 µl 4 units/20 µl reaction RNase-free water variable Template RNA RNA, added at step 4 variable Up to 2 µg /20 µl reaction Total volume 20.0 µl

The appropriate volume of master mix was distributed to individual reaction tubes on ice. RNA was added to the individual reaction tubes and the reaction was vortexed for less than 5seconds. The volume of RNA added corresponded to <2 µg RNA (the volume of water added to the master mix was adjusted accordingly so that the total reaction volume after addition of RNA was 20 µl). Reactions were then incubated for 60 minutes at 37°C. Once RT was complete the cDNA samples were stored at -20°C.

31 2.7 PCR (POLYMERASE CHAIN REACTION) PCR was performed to amplify the cDNA of interest.

Primers: Unlabelled oligonucleotide primers were designed for each of the six genes studied, based on the known cDNA sequence of each gene. The primers used are listed in Table 9. Primers were designed to be about 20bp long. Regions of cDNA containing multiple repeats were avoided. Primers were designed so that the ratio of G/C compared to A/T was close to 50%, and so that both forward and reverse primers had similar melting temperatures (Tm). Primers successfully used in previously published studies were also used when possible, as acknowledged in Table 9. Primers were designed to amplify overlapping fragments of cDNA so that together they spanned the entire coding area of each of the six genes. These cDNA amplicons ranged in size from 300-600 base pairs. This is the optimal size for dHPLC. For each gene there were between two and five amplicons. Primers were diluted to a working concentration of 10pmol/µl.

PCR reaction mix: 50 µl PCR reactions were performed. A PCR master mix was made using the components listed in Table 8. Platinum PCR Super Mix (Invitrogen, Catalogue No. 11306-016) was used which contains: anti-Taq DNA polymerase antibody, Magnesium (1.65mM MgCl2), deoxyribonucleotide triphosphates and recombinant Taq DNA polymerase.

Table 8: PCR master mix components

Reagent Volume per reaction Platinum PCR Super Mix 46 µl Primer Forward Primer 1.25 µl (250mM final concentration) Reverse Primer 1.25 µl (250mM final concentration) Template cDNA 1-2.0 µl Total Volume 49.5-50.5 µl

The number of reactions was formulated and the volume of master mix adjusted accordingly. 48.5 µl of master mix was distributed to each reaction tube and 1-2 µl of cDNA was then added. No DNA was added to one tube, the blank. This reaction would not be expected to produce a PCR product so no band should be seen on polyacrylamide gel. If a band was present in the blank lane, contamination of the PCR reaction has

32 occurred, and all samples were discarded. A control sample was also amplified for each PCR.

50 µl PCR reactions were chosen as opposed to a smaller volume as enough sample was needed for both dHPLC and sequencing. The 50 µl PCR sample was used as follows: 3- 5 µl for the polyacrylamide gel, 20 µl for dHPLC, a further 10 µl (+10 µl control DNA) for dHPLC and 10-15 µl for purification and sequencing.

PCR Reaction: Several steps were undertaken in PCR thermocycling. There was an initial step of 94°C for 3 minutes, to completely denature the template and activate the enzyme (Taq). Following this there was 35 cycles of PCR amplification which involved 1. Denaturation 94°C 30 seconds 2. Annealing Variable temperature (54 - 62°C) 30 seconds 3. Extension 72°C 1 minute A final 10 minute extension at 72°C completed the PCR program.

PCR optimization: For each amplicon, the PCR was optimized by trials of differing annealing temperatures, quantity of cDNA and cycle number. The PCR reaction was considered optimized when there was a single clean band of the expected base pair size on polyacrylamide gel electrophoresis (Figure 2). The PCR primers used and the optimised annealing temperatures are listed in Tables 9 and 10.

33

Table 9: Oligonucleotide primers used in PCR and sequencing.

Gene Forward Primer 5'- 3' Reverse Primer 5'- 3' NDUFV1F1- NDUFV1R1- NDUFV1 GAAGGTGACAGCGTGAGGTG ATACTTGGGCCTGCCATCTGAG NDUFV1F2- NDUFV1R2- AAGTGGAGCTTCATGAATAAGCC GACTCGATGAGCGCTGTCTC NDUFV1F3- NDUFV1R3- GAATGCTTGTGGCTCTGGCTATG GGCACAGACATCTCCTCCTCCA NDUFV1F4- NDUFV1R4- ATCTCTGGCCATGTCAACCAC CATCACCTTGTTCATCCAGTCC NDUFV1F5- NDUFV1R5- TATAAGCACGAGAGCTGTGGCC GGCAGCACTCGCTTTATTGTCC *NDUFS1F1- *NDUFS1R1- NDUFS1 TAGCAGAACAGCCTCCGCGG AATCGGCTCCTATCATTTCCAAA *NDUFS1F2- *NDUFS1R2- AATCACCCATTGGACTGTCCT GGCAAATCTGGTTTTATCAGA *NDUFS1F3- *NDUFS1R3- AGAACTGGAGAAGTGATGAGGA TAAGTCATTATGCAGCCAGCTC *NDUFS1F4- *NDUFS1R4- GGCAGATGTTGTTCTTCTGGTT TCCATCTGCTCCCAGGAGAAA *NDUFS1F5- *NDUFS1R5- CTTGGCTATAAGCCTGGGGTG AACTGGGATCCTAGTAGAAGCT NDUFS2F1- NDUFS2R1-2- NDUFS2 CTGTGCGAATAGGTGAGAAGC GTCCACATCATTCCAAGGTGG NDUFS2F2- NDUFS2R2-2- TGTGGAATGGGCACAGCAGT AGCAACTTCTCCACAGCTAGAG NDUFS2F3- NDUFS2R3-2- CCATGATGTGTAACGAACAGGC GTCCGATTTCGCCAGATCCTA NDUFS2F4- NDUFS2R4-2- TGAGTTGGAGGAGTTGCTGAC GTGATGAATCAGTGACTCCA NDUFS2F5- NDUFS2R5-2- CCTAAGCGAGCAGAGATGAA CCACAGAAGAAGCTGATAGGC NDUFS4F1- NDUFS4R1- NDUFS4 CCTGGCGTTTGCCTGCAG AAGGATTTTCCCATCGCTCCC NDUFS4F2- NDUFS4R2- TCGCAATAACATGCAGTCTGG AAGCAGAGATATAGTCAGTGCC *NDUFS7F1- *NDUFS7R1- NDUFS7 CTGAAGGCCGAGGCCAAG GTTGACGAGGTCATCCAGC NDUFS7F2- NDUFS7R2- GTTCTCTGTGGCCCATGACC CCGAGTAGGAATAGTGGTAG *NDUFS7F3- *NDUFS7R3- CTATTCCTACTCGGTGGTG CCTCACGGGACACAAGCAG *NDUFS7F2B- *NDUFS7R2B2- AGAGCCGTGGCTCCCAAACC ATGTCCACGGGCACGATGCG *NDUFS8F1- NDUFS8R1- NDUFS8 TGGCCGAATGGCAGCGTC AGTTGATGGTGGCCGGTTCC NDUFS8F2- *NDUFS8R2- CCTACAAGTATGTGAACATGC GATGTCATAGCGGGTGGTCC *NDUFS8F3- *NDUFS8R3- CATTGCCTGCAAGCTCTGCG TTTTATTGGGCAGCAGGGGC Footnote: *Primers from Benit et al. 2001

34 Table 10: Optimised PCR conditions and amplicon size

Gene Amplicon Primers Amplicon PCR size annealing (base pairs) temperature NDUFS4 Amplicon 1 NDUFS4F1/R1 353 57/58 Amplicon 2 NDUFS4F2/R2 305 54 NDUFV1 Amplicon 1 NDUFV1F1/R1 375 55 Amplicon 2 NDUFV1F2/R2 353 55 Amplicon 3 NDUFV1F3/R3 333 57 Amplicon 4 NDUFV1F4/R4 357 55 Amplicon 5 NDUFV1F5/R5 344 55 NDUFS8 Amplicon 1 NDUFS8F1/R1 337 58 Amplicon 2 NDUFS8F2/R2 332 58 Amplicon 3 NDUFS8F3/R3 338 60 NDUFS7 Amplicon 1 NDUFS7F1/R1 309 58 Amplicon 2 NDUFS7F2/R2 267 55 Amplicon 3 NDUFS7F3/R3 254 55 NDUFS2 Amplicon 1 NDUFS2F1/R1 298 58 Amplicon 2 NDUFS2F2/R2 357 58 Amplicon 3 NDUFS2F3/R3 388 60 Amplicon 4 NDUFS2F4/R4 376 54 Amplicon 5 NDUFS2F5/R5 346 55 NDUFS1 Amplicon 1 NDUFS1F1/R1 495 58 Amplicon 2 NDUFS1F2/R2 513 62 Amplicon 3 NDUFS1F3/R3 486 61 Amplicon 4 NDUFS1F4/R4 469 60 Amplicon 5 NDUFS1F5/R5 625 60

2.8 DENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS Denaturing Polyacrylamide gel electrophoresis with silver staining was used to visualise the double stranded DNA (PCR products). PCR products were run on polyacrylamide gels to check that DNA amplification was successful and to check that the PCR product was the expected size (Figure 2).

TAE Buffer (50x) was made by adding Tris-HCL 968.0g, Na2EDTA 74.4g, Glacial Acetic acid 270 ml and dH20 to a total volume of 4L. A 6% polyacrylamide gel was made by mixing 600 µl 50xTAE buffer (see below), 2.25 ml 40% Acrylamide solution

(19:1, acrylamide: bis-acrylamide), 12 ml dH2O, 20 µl TEMED and 100 µl 10% ammonium persulphate solution.

35 Two glass plates, separated by spacers, were assembled vertically with clamps, in a casting stand. The 6% polyacrylamide gel was immediately poured between the plates, and a comb with the appropriate number of wells was inserted. The gel was left for 30 minutes to polymerise. The comb was removed and the wells washed out with 1xTAE. One drop of loading buffer was mixed with 4 µl of PCR product and loaded into the gel wells with a micropipette. Molecular weight marker (pUC18, Hae III digest, Sigma, product no. D6293) was added to the first well. The electrophoresis tank was filled with 1xTAE and the polyacrylamide gel inserted. The gel was run for 45 minutes at 300V.

After electrophoresis, the DNA was silver stained. The gel was carefully removed from the glass plates and placed into a large plastic container with 200 ml of 10% ethanol for five minutes. This fixed the DNA in the gel. The ethanol was decanted and 200 ml of 1% nitric acid solution was added and left for three minutes. The nitric acid was discarded and the gel washed briefly in MilliQ water. 400 ml 10mM AgNO3 solution was then added and the gel left for 20 minutes on an agitator. After the silver was decanted, the gel was washed briefly in 200 ml 0.5% formaldehyde solution. It was then developed using 400 ml of 0.1M NaOH / 0.15% formaldehyde solution. The gel was swirled around in the developer until the desired signal strength was obtained. The developer was then decanted and 200 ml glacial acetic acid solution added for five minutes. The gel was then washed in 200 ml 10% ethanol for five minutes and then dried using gel drying frames and cellophane film. A permanent record of the gel was produced (Figure 2).

The gel was examined. The presence of extra bands was an indication to repeat the PCR and optimize the PCR conditions further. However, if the extra bands were very faint compared to the desired PCR product band, these samples were sometimes accepted for dHPLC screening. The presence on dHPLC of a single well defined peak at the expected retention time for amplicon size was a further indication that the desired PCR product was obtained.

36 Figure 2: Polyacrylamide gel electrophoresis with silver staining.

NDUFV1A5 NDUFV1A3 Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

MW 17 21 25 28 C4 b C4 C4 C4 C4 C4 C4 C4 b bp 587 458 434 298 267 257

174

102

80

Footnote: The PCR products are for NDUFV1 Amplicon 5(A5) and Amplicon 3 (A3). Lane 1 contains the Molecular Weight marker (MW) pUC18 Hae III digest which confirms that Amplicon 5 is 344 base pairs (bp) and Amplicon 3 is 333 bp in size. Lanes 3-6 contain patient PCR samples as indicated by the patient ID number. C4 indicates the Control 4 sample. The blank (b) is the PCR reaction where no DNA is added.

2.9 DENATURING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Denaturing high performance liquid chromatography (dHPLC) was the mutation screening method selected for this project, as described in detail below. Heteroduplexes were formed after PCR amplification, by heating and slow cooling of patient and normal control PCR products. The melting temperature for the PCR product was calculated and assessed. The amplicon was then analysed by dHPLC using the Varian Helix dHPLC system and mutations were identified by variations in retention time profiles.

Standford dHPLC Melt Program: Prior to running samples by dHPLC, the recommended dHPLC temperature was calculated using the Stanford dHPLC melt program (http://insertion.stanford.edu/melt.html) (Table 11). At this web site, the amplicon sequence was entered and the recommended dHPLC temperature(s) calculated using a previously published algorithm (Oefner and Underhill 1998, Jones et al. 1999). The melting temperatures are those at which the sites are closed in 50% of the fragment (Xiao and Oefner 2001). Depending on the nature of the amplicon sequence (length,

37 G/C content), one or several temperatures were recommended (Table 11). For some amplicons, two different dHPLC temperatures were recommended as different sections of DNA within the same amplicon had different melt profiles because of their sequence content. When a number of temperatures were recommended, dHPLC analysis was performed at the highest temperature. However, if there was a big difference (>3-40C) between two recommended temperatures, both temperatures were tested (Xiao and Oefner 2001). If the lower melt temperature only applied to a few base pairs at the beginning or end of the amplicon (sequence that would also be covered by analysis of adjacent amplicons) the lower temperature was not used.

Melt curves for each amplicon: The Stanford dHPLC melt program recommended temperature was used as a guideline to determine the best melt temperature for detecting mutations for each amplicon. However, the final optimised temperature was confirmed by melt curves for each amplicon, which can be seen in Appendix 2, Figures 18-23. The final temperatures used for dHPLC are listed in Table 11.

A control sample for each amplicon was run at a non denaturing dHPLC temperature of 50°C. Homoduplexes and heteroduplexes have the same chromatogram profile at this temperature and a single well defined peak is seen, at the retention time predicted by the amplicon size. The control sample was then run at a number of different temperatures, above and below the Stanford recommended temperature. Higher temperatures cause partial or complete denaturation and the DNA has a shorter retention time. The dHPLC temperature chosen for each amplicon was the partial denaturing temperature, where the first significant shift in the peak to the left of the 50°C curve, was seen. That is, the first significant reduction in retention time.

Heteroduplex DNA molecules denature more readily than homoduplexes, and hence have shorter retention times. The optimised dHPLC temperature is the temperature at which a heteroduplex is more denatured than a homoduplex, and consequently has a different chromatographic profile. As there were no positive controls available for study, the temperature was chosen empirically, based on the reduction in retention time, rather than on confirmation of mutation detection.

38 In most cases, the Stanford recommended melt temperatures were identical to the temperature chosen on the basis of the individual melt curves (Table 11). However, for several amplicons with higher melt temperatures (above 620C), a reduction in retention time was not seen at the Stanford recommended temperature. The temperature that caused the first significant left shift was one or two degrees higher than the Stanford recommended temperature (Table 11), as demonstrated by the melt curves for each amplicon (Appendix 2, Figures 18-23). For these amplicons, one temperature close to the Stanford recommended temperature was used, in addition to the optimum temperature suggested by the melt curve. Other investigators have also noted that the Stanford calculator underestimates the melt temperature for some amplicons. The reason for this is currently unknown (Xiao and Oefner 2001).

Table 11: dHPLC temperatures used for each amplicon

Gene Amplicon Stanford dHPLC recommended temperature dHPLC temp (°C) used (°C) NDUFV1 Amplicon 1 59, 64 64, 66 Amplicon 2 62 64, 65 Amplicon 3 63 63, 64 Amplicon 4 62 62, 64 Amplicon 5 53, 58, 63 65 NDUFS1 Amplicon 1 60 60 Amplicon 2 59 58.5, 59 Amplicon 3 60 59, 60 Amplicon 4 59 59 Amplicon 5 59 58.5, 59 NDUFS2 Amplicon 1 63 63 Amplicon 2 61 61 Amplicon 3 61 61 Amplicon 4 60 60 Amplicon 5 60 60 NDUFS4 Amplicon 1 57, 62 59, 62 Amplicon 2 58 58 NDUFS7 Amplicon 1 64 66, 66.5 Amplicon 2 +2B 64 66 Amplicon 3 59, 64 66.5 NDUFS8 Amplicon 1 63 64, 66 Amplicon 2 58, 63 64, 66.5 Amplicon 3 53, 58, 63 64, 65

39 Denaturation and Reannealing of PCR samples: Prior to the dHPLC run, all PCR samples underwent denaturation and slow re-annealing using the following thermocycler program; 95°C for three minutes, then two minutes at each of the following temperatures: 94°C, 92°C, 90°C, 88°C, 86°C, 84°C, 82°C, 80°C, 78°C, 76°C, 74°C, 72°C, 70°C, 68°C, 66°C, 64°C, 62°C and 60°C. The program was completed with 1 minute at 26°C. Ramp speed used 02.

Denaturing and slowly re annealing the DNA strands, enables DNA strands with a mutation to reanneal with wild type DNA strands (Figure 3). In this way a mismatch, or heteroduplex is created. For samples with heterozygous mutations, denaturation and reannealing generates four dsDNA species (two homoduplexes with perfectly matched DNA strands and two heteroduplexes with mismatched DNA sequences) Heteroduplexes can then be detected by dHPLC.

Figure 3: Heterozygous mutations identified by dHPLC. After PCR there are two double stranded DNA species, one with the normal DNA sequence (wild type) and one with the mutation. Denaturing and slowly re-annealing the PCR products generates four dsDNA species. Heteroduplexes are created, and can then be detected by dHPLC.

PCR Product

Mutant Heteroduplexes I I I I I I TA TC GA I I I I I I dHPLC and denature and and re anneal

Wild type Homoduplexes I I I I I I GC TA GC I I I I I I

However, with this method, homozygous mutations would not be detected by dHPLC. As the two DNA stands are identical, denaturation and re-annealing would not result in a mismatch. Therefore, all PCR samples were mixed with control (wild type) DNA specific for that amplicon. An equal amount of patient PCR product and control PCR

40 product were combined (10 µl of each). If the patient had a PCR product band on polyacrylamide gel that was weaker in strength than control PCR product bands, then 15 µl of patient DNA was mixed with 10 µl of control DNA. The mixed sample then underwent denaturation and slow re-annealing prior to dHPLC. This allows the detection of homozygous mutations by dHPLC.

For each amplicon, all samples were run by dHPLC twice; with and without the addition of control DNA. The samples could have been run only once (with the addition of control DNA) and both homozygous and heterozygous mutations should be detected. However, to ensure that heterozygous mutations were not missed because of over dilution with control DNA, the samples were run twice. On review of results however, this degree of dilution did not affect the detection of heterozygous mutations. dHPLC: Patient samples were run on the VARIAN Prostar Helix dHPLC system. This system runs 96 samples per plate, and each sample takes around 10 minutes. Three to five µl DNA (PCR product) is taken up by an autosampler and then carried onto and through a column by a high pressure flow of buffer (mobile phase). A stationary phase is contained in a column which is located within an oven. DNA is retained on the column through a hydrophobic interaction. An increasing gradient of organic solvent causes small DNA molecules and heteroduplexes to elute before larger molecules in a process called ion- pair reversed-phase liquid chromatography.

The Varian Helix column 75 x 3mm (product no. 392613001) used in this project produces a polymer-coated 3-micron wide pore alkylated silica based stationary phase. The mobile phase consists of two buffers. Buffer A is composed of 100mM TEAA (triethylammonium acetate), pH 7 and 0.1mM EDTA. Buffer B is composed of 100mM TEAA, pH 7, 0.1mM EDTA and 25%(v/v) acetonitrile. Buffer A ensures that that the DNA binds to the column, while Buffer B is used to elute the samples. The flow rate of buffers is kept constant, while the proportion of Buffer B increases over time.

The triethylammonium ion is amphiphilic (as one end of the molecule is hydrophilic and the other is hydrophobic) and the mobile phase also contains a small hydrophilic counter ion (acetate). (Xiao and Oefner 2001). The ion pairing agent in the buffer covers

41 the dsDNA in a hydrophobic layer and binds the DNA to the hydrophobic column media. The positively charged triethylammonium ions are adsorbed at the interface between the non polar, hydrophobic stationary phase and the hydroorganic mobile phase, resulting in a positive surface potential (Xiao and Oefner 2001). The DNA is retained on the column because of an interaction between this positive surface potential and the negative surface potential of DNA. This negative surface potential of DNA is generated by the dissociated phosphodiester groups of the sugar-phosphate backbone of DNA, making the outer surface of dsDNA highly hydrophilic. The number of hydrophobic ion pairs attached to the DNA is proportional to the length of the DNA, so dsDNA is retained according to length (Xiao and Oefner 2001). The elution of the dsDNA from the column is achieved by an increase in the concentration of organic solvent (acetonitrile) which causes the dsDNA and the amphiphilic ions to leave the column (Xiao and Oefner 2001). The DNA leaves the column in order of increasing length. As they leave the column, DNA species are detected by UV absorbency at 260nm. The detection of different DNA species generates a series of peaks resolved over time, called the chromatogram.

Validation of the Varian Helix dHPLC System: Two validation procedures were performed to monitor the quality of dHPLC. Firstly, a molecular weight marker, pUC18 HaeIII (Sigma Cat. No. 6293) was run at the beginning and end of every run to ensure that the column was working reliably. The chromatogram generated from pUC18 HaeIII is shown in Figure 4. The peaks, from left to right, represent increasingly larger fragments of double stranded DNA and the size of the fragment in base pairs (bp) is indicated. The 257 and 267 bp peaks make a doublet that is used to assess the resolution of the system. As the column gets towards the end of its useful life span, this doublet looses resolution, and a column change is needed.

The second validation procedure utilizes the DYS271 mutation standard (Varian part no. 393566301). At the dHPLC temperature of 550C, a specific heteroduplex chromatogram pattern is expected. The DYS271 mutation standard was assessed with each new column.

42 Figure 4: dHPLC chromatogram of the pUC HaeIII molecular weight marker

Footnote: the x axis shows the time in minutes, the y axis the mVolts. bp- base pairs.

Analysis of dHPLC results: The VARIAN Star Reviewer software package was used for dHPLC result analysis (Varian Australia Pty Ltd, Mulgrave, Victoria). dHPLC yielded large numbers of patient results for review. The analysis software package was able to group similar chromatographic profiles together. By doing this it separated heteroduplex samples from homoduplex samples in a two dimensional cluster map. Data points on the cluster map were also linked to normalised chromatographic profiles, allowing rapid comparison of all chromatograms within a group. The chromatogram groups were then labeled eg. homoduplex, heteroduplex. All chromatograms were subsequently reviewed individually, to ensure that they were appropriately labeled. All samples with an abnormal chromatographic profile by dHPLC were considered for DNA sequencing.

For each amplicon, there was a characteristic chromatographic profile obtained at the optimised temperature. For homoduplexes this was usually a single well defined peak. Mutation detection relied on finding a change in this chromatographic profile. At the

43 optimised temperature, heteroduplexes will begin to denature, while the more stable homoduplexes remain intact. Heteroduplexes eluted earlier, and were detected by a reduced retention time as well as by the presence of multiple peaks in the chromatographic profile.

Whether the heteroduplex produces one or two additional peaks depended on several factors. The sequence either side of the mismatch can influence the stability of the heteroduplex (Ke and Wartell 1993). For example, GC base pairs form three hydrogen bonds and so are more stable than AT base pairs that only form two hydrogen bonds. Also the hydrogen bonding between non Watson-Crick base pairs (eg. GA, GT) can influence the stability of the mismatch (Xiao and Oefner 2001). Another factor may be the composition of the denatured single stranded DNA, which is retained according to its base pair content rather than its length (Xiao and Oefner 2001).

If there is more than one mismatch in the amplicon, then more than two additional peaks may be seen (Xiao and Oefner 2001). If the mismatch is in an area of the amplicon that melts at a low temperature, then four peaks may be seen, as the homoduplexes have also started to denature. The four peaks represent the two homoduplex species and the two heteroduplex species. The order of elution of the different DNA species is determined by the degree of stability of each species.

As different mismatches have different degrees of instability, different mutations usually had different chromatographic profiles. However, sometimes different mutations will not be distinguished by dHPLC chromatographic profile (Xiao and Oefner 2001). Most mutations will be evident over a range of temperatures, 4-110C in one study (Jones et al. 1999). Again, however, some mutations will only be detected at a single temperature (Xiao and Oefner 2001) making temperature optimization critical for mutation detection.

Sensitivity and specificity for dHPLC consistently exceeds 96% and dHPLC can detect SNPs, small deletions and small insertions (Xiao and Oefner 2001).

44 2.10 PCR PRODUCT PURIFICATION

The QIAquick PCR purification kit (QIAGEN cat. No. 28180) was used to purify PCR products. The aim was to remove any primers, nucleotides, polymerase and salts from the PCR sample prior to sequencing. By adding this step, the sequencing results were of better quality with less background contamination. DNA was purified according to a protocol described by the manufacturer (QIAquick Spin Handbook, March 2001) and this protocol is summarised below. The method uses a column with a silca-gel membrane that selectively binds the DNA, while contaminants are removed.

50 µl of Buffer PB (a prepared buffer supplied with the purification kit) was added to 10 µl of PCR product and mixed. This buffer optimises the salt concentration and pH for absorption of DNA (larger than 100bp) onto the column membrane. The sample was then applied to the column provided, which was placed within a two ml collection tube. This tube was centrifuged for 60 seconds at 10,000 g and the flow-through was discarded. To wash contaminants from the column, 0.75 ml of BufferPE (a prepared buffer supplied by the purification kit to which ethanol was added) was applied to the column. It was centrifuged for 60 seconds at 10,000 x g. Flow-through was discarded and the sample spun for another 60 seconds at 10,000 g. The column was then placed in a clean 1.5 ml microcentrifuge tube. The DNA was eluted by addition of 30 µl of water to the centre of the column. The sample was left to stand for 1 minute and then centrifuged for one minute at 10,000 g. The eluted DNA sample was stored at 4oC.

2.11 DNA CYCLE SEQUENCING

Amplicons with abnormal profiles by dHPLC underwent direct DNA sequencing. All mutations were confirmed with sequencing in both directions. If a large number of samples in one amplicon were heteroduplexes, and the dHPLC chromatogram was identical, only some of the samples were selected for sequencing. A selection of samples with normal dHPLC profiles were also sequenced to provide comparison. If a mutation was found in one amplicon, then remaining amplicons may be sequenced to identify the second mutation. For NDUFS4, NDUFS7 and NDUFS8, Patients 1-10 were

45 routinely sequenced prior to the availability of dHPLC. Hence, for many amplicons there are samples sequenced that had normal dHPLC results.

ABI Prism Big Dye Terminator cycle sequencing ready reaction kit, supplied by Applied Biosystems (part number 4314417) was used for cDNA sequencing. The reaction products were analysed on the ABI Prism 310 DNA sequencer. The primers used for sequencing were the same as those used for PCR (Table 9). For NDUFS1, the amplicons were too long (469-625 base pairs) to sequence in one section. For these amplicons, additional primers were designed to sequence the PCR products in smaller sections. These primers are listed in Table 12. For the NDUFV1 gene, two mutations were identified that were either close to a primer, or in section of double sequence due to the presence of a normal splice variant. For this reason, new primers were designed and used for PCR and sequencing. These primers are also listed in Table 12.

Table 12: Additional primers used for PCR and sequencing

Gene Forward Primer 5'- 3' Reverse Primer 5'- 3' NDUFV1 NDUFV1F2M- NDUFV1R2M- GGCGAGATCAAGACATCGG CAGCAGCTTGTGAGGATCAT NDUFV1F4M- NDUFV1R4M- TGAGACGGTGCTGATGGACT ACAGGGAGTCGATCTCGGC NDUFV1F5M- AAGCCATCGCCCGCCTCATT NDUFS1 NDUFS1F1M- NDUFS1R1M- TCCCTCGATTCTGTTATCA ACAAGGCACATCCTGCAGT NDUFS1F2M- NDUFS1R2M- GCAAGTTGGCACATACATTG CCAGACAGTTCAGACATGAAC NDUFS1F3M- NDUFS1R3M- CTTGGTGGATGCTGAAGCC GGTGTCAGAGTCCACTCTAT NDUFS1F4M- NDUFS1R4M- TAGGCAGTTCTGCACTCCA GCAAGAATTGCTGCTCCATC NDUFS1F5M- NDUFS1R5M- ACCTCCTGGCTTGGCAAGA CCTTACTTGATCCAGAGTATC

Sequencing reaction: The fluorescent tags used in the sequencing reaction are light sensitive, and so exposure to the light was minimised. The sequencing reaction mix was made using the following ingredients (Table 13).

46 Table 13: Sequencing reaction mix

Reagent Volume per reaction sterile water 7.7 µl terminator ready mix 1 µl *primer (10 pmol/µl) 0.3 µl aliquot 9 µl PCR product 1 µl Footnote: * forward or reverse primer

Nine µl of sequencing mix was dispensed into a PCR tube and 1 µl of purified PCR product was added. One drop of paraffin oil was applied (to minimise evaporation of sample) and the tube was sealed. The sequencing reaction used the following thermocycler program (Table 14).

Table 14: thermocycler program for the sequencing reaction

Step Cycle 1 Cycle 2 Cycle 3 1 940C for 0.10 940C for 0.20 260C 0.05 2 500C for 0.05 3 600C for 4.00 Repeats 1 25 1

Removal of unincorporated dye terminators: For each sequencing reaction a 1.5ml microcentrifuge tube was prepared containing 1 µl 3M sodium acetate (pH 4.6) and 20 µl 95% Ethanol. Using a pipette the entire sequencing reaction (excluding the oil) was aspirated and then mixed thoroughly with the ethanol and sodium acetate. The sample was then vortexed and left at room temperature (protected from light) for one hour, to allow precipitation. The tube was then spun in a microcentrifuge for 20 minutes at maximum speed (13,500rpm). The supernatant (containing the unincorporated dye terminators) was then aspirated and discarded. The DNA pellet was rinsed by adding 100 µl of 70% ethanol, followed by a brief vortex. The sample was then centrifuged at maximum speed for five minutes and the supernatant carefully aspirated and discarded. The pellet was then dried in a vacuum centrifuge at 60°C for 10-15 minutes.

Sequencing: 15 µl of highly deionised formamide (Hi Di Formimide, ABI 4311320, Applied Biosystems, California, USA) was added to the dried DNA pellet and the sample was vortexed to resuspend the DNA. The sample was transferred to a 0.2 ml

47 PCR reaction strip tube and denatured at 950C for three minutes, then chilled on ice. The sample was then stored, protected from light. The sample was run on the ABI Prism 310 sequencer. Sequencing data was viewed using the ABI Prism 310 sequence analysis software. All sequencing was printed onto paper and analysis for mutations was performed manually, by comparison to known reference sequences from Genbank for all six genes.

For each mutation identified in this study, at least fifty anonymous controls were sequenced. The sequencing of these controls was performed in Dr David Thorburn’s laboratory in Melbourne, by a scientist under his supervision. In addition, the genomic DNA sequencing of family members of children with identified mutations was also performed in this laboratory, as was genomic sequencing of the NDUFS2 and NDUFS4 gene in patients 17 and 26 respectively.

48 3. RESULTS

3.1 SUMMARY OF MUTATIONS IDENTIFIED

Eight different mutations in complex I subunit genes were identified (Table 15). Mutations were found in six of the 34 (18%) patients with confirmed complex I deficiency. Both mutations were identified in five patients and a single mutation was identified in one patient. All mutations were novel. One mutation in NDUFV1 (R386H) was identified in two apparently unrelated patients. The NDUFS4 mutation c.221delC was identified in two Lebanese patients. Mutations were found in three of the six genes studied; NDUFV1 NDUFS2 and NDUFS4. Two patients are compound heterozygotes for mutations in NDUFV1. Two patients are compound heterozygotes for NDUFS2 mutations. One patient is homozygous for an NDUFS4 mutation, while another is heterozygous for this same mutation (Table 15).

The dHPLC results and sequencing results that led to these findings are described in detail below. Samples from patients 1-37 are identified by their Patient number. Patient samples 1-38 and 29-37 often underwent dHPLC on different runs as samples 29-37 were not available at the beginning of the project.

Table 15: Complex I subunit gene mutations

Gene Patient Id Mutation 1 Mutation 2 NDUFV1 Patient 13 c.331A>G, K111E c.1157G>A, R386H (exon 4) heterozygous (exon 8) heterozygous Patient 27 c.755C>G, P252R c.1157G>A, R386H (exon 6) heterozygous (exon 8) heterozygous NDUFS2 Patient 17 IVS9+4A>G (intron 9)1 c.875T>C, M292T heterozygous (exon 10) heterozygous Patient 25 c.661C>T, R221X c.998G>A, R333Q (exon 7) heterozygous (exon 11) heterozygous NDUFS4 Patient 262 c.221delC c.221delC (exon3) homozygous (exon3) homozygous Patient 19 c.221delC not found (exon 3) heterozygous Footnote: 1 this mutation identified in gDNA. In cDNA this allele was absent presumably due to nonsense mediated mRNA decay. 2this patient also has a NDUFS4 splice variant identified as described below.

49 3.2 dHPLC RESULTS

3.2.1 NDUFV1 dHPLC results The results of dHPLC analysis of NDUFV1 are summarized in Table 16. Amplicon 1 was analysed at two temperatures, 64°C and 66°C, with and without wild type DNA. All samples were homoduplexes. Amplicon 2 was analysed at two temperatures, 64°C and 65°C, with and without wild type DNA. Samples 13 and 15 were heteroduplexes at both temperatures, with and without the addition of wild type DNA. These two samples had different heteroduplex profiles, suggesting different mutations (Appendix 4, Figure 32). Samples 11 and 20 were homoduplexes that were slightly broader than other homoduplexes. Amplicon 3 was analysed at two temperatures, 63°C and 64°C, with and without wild type DNA. Sample 27 was a heteroduplex and all other samples homoduplexes (Appendix 4, Figure 33).

Amplicon 4 was analysed at two temperatures, 62°C and 64°C, with and without wild type DNA. Samples 13 and 27 were heteroduplexes with exactly the same chromatographic profile at both temperatures tested (Appendix 4, Figure 31). These two samples were clearly different from all other samples. The analysis of other samples was complicated by the presence of two additional peaks immediately prior to the main peak (Appendix 4, Figure 31). The height of these additional peaks varied and accounted for the different classifications of samples as homoduplexes (additional peaks were very low amplitude or absent), possible homoduplexes (the additional peaks were of moderate height) or possible heteroduplexes (additional peaks were high) (Table 16). There was a continuum between these three categories with no definite distinction between the three groups. The finding of a splice variant in these samples explains these findings and this is discussed further in the context of the sequencing results for NDUFV1.

Amplicon 5 was analysed at 65°C, with and without wild type DNA. Samples 13 and 27 which were heteroduplexes in amplicon 4 were also heteroduplexes with identical profiles in amplicon 5. The mutation identified in both Patient 13 and 27 is in the area of overlap between amplicons 4 and 5. All other samples were homoduplexes.

50 Table 16: NDUFV1 dHPLC results

NDUFV1 dHPLC Samples run Patients with similar dHPLC profile Amplicon temp dHPLC profiles (Patient ID ) 1 64 1-37 +/-w all patients homoduplex 66 1-37 +w all patients homoduplex 2 64 1-37 -w 13 heteroduplex 1 15 heteroduplex 2 all other patients homoduplex 1-37 +w 13 ?heteroduplex 15 heteroduplex 1 11, 20 ?homoduplex all other patients homoduplex 65 1-28 +/-w 13 heteroduplex 1 15 heteroduplex 2 all other patients homoduplex 3 63 1-28 +w 27 ?homoduplex all other patients homoduplex 29-37 +/-w all patients homoduplex 64 1-28 -w 27 heteroduplex 1 all other patients homoduplex 29-37 +/-w all other patients homoduplex 4 62 1-37 +w 13, 27 heteroduplex 1 12, 28 ?homoduplex 8, 9, 15, 16, 17, 21, 22, ?heteroduplex 24, 25, 29 all other patients homoduplex 1-37 -w 13, 27 heteroduplex 1 8, 9, 15, 24, 25, 29 ?heteroduplex 2, 5, 16, 17, 19, 20, 21, ?homoduplex 22, 26, 34, 36, 37 all other patients homoduplex 64 1-37 -w 13, 27 heteroduplex 1 5, 8, 9, 15, 16, 21, 22, ?homoduplex 24, 25, 26, 29, 36, 37 all other patients homoduplex 1-37 +w 13, 27 heteroduplex 1 8, 9, 15, 16, 21, 22, 24, ?homoduplex 25, 29 all other patients homoduplex 5 65 1-37+/ -w 13, 27 heteroduplex 1 all other patients homoduplex

Footnote: The distinction between heteroduplex 1, 2 or 3 only applies to that particular dHPLC run at that temperature and distinguishes different types of heteroduplex profiles. ?homoduplex: similar dHPLC profile to homoduplexes, but some minor difference in shape, retention time or amplitude. ?heteroduplex: not a definite multipeak heteroduplex, but some heteroduplex characteristics such as reduced retention time or broader curve. C5: Control 5, w: wild type DNA, +/- w: samples run both with and without wild type DNA.

51 3.2.2 NDUFS1 dHPLC results The results of dHPLC analysis of NDUFS1 are summarised in Table 17. Amplicon 1 was analysed at 60°C. Four patient samples (18, 30, 32 and 36) had the same heteroduplex profile, with and without addition of wild type DNA. The remaining samples were homoduplexes.

Amplicon 2 was analysed by dHPLC at two temperatures, 58.5°C and 59°C. The same four samples (18, 30, 32 and 36) that were heteroduplexes in amplicon 1 were also heteroduplexes in amplicon 2, as a polymorphism was present in the area of overlap between amplicon 1 and 2 (see sequencing results NDUFS1). The remaining results for amplicon 2 were complicated as all samples had broad multi-peak profiles and this made interpretation difficult. Samples 1, 11, 12, 14, 23, 28, 3, 7 and Control 5 were homoduplexes with a less prominent third peak on the left shoulder of the curve. Samples 2, 4, 6, 8 and 29 had an extra shoulder or indentation, on the left of the main peak, though again they were very similar to homoduplex curves. At 59°C with the addition of wild type DNA, samples 1, 8, 29, 33 and 35 were homoduplexes but they had a more prominent indentation on the left side of the main peak. None of these minor differences were associated with a sequence variant (see sequencing results NDUFS1).

Amplicon 3 was analysed at 59°C and 60°C. Results were complicated by the presence of two polymorphisms in this amplicon (c.966T>G and c.1251A>G), as described in the sequencing results. At 59°C (without addition of wild type DNA) samples 1, 4, 6, 9, 13, 14, 15, 24, 28, 33, 34 and 35 were heteroduplexes with identical chromatographic patterns. Samples 17, 21, 29 and 32 were also heteroduplexes with their own specific profile. Samples 27 and 30 had a different heteroduplex profile again. The remaining samples were homoduplexes.

With the addition of wild type DNA (Control 5), all samples were heteroduplexes as control 5 was itself a heteroduplex. Samples 2, 3, 5, 7, 8, 12, 19, 20, 23, 26 and 31 were heteroduplexes of the same type. Samples C5, 1, 4, 6, 9, 10, 11, 13, 14, 15, 16, 17, 18, 21, 22, 24, 25, 27, 28, 29, 30, 32, 33, 34, 35, 36 and 37 were heteroduplexes of the same type.

52 At 60°C, amplicon 3 was assessed with the addition of a different wild type DNA (Control 2), which had a homoduplex profile. This was an attempt to clarify dHPLC results which were all heteroduplexes when the heteroduplex control (C5) was added to each of the patient samples. With addition of wild type DNA (C2), samples 1, 4, 6, 9, 14, 28, 34, and 35 were heteroduplexes. Samples 21, 29, 32 and 36 became heteroduplexes, having been homoduplexes without the addition of wild type DNA. Samples 27 and 30 were again heteroduplexes with the same distinct pattern. Samples 13, 15, 24 and 33 were possible heteroduplexes. Sample 31 was a homoduplex without the addition of wild type DNA, but with the addition of control 2 DNA this sample became a heteroduplex, quite different from other heteroduplexes for this amplicon.

Amplicon 4 was assessed at 59°C. The polymorphism c.1251A>G is in the area of overlap between amplicon 3 and 4 and explains the large number of heteroduplexes identified in amplicon 4 (see sequencing results). Without wild type DNA, fifteen patient samples (1, 4, 6, 9, 13, 14, 15, 16, 24, 27, 28, 30, 33, 34, 35, and C5) were heteroduplexes with the same dHPLC chromatographic profile. The remaining samples were homoduplexes, though the curves for samples 2, 3, 5, 8 and 10 had a slightly more prominent left shoulder. Wild type DNA (control 5) was itself a heteroduplex for this amplicon. When mixed with the patient samples, all samples were heteroduplexes. Therefore, the samples were run again with the addition of a different wild type DNA (control 2), which was a homoduplex for amplicon 4. With this, the same 15 patient samples were heteroduplexes, though they were now divided into a number of groups according to heteroduplex profile. Samples 1, 4, 6, 9 13, 14 and 15 showed a similar heteroduplex profile, that was different to the heteroduplex profile seen in samples 24, 27, 28, 30, 34 and 35. Sample 33 had a different profile to all other heteroduplexes. Sample 16 was probably over diluted with wild type DNA as it now looked like a homoduplex. Sample 31, which was a homoduplex without addition of wild type DNA, now became a heteroduplex with its own distinctive profile. The remaining samples were homoduplexes.

Amplicon 5 was analysed at 59°C, with and without the addition of wild type DNA. All samples were homoduplexes, though sample 15 had a slightly broader curve.

53 Table 17: NDUFS1 dHPLC results NDUFS1 dHPLC Samples Patients with similar dHPLC dHPLC profile Amplicon temp run profiles (Patient ID ) 1 60 1-37+/-w 18, 30, 32, 36 heteroduplex all other samples homoduplex 2 58.5 1-37-w 18, 30, 32, 36 heteroduplex 2, 4, 6, 8, 29 ?heteroduplex 1, 3, 7, 11, 12, 14, 23, 28, C5 homoduplex 1 all other samples homoduplex 2 1-37+w 18, 30, 32, 36 heteroduplex 8 ?homoduplex all other samples homoduplex 59 1-37+w (C5) 18, 30, 32, 36 heteroduplex 1, 8, 29, 33, 35 ?homoduplex all other samples homoduplex 3 59 1-37-w 1, 4, 6, 9, 13, 14, 15, 24, 28, 33, heteroduplex 1 34, 35 17, 21, 29, 32 heteroduplex 2 27, 30 heteroduplex 3 all other samples homoduplex 1-37+w (C5) 2, 3, 5, 7, 8, 12, 19, 20, 23, 26,31 heteroduplex 1 C5, 1, 4, 6, 9, 10, 11, 13, 14, 15, heteroduplex 2 16, 17, 18, 21, 22, 24, 25, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37 60 1-37+w (C2) 1, 4, 6, 9, 14, 28, 34, 35 heteroduplex 1 21, 29, 32, 36 heteroduplex 2 27, 30 heteroduplex 3 13, 15, 24, 33 ?heteroduplex 31 heteroduplex 4 (homoduplex -w) all other samples and C2 homoduplex 4 59 1-37-w 1, 4, 6, 9, 13, 14, 15, 16, 24, 27, heteroduplex 2 28, 30, 33, 34, 35, C5 (all the same) 2, 3, 5, 8, 10 ?homoduplex all other samples homoduplex 1-37+w (C2) 1, 4, 6, 9, 13, 14, 15 heteroduplex 1 24, 27, 28, 30, 31,34, 35 heteroduplex 2 31 heteroduplex (homoduplex -w) 16 homoduplex 33 heteroduplex 3 all other samples homoduplex 1-37+w (C5) all samples heteroduplex 5 59 1-37+/-w 15 ?homoduplex all other samples homoduplex

Footnote: The distinction between a heteroduplex 1, 2, 3 or 4 or a homoduplex 1, 2 or 3 only applies to that particular dHPLC run at that temperature and distinguishes different chromatographic profiles. ?homoduplex: similar dHPLC profile to homoduplexes, but some minor difference in shape, retention time or amplitude. ?heteroduplex: not a definite multipeak heteroduplex, but some heteroduplex characteristics such as reduced retention time or broader curve. C5: Control 5, C2: Control 2, w: wild type DNA, +/- w: samples run both with and without wild type DNA.

54 3.2.3 NDUFS2 dHPLC results

The results of dHPLC for all five amplicons of NDUFS2 are summarised in Table 18. For amplicon 1 and 2 all samples were homoduplexes. For amplicon 3, Patient 25 had an abnormal dHPLC profile, with and without the addition of wild type DNA. Without wild type DNA, the dHPLC chromatogram was a definite heteroduplex (Appendix 4, Figure 34). In amplicon 3, without addition of wild type DNA, five patients (5, 13, 16, 33 and 34) had a slightly different dHPLC profile with a more prominent shoulder indentation in the left side of the peak. However the profile was very similar to the homoduplexes for this amplicon and subsequent sequencing did not detect any abnormalities in these samples.

For amplicon 4, Patient 17 was a heteroduplex only with the addition of wild type DNA indicating the presence of a homozygous mutation (Appendix 4, Figure 35). Four other patients (3, 14, 18, 22) as well as control 5, were heteroduplexes with and without the addition of wild type DNA (Appendix 4, Figure 35). All five samples had the same dHPLC chromatographic pattern, and the same SNP was identified by sequencing. As Control 5 was used when wild type DNA was added, all samples became heteroduplexes. Sample 17 still had a different pattern to all other samples tested.

In amplicon 5, samples 19 and 20 had a slightly increased retention time and the chromatographic peaks were broader and of lower amplitude. Sample 33 had a low amplitude abnormal peak.

55 Table 18: NDUFS2 dHPLC results

NDUFS2 dHPLC Samples run Patients with dHPLC profile Amplicon temp similar dHPLC profiles (Patient ID ) 1 63 1-37, C5 +/-w all patients homoduplex 2 61 1-37, C5 +/-w all patients homoduplex 3 61 1-37, C5 +/-w 25 heteroduplex (-w), ?homoduplex (+w) 5, 13, 16, 33, 34 ?homoduplex (-w), homoduplex (+w) all other patients homoduplex 4 60 1-37, C5 +/-w 17 homoduplex (-w), heteroduplex 1 (+w) C5, 3, 14, 18, 22 heteroduplex 2 (+/-w) (all the same) all other patients homoduplex (-w) 5 60 1-37, C5 +/-w 19, 20 increased retention time, broad peak 33 low amplitude, abnormal peak all other patients homoduplex Footnote: ?homoduplex: similar dHPLC profile to homoduplexes, but some minor difference in shape, retention time or amplitude. ?heteroduplex: not a definite multipeak heteroduplex, but some heteroduplex characteristics such as reduced retention time or broader curve. C5: Control 5, C2: Control 2, w: wild type DNA, +/- w: samples run both with and without wild type DNA.

3.2.4 NDUFS4 dHPLC results The results of dHPLC analysis of NDUFS4 are summarized in Table 19 below. At 59°C, six samples (4, 19, 22, 23, 26 and 33) were heteroduplexes (Appendix 4, Figure 36) though they were not all identical, suggesting that different sequence variants may be present. Samples 4, 23 and 33 had the same chromatographic profile, but 19, 22 and 26 were all different. All other samples were homoduplexes. At 62°C, the results were less clear. Patients 19 and 26 were still heteroduplexes. Samples 22 and 33 were still abnormal with broad peaks, though not multiple peaks. Patients 4 and 23 were now assessed as homoduplexes. Samples 2, 8, 9, 13, 15, 16, 21, 24, 27, 30, 34, 35, and 36 were homoduplexes with a slightly broader chromatographic peak.

Amplicon 2 of NDUFS4 was analysed at the dHPLC temperature of 58°C. All samples were classified as heteroduplexes, as the peaks were very broad and bifid. This was likely to be due to extensive DNA denaturation at 58°C, rather than due to the presence of a mutation or polymorphism. With and without the addition of wild type DNA, samples 1, 4, 12, 14, 18, 19, 21, 23, 25, 27, 28 and 33 had a different heteroduplex

56 profile when compared to the other patients and the control. Samples 11, 13, and 32 also had a different heteroduplex pattern, but only when wild type DNA was added.

Table 19: NDUFS4 dHPLC results

NDUFS4 dHPLC Samples run Patients with similar dHPLC profile Amplicon temp dHPLC profiles (Patient ID ) 1 59 1-37, C5, +/-w 19, 26 heteroduplex 1 4, 23, 33 heteroduplex 2 22 heteroduplex 3 all other patients, C5 homoduplex 62 1-37, C5 -w 22, 26 ?heteroduplex 33 ?heteroduplex 19 heteroduplex 1 2, 8, 9, 13, 15, 16, 21, 24, ?homoduplex 27, 30, 34, 35, 36 all other patients, C5 homoduplex 1-37, C5 +w 26 heteroduplex 1 8, 9, 22 ?homoduplex 19 ?heteroduplex all other patients, C5 homoduplex 2 58 1-37, C5 -w 1, 4, 12, 14, 18, 19, 21, heteroduplex 2 23, 25, 27, 28, 33 all other patients, C5 heteroduplex 1 1-37, C5 +w 1, 4, 12, 14, 18, 19, 21, heteroduplex 2 23, 25, 27, 28, 33 C5 heteroduplex 4 11, 13, 32 heteroduplex 3 all other patients heteroduplex 1 Footnote: The distinction between a heteroduplex 1, 2, 3 or 4 only applies to that particular dHPLC run at that temperature and distinguishes different types of heterduplex profiles. ?homoduplex: similar dHPLC profile to homoduplexes, but some minor difference in shape, retention time or amplitude.?heteroduplex: not a definite multipeak heteroduplex, but some heteroduplex characteristics such as reduced retention time or broader curve. C5: Control 5, w: wild type DNA, +/- w: samples run both with and without wild type DNA.

3.2.5 NDUFS7 dHPLC results The results of dHPLC analysis of NDUFS7 are summarized in Table 20. Amplicon 1 was analyzed at 66°C and 66.5°C. All samples were classified as homoduplexes, though samples C5, 1, 2, 5, 6, 11, 17, 18, 19, 21, 22, 24, 30, 34, 35 and 36 had a different homoduplex profile with a broader curve. When mixed with control DNA (C5) all samples became heteroduplexes, with the same pattern. Amplicon 2 was analyzed twice, using two different sets of primers. All patient samples were homoduplexes. For amplicon 3 all samples were homoduplexes when analysed at 66.5°C. However, Sample 8 had a different profile with a high prominent extra shoulder.

57 Table 20: NDUFS7 dHPLC results

NDUFS7 dHPLC Samples Patients with dHPLC profile Amplicon temp run similar dHPLC profiles (Patient ID ) 1 66 1-37 -w C5, 1, 2, 5, 6, 11, 17, ?homoduplex 18, 19, 21, 22, 24, 30, 34, 35, 36 all other patients homoduplex 1-37 +w all patients homoduplex 66.5 1-37 +w 2 ?homoduplex all other patients homoduplex 2* 66 1-37+/-w all patients homoduplex 3 66.5 1-37 +/-w 8 ?homoduplex all other patients homoduplex Footnote: ?homoduplex: similar dHPLC profile to homoduplexes, but some minor difference in shape, retention time or amplitude. C5: Control 5, w: wild type DNA,+/- w: samples run both with and without wild type DNA. NDUFS7 F2B/R2B are primers described by Benit et al. (2001). *Amplicon 2 was analysed with two sets of primers (NDUFS7 F2/R2 and F2B/R2B).

3.2.6 NDUFS8 dHPLC results The results of dHPLC analysis of NDUFS8 are summarised in Table 21. Amplicon 1 was analysed at two temperatures, 64°C and 66°C, with and without wild type DNA. All samples were homoduplexes. Sample 1 was a homoduplex with a slightly different shaped curve. Amplicon 2 was analysed at two temperatures, 64°C and either 66°C or 66.5°C. The different higher temperature (66°C or 66.5°C) was used on different runs as the samples were run on different days and the melt curve indicated a slightly different optimal temperature on each of these days. All samples were homoduplexes.

Amplicon 3 was analysed at two temperatures, 64°C and 65°C, with and without wild type DNA. Without wild type DNA, nine samples (3, 4, 9, 12, 18, 19, 21, 27, 31) were heteroduplexes and the remaining patient samples were homoduplexes. The controls C3 and C5 were a different type of heteroduplex from the patient samples. When mixed with wild type DNA (C3 or C5) all samples were heteroduplexes. However, the chromatographic profile was different between those samples that were homoduplexes and the nine heteroduplex samples 3, 4, 9, 12, 16, 18, 19, 21, 27 and 31. At 64°C, sample 16 became a heteroduplex (like samples 3, 4, 9, 12, 16, 18, 19, 21, 27 and 31) with the addition of wild type DNA, suggesting a homozygous DNA change. At 65°C, this change to a heteroduplex was not seen when wild type DNA was added.

58

Table 21: NDUFS8 dHPLC results

NDUFS8 dHPLC Samples Patients with similar dHPLC profile Amplicon temp run dHPLC profiles (Patient ID ) 1 64 1-37 -w 1 ?homoduplex all other patients homoduplex 1-37 +w all patients homoduplex 66 1-37 +/-w all patients homoduplex 2 64 1-37 +/ -w all patients homoduplex 66 29-37+/-w all patients homoduplex 66.5 1-28 +w all patients homoduplex 3 64 1-37 -w 3, 4, 9, 18, 19, 31 heteroduplex 1 12, 21, 27 ?heteroduplex Controls (C3, C5) heteroduplex 2 all other patients homoduplex 1-37 +w 9, 12, 16, 18, 19, 21, 27, 31 heteroduplex 2

(C3 for 1- 3, 4 heteroduplex 3

28, C5 for C3, C5 heteroduplex 4 29-37) Remaining samples heteroduplex 1 65 1-37 -w 3, 4, 9, 12, 18, 19, 21, 27, 31 heteroduplex 2 C3 heteroduplex 3 1-37 +w (C3 3, 4, 9, 12, 18, 19, 21, 27, 31 heteroduplex 2 for 1-28, C5 C5 heteroduplex 3 for 29-37) Remaining samples heteroduplex 1 Footnote: The distinction between a heteroduplex 1, 2, 3 or 4 only applies to that particular dHPLC run at that temperature and distinguishes different types of heteroduplex profiles. ?homoduplex: similar dHPLC profile to homoduplexes, but some minor difference in shape, retention time or amplitude.?heteroduplex: not a definite multipeak heteroduplex, but some heteroduplex characteristics such as reduced retention time or broader curve. C3: control 3, C5: control 5, w: wild type DNA,+/- w: samples run both with and without wild type DNA.

59 3.3 SEQUENCING RESULTS

3.3.1 NDUFV1 sequencing results The sequencing results for NDUFV1 are summarized in Table 22 and described in more detail below.

Table 22: NDUFV1 sequencing results

NDUFV1 dHPLC All samples Sequencing result Comment Amplicon abnormals sequenced (similar dHPLC profiles grouped together) A2 13 13 c.331A>G, K111E missense mutation 15 15 c.439C>T, R147W polymorphism c.549 C>G, G183G polymorphism 11, 20, C5 11, 20 no mutation A3 27 27 c.755C>G, P252R missense mutation 17 no mutation A4 13, 27 13, 27 c.1157G>A, R386H missense mutation 2, 5, 8, 9, 12, 15, 15 c.1056T>C, A352A polymorphism 16, 17, 19, 20, 21, 8, 12, 16, no mutation / normal splice 22, 24, 25, 26, 28, 17, 21, 22, polymorphism variant present 29, 34, 36, 37 25, 36 A5 13, 27 13, 27 c.1157G>A, R386H missense mutation

NDUFV1 mutations

Patient 13: Two missense mutations in NDUFV1 were identified in Patient 13. The first mutation in Patient 13 was identified by dHPLC screening of amplicon 2 and confirmed on sequencing. The mutation is an A>G change at position 331 of coding DNA, in exon 4. This mutation leads to an amino acid change in codon 111 from lysine to glutamate (K111E). Both amino acids are large polar amino acids. The mutation was confirmed by sequencing in both directions. As the mutation was very close to the primer NDUFV1 F2, the mutation was not seen clearly with sequencing using this primer. The sequencing was repeated using specifically designed primers NDUFV1 F2M and NDUFV1 R2M (Appendix 3, Figure 25). This mutation was not seen in 100 control chromosomes.

The Lysine at position 111 of human NDUFV1 is conserved across a number of species and is the fifth of 24 consecutive conserved amino acids in this position of the

60 homologous genes of M. musculus, B. taurus, X. laevis, D. melanogaster, and C. elegans. It is not conserved in E. coli or S. pombe, though they have the similar amino acid arginine in this position (Figure 6).

The second mutation in Patient 13 was identified by an abnormal dHPLC profile in both amplicons 4 and 5 as the mutation is in the area of overlap between the two amplicons. The mutation is a G>A substitution at position 1157 of the coding DNA, in exon 8. This mutation leads to an amino acid change from an arginine to a histidine at codon 386 (R386H). Arginine is a large polar amino acid and histidine is of intermediate polarity. The mutation was confirmed by sequencing in both directions and was seen most clearly with sequencing primers NDUFV1 R5 and NDUFV1 F5M (Appendix 3, Figure 24). This mutation was not present in 104 control chromosomes. This arginine at position 386 of human NDUFV1 is highly conserved and is the fifth of five consecutive conserved amino acids (Figure 6). It is located within the consensus motif for the iron sulfur binding site, E-S-C-G-x-C-x-P-C-R-x-G (PROSITE PS00645, http://www.expasy.org/prosite) (Figure 5) where the three side chains of the cysteine residues are putative ligands for the tetra iron-sulfur complex (Schuelke et al. 1998). The R386H mutation may interrupt normal binding of the iron-sulfur complex. Parental samples were not available.

Patient 27: Two missense mutations in NDUFV1 were identified by dHPLC screening and confirmed with sequencing in Patient 27. The first mutation is the G>A substitution at position 1157 of coding that changes the amino acid at codon 386 from an arginine to a histidine (R386H). This is the same missense mutation that was found in patient 13. This mutation was not seen in 104 control chromosomes and was heterozygous in Patient 27 and his father.

The second mutation in Patient 27 was detected by dHPLC screening of amplicon 3. The mutation, a C>G change at position 755 of coding (exon 6) leads to a change in the amino acid at codon 252 from a small non polar proline to a large polar arginine (P252R). Proline normally causes a kink in the protein structure and this would be lost when another amino acid is substituted. The mutation was confirmed by sequencing in

61 both directions (Appendix 3, Figure 26). The mutation was found to be heterozygous in the patient’s mother and was not present in 102 control chromosomes.

The proline at position 252 of human NDUFV1 is highly conserved (Figure 6). It is the first of eight consecutive amino acids fully conserved in the homologous proteins of M. musculus, B. taurus, X. laevis, D. melanogaster, and C. elegans. It is also conserved in E. coli, S. pombe and A. thalania.

Figure 5: The NDUFV1 amino acid sequence.

MLATRRLLGWSLPARVSVRFSGDTTAPKKTSFGSLKDEDRIFTNLYGRHDWRLKGSL K111E SRGDWYKTKEILLKGPDWILGEIKTSGLRGRGGAGFPTGLKWSFMNKPSDGRPKYLV R147W VNADEGEPGTCKDREILRHDPHKLLEGCLVGGRAMGARAAYIYIRGEFYNEASNLQV

AIREAYEAGLIGKNACGSGYDFDVFVVRGAGAYICGEETALIESIEGKQGKPRLKPPFP P252R ADVGVFGCPTTVANVETVAVSPTICRRGGTWFAGFGRERNSGTKLFNISGHVNHPCT

VEEEMSVPLKELIEKHAGGVTGGWDNLLAVIPGGSSTPLIPKSVCETVLMDFDALVQA R386H QTGLGTAAVIVMDRSTDIVKAIARLIEFYKHESCGQCTPCREGVDWMNKVMARFVRG

DARPAEIDSLWEISKQIEGHTICALGDGAAWPVQGLIRHFRPELEERMQRFAQQHQAR

QAAS

Footnote: The location of the putative binding sites, mitochondrial import sequence and mutations identified are indicated (modified in part from Schuelke et al. 1998) MLATRRLLGWSLPARVSVRF is the mitochondrial import sequence. The mutations identified in this study are bold face, shown above the normal sequence. GRGGAGFPTGLKWSFMNKPSD indicates the NADH binding site, with invariant amino acids highlighted. GAGAYICGEETALIES represents the putative FMN binding site, with invariant amino acids highlighted. G-[AM]-G-[AR]-Y-[LIVM]-C-G-[DE](2)-[STA](2)-[LIM](2)-[EN]- S (PROSITE PS006444) is the consensus sequence for this binding site. ESCGQCTPCREG indicates the iron sulphur binding site.

62 Figure 6: Conservation of NDUFV1 amino acids. Mutations and polymorphisms are indicated.

K111E R147W

Homo sapiens KWSFMNKP---SDGRPK--YLVVNADEGEPGTCKDREILRHDPHKLLEGCLVGGRAPTGLKWS 155 B. taurus KWSFMNKP---SDGRPK--YLVVNADEGEPGTCKDREIIRHDPHKLVEGCLVGGRAPTGLKWS 155 M. musculus KWSFMNKP---SDGRPK--YLVVNADEGEPGTCKDREIMRHDPHKLVEGCLVGGRAPTGLKWS 155 X. laevis KWSFMNKP---SDGRPK--YLVVNADEGEPGTCKDREIMRHDPHKLVEGCLVAGRSPTGLKWS 161 D. melanogaster KWSFMNKP---GDGRPK--YLVVNADEGEPGTCKDREIMRHDPHKLVEGCLIAGRAPSGMKWS 170 A gambiae KWSFMNKP---SDGRPK--YLVVNADEGEPGTCKDREIMRHDPHKLIEGCLIAGRAPTGMKWS 182 C elegans KWGFMNKP---FDGRPK--YLVVNADEGEPGTCKDREIMRHDPHKLIEGCLIGGVAPSGMKWG 166 N. crassa KWSFMNFKDWDKDDKPR--YLVVNADEGEPGTCKDREIMRKDPHKLVEGCLVAGRAPSGLKWS 167 E. coli KWSLMPKD---ESMNIR--YLLCNADEMEPGTYKDRLLMEQLPHLLVEGMLISAFASTGLKWS 129 A. thalania KWSFMPKV---SDGRPS--YLVVNADESEPGTCKDREIMRHDPHKLLEGCLIAGVGPSGLKWS 178

P252R

Homo sapiens PTTVANVETVAVSPTICRRGGTWFAGFGRE 268 B. taurus PTTVANVETVAVSPTICRRGGAWFASFGRE 268 M. musculus PTTVANVETVAVSPTICRRGGTWFAGFGRE 268 X. laevis PTTVANVETVAVAPTICRRGGSWFASFGRE 274 D. melanogaster PTTVTNVETVAVAPTICRRGGVWFASFGRT 283 A. gambiae PTTVSNVETVAVAPTICRRGGTWFASFGRT 295 C. elegans PTTVTNVETVAVAPTICRRGGDWFASFGRE 279 N. crassa PSTVANVETVAVAPTICRRGGNWFAGFGRE 280 E. coli PTCVNNVETLCNVPAILANGVEWYQNISKS 242 A. thalania PTTVTNVETVAVSPTILRRGPEWFSSFGRK 291

R386H

Homo sapiens LIEFYKHESCGQCTPCREGVDWMNKVMARFVRG 402 B. taurus LIEFYKHESCGQCTPCREGVDWMNKVMARFVRG 402 M. musculus LIEFYKHESCGQCTPCREGVDWMNKVMARFVKG 402 X. laevis LIEFYKHESCGQCTPCREGVDWMNKVMWRMCRG 408 D. melanogaster LISFYKHESCGQCTPCREGIGWMNKIMTRFVKG 417 A. gambiae LIMFYKHESCGQCTPCREGIAWMNKIMHRFVTG 422 C. elegans LSLFYKHESCGQCTPCREGCNWLNKMMWRFVDG 413 N. crassa LSHFYRHESCGQCTPCREGSKWTEQIMKRFEKG 414 E. coli LEEFFARESCGWCTPCRDGLPWSVKILRALERG 374 A. thalania LSYFYKHESCGQCTPCREGTGWLWMIMERMKVG 425

Footnote: The comparative sequences for other species were obtained from the NCBI Conserved Domain Database (CDD). The sequences of the human complex I genes were blasted against the homologous genes from other species at the NCBI website, Homologene.

NDUFV1 sequence variants of uncertain significance The heterozygous NDUFV1 sequence variant c.439C>T identified in Patient 15, results in an amino acid change from the large polar arginine to tryptophan which is of intermediate polarity, at codon 147 (R147W). The Grantham chemical difference is 101 (see Discussion for definition of Grantham chemical difference) (Grantham 1974). This mutation was not seen in 104 control chromosomes. A second mutation was not identified in Patient 15 by dHPLC screening. Arg147 in human NDUFV1 is not

63 conserved across all species and is located in a region of the NDUFV1 gene that is not highly conserved (Figure 6). A heteroplasmic pathogenic mutation in mtDNA subunit gene ND5 (12706T>C), previously reported in two other complex I Leigh disease cases (Taylor et al. 2002), has subsequently been identified in Patient 15 (Dr D. Thorburn). Therefore the R147W missense change is not considered to be of pathogenic significance in Patient 15. It may represent a rare, benign polymorphism. Alternatively, if it is a pathogenic mutation it does not appear to be the cause of complex I deficiency in Patient 15.

NDUFV1 polymorphisms Polymorphisms identified in NDUFV1 are listed in Table 23. All three polymorphisms were identified in Patient 15. The sequence variants c.549C>G (G183G) and c.439C>T (R147W) were both in amplicon 2, so one or both may have contributed to the abnormal dHPLC profile (Appendix 4, Figure 32). The polymorphism c.1056T>C, was in amplicon 4 and was not identified by dHPLC (see Discussion).

Table 23: NDUFV1 polymorphisms

Patient Polymorphism dHPLC Amplicon Exon ID identified 15 c.439C>T, R147W, heterozygous yes 2 4 15 c.549C>G, G183G, heterozygous yes 2 5 15 c.1056T>C, A352A, heterozygous no 4 7

NDUFV1 splice variant A normal NDUFV1 splice variant was identified in amplicon 4. The splice variant is 82 base pairs (bp) shorter than the normal transcript due to skipping of exon 8. An admixture of the spice variant and normally spliced NDUFV1 was identified in most patients by dHPLC and/or sequencing of amplicon 4 (Appendix 4, Figure 31). The presence of the splice variant resulted in extra peaks in the dHPLC profile, at a shorter retention time than the main peak. The additional peaks were located on the dHPLC chromatogram at the correct location for DNA which is 82bp shorter than the expected amplicon 4 size of 357bp.There were two additional peaks (rather than one) just prior to the main peak and this may be due to greater denaturation of the shorter splice variant at the temperatures tested. The quantity of splice variant was judged by the relative height of the sequencing peaks of splice variant compared to those of the superimposed normal

64 sequence, as well as by the height of the extra peaks on dHPLC. In sequenced samples with large quantities of splice variant present (Patients 8, 15, 25) both extra dHPLC peaks are high. In samples with minimal or absent splice variant on sequencing (patients 12, 17), the additional peaks on dHPLC profile were low or absent.

3.3.2 NDUFS1 sequencing results The sequencing results for NDUFS1 are summarized in Table 24. No mutations were identified in NDUFS1. For a number of NDUFS1 amplicons, homoduplexes were identified that had slight differences in curve shape. No mutations or polymorphisms were identified on sequencing to explain these minor differences. These included samples 1, 3, 7, 11, 12, 14, 23, 28, 33 and 35 in Amplicon 2, samples 2, 3, 5, 8, 10 in amplicon 3 and sample 15 in amplicon 5 (Table 24).

Table 24: NDUFS1 sequencing results

NDUFS1 dHPLC abnormals All samples Sequencing Comment amplicon (similar dHPLC sequenced result profiles grouped together) A1 18, 30, 32, 36 18, 30, 32, 36 het c.414T>C, Polymorphism D138D c.414T>C explains 22 normal dHPLC results A2 18, 30, 32, 36 18, 30, 32, 36 het c.414T>C, Polymorphism D138D c.414T>C explains 2, 4, 6, 8, 29 2, 4, 6, 8, 29 no mutation/ dHPLC results 1, 3, 7, 11, 12, 14, 23, 1, 7, 14, 33, 35 polymorphism 28, 33, 35, C5 22 A3 1, 4, 6, 9, 13, 14, 15, 14, 34 c.966T>G(TG) Polymorphisms 24, 28, 33, 34, 35 c.1251A>G(AG) 1. c.966T>G (A322A) 17, 21, 29, 32 21, 29, 32 c.966 T>G(TG) 2. c.1251A>G (R417R) c.1251A>G(GG) The various 27, 30 27, 30 c.966 T>G(TT) combinations of these c.1251A>G(AG) two polymorphisms explains the dHPLC 31 31 c.966 T>G(TT) results. c.1251A>G(GG) 36 36 c.966 T>G(TT) c.1251A>G(AA) A4 1, 4, 6, 9, 13, 14, 15, 1, 9, 14, 16, c.1251A>G(AG) Polymorphism A>G 16, 24, 27, 28, 30, 34, 24, 27, 28, 30 1251 (R417R) explains 35 dHPLC results 2, 3, 5, 8, 10 5 c.1251A>G(AA) 31 31 c.1251A>G(GG) 36 c.1251A>G(AA) A5 15 15 normal Footnote: het: heterozygous, hom: homozygous

65 NDUFS1 Polymorphisms The polymorphisms identified in NDUFS1 are listed in Table 25. None of these SNPs resulted in a change to the amino acid. The polymorphism c.414T>C (D138D) was located in the overlap area between amplicon 1 and 2 and was identified by dHPLC and sequencing in both amplicons in four patients (Patients 18, 30, 32, 36).

Amplicon 3 of NDUFS1 contained two polymorphisms which complicated dHPLC results. The polymorphism c.966T>G (A322A) was in amplicon 3 and the polymorphism c.1251A>G (R417R) was in the overlap area between amplicon 3 and 4. The various combinations of these two polymorphisms explained the large number of different dHPLC heteroduplexes in amplicon 3 (Table 24). Fifteen samples in amplicon 4 were heteroduplexes (without addition of wild type DNA). Eight of these samples were sequenced and were heterozygous for the c.1251A>G polymorphism. Patient 31 became a heteroduplex with addition of wild type DNA, and was found to be homozygous GG for the c.1251A>G polymorphism.

Table 25: NDUFS1 polymorphisms Polymorphism Patient ID Homozygous or dHPLC Amplicon heterozygous identified c.414T>C, D138D 32, 36, 18, 30 heterozygous TC yes 1, 2 c.966T>G, A322A 14, 29, 32, 21, 34 heterozygous TG yes 3 c.1251A>G, R417R 30, 27, 14, 34 heterozygous AG yes 3, 4 31 homozygous GG yes

3.3.3 NDUFS2 sequencing results NDUFS2 sequencing results are summarized in Table 26.

As for NDUFS1, a number of homoduplex samples with slight variations in curve shape were identified, such as Patients 5, 13, 16, 33 and 34 in amplicon 3 and Patients 19 and 20 in amplicon 5 (Table 26). No sequence variants were identified by sequencing.

66 Table 26: NDUFS2 sequencing results

NDUFS2 dHPLC abnormals All samples Sequencing Comment Amplicon (similar dHPLC sequenced result profiles grouped together) A1 nil 25 no mutations A2 nil 25 no mutations A3 25 25 het c.661C>T, nonsense R221X mutation 5, 13, 16, 33, 34 5, 13, 16, 33, no mutations 34, 35 A4 17 17 hom c.875T>C, homozygous M292T missense mutation C5, 3, 14, 18, 22 C5, 3, 14, 18, het c.1054C>G, polymorphism 22 P352A 25 het c.998G>A, heterozygous R333Q missense mutation 4, 33 no mutation A5 19, 20 19, 20, 26 no mutation 33 33, 25 het c.1290C>T, polymorphism A430A Footnote: het: heterozygous, hom: homozygous

NDUFS2 mutations

Patient 25: Patient 25 is a compound heterozygote for two NDUFS2 mutations. The first mutation was identified in amplicon 3 by dHPLC screening. The mutation is a heterozygous C>T change at position 661 of coding, which leads to the formation of a premature stop codon, R221X (Appendix 3, Figure 28). The allele with the stop mutation is in much smaller quantity than the second allele, most likely due to NMD. This is shown by a much lower sequencing peak for the stop mutation compared to the normal sequence (Appendix 3, Figure 28).

The second mutation is a missense mutation in amplicon 4. This amplicon was sequenced because the first mutation had been identified in amplicon 3. This mutation was not detected by dHPLC screening even though a polymorphism (c.1054C>G, P352A) was clearly identified in this amplicon by dHPLC (see Discussion).The mutation is a heterozygous G>A at position 998 of coding. This leads to an amino acid change from the highly conserved arginine to glutamine at codon 333 (R333Q). This heterozygous mutation was confirmed by sequencing in both directions. The allele with

67 the missense mutation appears to be in much greater quantity than the allele containing the stop mutation in amplicon 3, as determined by the height of the DNA sequencing peaks at position 998 (Appendix 3, Figure 29), assuming they are on different alleles. Both arginine and glutamine are large polar amino acids. However, the finding of a second mutation in this patient, suggests that this mutation is pathogenic. The remainder of NDUFS2 was sequenced in patient 25 and no further mutations were identified.

The arginine at position 333 of the human NDUFS2 is highly conserved across different species. It is conserved in M. musculus (13th of 141 consecutive conserved amino acids) and B. taurus (13th of 20 consecutive conserved amino acids). It is also conserved in X. tropicalis, D. melanogaster, C. elegans, N. crassa and A. thalania. It is not conserved in E. coli (Figure 7).

Patient 17: This patient had an apparent homozygous mutation in NDUFS2 in studies of cDNA. dHPLC screening identified a heteroduplex in amplicon 4 of NDUFS2 only after addition of wild type DNA, as would be expected for a homozygous mutation. On sequencing cDNA, the mutation was a homozygous T>C at position 875 coding (Appendix 3, Figure 27). This changes the amino acid at codon 292 from methionine to threonine (M292T). Methionine is a large non polar amino acid and threonine is a small non polar amino acid. The methionine at position 292 of human NDUFS2 is conserved throughout most species (Figure 7). It is conserved in the M. musculus and B. taurus where it is the 10th of 37 consecutive conserved amino acid. This amino acid is also conserved in X. tropicalis, D. melanogaster, C. elegans, N. crassa and A. thaliana. It is not conserved in E. coli (Figure 7).

Sequencing of genomic DNA showed that the M292T mutation was in fact heterozygous. As the second allele was lost in cDNA, it is likely that this allele was lost due to mRNA decay, despite that fact that the cells were grown in cycloheximide. The other NDUFS2 amplicons were screened by dHPLC and were normal. The NDUFS2 gene was subsequently sequenced in gDNA and the second mutation, a heterozygous splice site mutation IVS9+4A>G was identified (c.780+4 A>G). This mutation would be predicted to obliterate the normal exon 9 donor splice site using the splice prediction program http://www.fruitfly.org/cgi-bin/seq_tools/splice.pl.

68 Figure 7: Conservation of amino acids of a segment of NDUFS2

M292T R333Q

Homo sapiens LNYGFSGVMLRGSGIQWDLRKTQPYDVYDQVEFDVPVGS-RGDCYDRYLCRVEEMRQSLR 342 B. taurus LNYGFSGVMLRGSGIQWDLRKTQPYDVYDQVEFDVPIGS-RGDCYDRYLCRVEEMRQSIR 307 M. musculus LNYGFSGVMLRGSGIQWDLRKTQPYDVYDQVEFDVPIGS-RGDCYDRYLCRVEEMRQSLR 342 X. tropicalis LNYGFSGVMLRGSGIQWDLRKSQPYDVYDQVEFDVPIGS-RGDCYDRYLCRVEEMRQSMR 338 D. melanogaster LNYGFSGVMLRGSGIKWDLRKQQPYDAYNLVNFDVPIGT-KGDCYDRYLCRVEEMRQSLR 347 A. gambiae LNYGFSGVMLRGSGIKWDLRKVQPYDAYDQMEFDVPIGT-KGDCYDRYLCRIEEMRQSLR 318 C. elegans LNWGFSGVMVRGSGIKQDVRKTEPYDAYADMEFDVPIGT-KGDCYDRYLCRVEEMRQSLN 353 N. crassa LNLSFTGVMLRGSGVPWDIRKSQPYDAYDQVEFDVPVGI-NGDCYDRYLCRMEEFRQSLR 357 E. coli LEWGTTGAGLRATGIDFDVRKARPYSGYENFDFEIPVGGGVSDCYTRVMLKVEELRQSLR 286 A. thalania INWGLSGPMLRASGIPWDLRKIDRYESYDEFEWEIQWQK-QGDSLARYLVRLSEMTESIK 273

Footnote: The comparative sequences for other species were obtained from the NCBI Conserved Domain Database (CDD). The sequences of the human complex I genes were blasted against the homologous genes from other species at the NCBI website, Homologene.

NDUFS2 Polymorphisms NDUFS2 polymorphisms are summarised in Table 27. No mutations were identified in NDUFS2.The unreported NDUFS2 polymorphism, c.1054C>G, (P352A) was identified in amplicon 4 by dHPLC and confirmed by sequencing in four patient samples (Patients 3, 14, 18 and 22) and one control (C5).This mutation leads to an amino acid change from a highly conserved proline to an alanine (Figure 8). However, both amino acids are small and non polar and the Grantham chemical difference is very low at 27 (Grantham 1974). In the humans, Proline352 is the first of two prolines in a row and the second proline is not conserved in a number of species (Figure 8). It may be that only one proline is needed to maintain NDUFS2 function. Patient 3 has this c.1054C>G sequence variant but also has subsequently had pathogenic mutations in another complex I subunit gene NDUFS6 identified (Kirby et al. 2004). The one control tested (C5) also has the c.1054C>G sequence variant. For all these reasons, the P352A change was considered a polymorphism.

69

Figure 8: NDUFS2 amino acid conservation and the polymorphism P352A

P352A Homo sapien IIAQCLNKMPPGEIKVDDAKVSPPKRAEMKTSMESLIHHFKLYTEGYQVPPGATYTAIEA 402 B. taurus IISQCLNKMPPGEIKVDDAKVSPPKRAEMKTSMESLIHHFKLYTEGYQVPPGATYTAIEA 367 M. musculus IIEQCLNKMPPGEIKVDDAKVSPPKRAEMKTSMESLIHHFKLYTEGYQVPPGATYTAIEA 402 X. tropicalis IILQCLNKMPEGEIKVDDAKVSPPKRSEMKRSMESLIHHFKLYTEGYQVPPGATYTAIEA 398 D. melanogaster IIDQCLNQMPAGEIKTDDAKVAPPSRSEMKTSMEALIHHFKLFTQGYQVPPGATYTAIEA 407 A. gambiae IIDQCLNRMPAGEIKTDDGKISPPSRTEMKQSMEALIHHFKLFTQGYQVPPGSTYTAVEA 378 C. elegens IVHQCLNKMPTGEIKSDDHKVVPPKRAEMKENMESLIHHFKFFTEGFQVPPGATYVPIEA 413 N. crassa IIHQCLNKMPAGPVRVEDYKISPPPRSAMKENMEALIHHFLLYTKGYAVPPGDTYSAIEA 417 E. coli ILEQCLNNMPEGPFKADHPLTTPPPKERTLQHIETLITHFLQVSWGPVMPANESFQMIEA 346 A. thalania IIQQALEGLPGGPYENLESRGFDRKRNPEWNDFEYRFISKKPSPT-FELSKQELYVRVEA 332

Footnote: The comparative sequences for other species were obtained from the NCBI Conserved Domain Database (CDD). The sequences of the human complex I genes were blasted against the homologous genes from other species at the NCBI website, Homologene.

Patient 33 had an abnormal dHPLC profile in amplicon 5 and the polymorphism c.1290C>T, A430A was identified by sequencing. Patient 25 was also found to have the c.1290C>T, A430A polymorphism, even though dHPLC for amplicon 5 was normal. This amplicon was sequenced when searching for the second mutation in Patient 25. Reasons that dHPLC failed to identify this SNP are considered in the Discussion.

Table 27: NDUFS2 Polymorphisms Polymorphism Patient ID dHPLC Amplicon identified c.1054C>G, P352A, heterozygous 3, 14, 18, 22, Control 5 yes 4 c.1290C>T, A430A , heterozygous 25, 33 no, yes 5

3.3.4 NDUFS4 sequencing results Sequencing results for NDUFS2 are summarized in Table 28.

70 Table 28: NDUFS4 sequencing results

NDUFS4 dHPLC Samples Sequencing result Comment Amplicon abnormals sequenced (similar dHPLC profiles grouped together) A1 19 19 1.het c.221delC 1. frame shift mutation 2. het c.12C>G 2. polymorphism 26 26 1. hom c.221delC 1. frame shift 2.172bp insertion of mutation intronic sequence at 2.splicing variant c.178 (between exon 2 and 3) 3. polymorphism 3. het c.12C>G 22 22 1. het c.158T>C 1. polymorphism (I53T) 2. het c.12C>G 2. polymorphism 4, 23, 33 4, 23, 33 polymorphisms c.198C>A 1. het c.198C>A polymorphism 2. het c.12C>G 2, 8, 9, 13, 15, 16, 1, 2, 3, 4, 5, 6, c.12C>G (V4V) polymorphism 21, 24, 27, 30, 34, 7, 8, 9, 10, 13, het CG: 1, 3, 4, 5, 6, 14, 17, 18, 19, 20, 35, 36 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 33, 35 18, 20, 21, 24, hom CC: 13 25, 27, 35 hom GG: 2, 7, 8, 9, 10, 15, 16, 24 A2 1, 4, 12, 14, 18, 1, 4, 18, 19, het c.312G>A c.312G>A, 19, 21, 23, 25, 27, 25, 28 (R104R) 28, 33 polymorphism 11, 13, 32 11, 13, 32 hom c.312G>A explains all 2, 3, 5, 6, 7, 8, no polymorphisms dHPLC findings 9, 10, 22, 26, 30, 36 Footnote: Prior to availability of dHPLC, 25 patients were sequenced for amplicon 1 and 10 patients for amplicon 2. het: heterozygous, hom: homozygous

NDUFS4 mutation Patient 26: Sample 26 was a dHPLC heteroduplex for NDUFS4 amplicon 1, with and without the addition of wild-type. DNA sequencing has shown that Patient 26 has a homozygous frame shift mutation, c.221delC in NDUFS4 (Appendix 3, Figure 30). This occurs in codon 74, and would lead to a translational frame shift and eventually a stop codon, 18 codons downstream (91X). This mutation would lead to a truncated protein and loss of the RVS phosphorylation consensus site. It may also be subjected to nonsense mediated mRNA decay (NMD) which has been reported in two other patients with premature stop codons in NDUFS4, who both failed to assemble normal complex I (Scacco et al. 2003). The c.221delC mutation is absent from 50 anonymous controls and 40 Lebanese controls.

71 In addition, Patient 26 also has a large intronic insertion in NDUFS4 due to a splicing variant. The presence of this insertion explains why the dHPLC heteroduplex was seen without the addition of wild type DNA. Exon 2 and Exon 3 normally join at nucleotides 177 and 178. In patient 26, there is an insertion of 172bp of intronic sequence between exon 2 and exon 3. This 172bp of intronic sequence is usually found in intron 2 (IVS2 +1194 to +1365). The sequencing results for Patient 26 for NDUFS4, using primer NDUFS4 F1, shows double sequence from position 179. Single sequence returns after the insertion, as demonstrated in Figure 9.

Figure 9: Patient 26 sequencing results for amplicon 1 of NDUFS4

Normal Exon1 Exon2 Exon3 (178-334) Allele (~40-98) (99-177) Exon1 Exon2 Insertion 172bp Exon3 Insert (~40-98) (99-177) (179-218) Allele Å single sequence Æ Ådouble sequenceÆ Åsingle seq.Æ

It is possible that the insertion is actually 173bp long and is inserted at position 177, or the insertion is 171 bp and is inserted at position 179 of the cDNA. This is because the intronic sequence and the cDNA both have two G’s at this point. However, it seems most likely that the insertion occurs between the two exons at position 178 (Figure 10).

Figure 10: The NDUFS4 exon 2 / exon 3 cDNA junction in Patient 26. The highlighted bases represent the insertion.

Exon 2 Exon 3 177 178 A A A A A T T G G A T A T C A C T A C T T T A A C T…………. A A A A A T T G G T T T A T C T T G T A C C A C T G………….

The 172 bp intronic insertion is normally located in intron 2 at position +1194 to +1365. The full insertion sequence is: (G)GTT TAT CTT GTA CCA CTG TGT TTT ACA TTC TGT GGT CTA GCC ACA CCA AGC CTG GAA CTT GCT ACT CTT TTT CCT GCT TCA AGG CAT TTG GAG TTC CTG TTC ACT CTA CCT GGA TGC ATT TTA ACT AAT CCT GAG GAC TTC ATC CGG TCA CCA TCT ACC TAT TCT TCA AAT G

72 The insertion of 172bp of intron in patient 26 is confirmed with sequencing in the reverse direction, NDUFS4 R1 (Figure 11).

Figure 11: Reverse sequencing of NDUFS4 amplicon 1 in Patient 26

Insertion Å double sequence Æ Allele Exon3 Insertion 172bp Exon2 (177- (285-178) Normal Exon3 Exon2 Exon1 UTR 5’ Allele (285-178) (177-99) (98-1) (-1-19)

Genomic DNA sequencing of NDUFS4 in Patient 26 did not identify a splice site mutation. For reasons presented in the Discussion, this NDUFS4 cDNA variant with the 172 base pair insertion between exons 2 and 3 is believed to be a normal splice variant, usually subjected to nonsense mediated mRNA decay (NMD). It is an alternatively spliced NDUFS4 that is predicted to generate an unstable mRNA, due to a translational frame shift and a premature stop codon 73 amino acids after the start of the insertion (132X). The 172bp insertion leads to a translational frame shift in exon 3, following the insertion. However, Patient 26 is homozygous for the exon 3 mutation c.221delC which would restore the frame, making the mRNA splice variant more stable than the wild type alternatively spliced mRNA. The premature stop codon would not be generated. Hence for Patient 26, the c.221delC mutation results in the generation of a somewhat more stable mRNA splice variant, containing both the 172bp insertion and the 1bp deletion. In summary, the pathogenic deletion c.221delC stabilises the mRNA splice variant in Patient 26. The significance of this splice variant is evaluated further in the Discussion.

Patient 19: Patient 19 is heterozygous for the NDUFS4 mutation c.221delC, the same mutation identified in Patient 26. The mutation was detected by dHPLC screening and confirmed by sequencing. The mutation was seen well using primer NDUFS4 R1 (Appendix 3, Figure 30). The splicing variant was not identified in Patient 19, but the quantity may have been too low to detect. Sequencing results with the forward primer (NDUFS4 F1) had persistent background artifact from position 179, and this may have been due to the presence of low levels of splice variant. NDUFS4, amplicon 2

73 was also sequenced in patient 19 and a second mutation was not identified. Patient 19 has a sister who died with Leigh syndrome and she is also heterozygous for the c.221delC mutation. A discussion of the significance of this mutation in patient 19 is included in the Discussion.

NDUFS4 sequence variants of unknown significance A single missense mutation was identified in Patient 22 in NDUFS4 by dHPLC and confirmed by sequencing. This was a T>C at position 158 of the coding resulting in a change in the amino acid at codon 53, from an isoleucine to a threonine (I53T). Isoleucine is a large non polar amino acid and threonine is a small non polar amino acid. Grantham chemical difference is 89. The isoleucine at position 53 in the human NDUFS4 gene is conserved in M. musculus, B. taurus and D. melanogaster. However, this amino acid is not conserved in C. elegans or A. thalania and there is not a NDUFS4 homologue in E. coli (Figure 12). The entire coding region of NDUFS4 was sequenced and a second mutation was not identified. This missense mutation would not lead to loss of the functionally important carboxl-terminal phosphorylation site. The effect on the structure of NDUFS4 is not known, nor whether the mutation would affect complex I assembly. However, as I53 is not highly conserved and a second mutation was not identified, it is most likely that this sequence variation is a polymorphism.

Figure 12: Amino acid conservation in NDUFS4

I53T

Homo sapiens ----RSLRTSTWRLAQDQTQDTQLITVDEKLDITT------LTGVPEEHIKT 74 B. taurus ----RSLSTSTWRLAQDQTRDTQLITVDEKLDITT------ITGVPEEHIKT 74 M. musculus ----RLLSTSTWKLADNQTRDTQLITVDEKLDITT------LTGVPEEHIKT 61 C. elegans ----RCLSTGKDNLPVTRSHDAKKVELNDILDKPSQKTPVKVSSDETMDIGGVPLDHQDA 96 D. melanogaster --LDPKTALARPEELEQRNKLSGKITVPTAVNLSP------ISGVPEEHIRE 81 A. gambiae PILDASVVLADAEER-ARDHLP-TITVPTKVDISP------ITGVPEEHVKE 104 A. thalania ------IAATLRRVARPFATDAVVESDYKRGEIGK------VSGIPEEHLS- 51

NDUFS4 polymorphisms Polymorphisms identified in NDUFS4 are recorded in Table 29. The common c.12C>G (V4V) polymorphism was identified in amplicon 1 by sequencing (see Discussion), but not by dHPLC. Of the 54 alleles sequenced, 20 were C and 34 were G.

74 Patients 4, 23 and 33 had the same type of heteroduplex profile in amplicon 1 and were found to have the same heterozygous polymorphism c.198C>A (G66G) (Table 28). In amplicon 2 of NDUFS4, the dHPLC results showed that 12 patients had the same heteroduplex profile. Six of these patients were sequenced and were found to have a heterozygous polymorphism c.312G>A (R104R). Three patients (11, 13, 32) became the same type of heteroduplex only with the addition of wild type DNA, and they were found to be homozygous AA for this polymorphism.

Table 29: NDUFS4 polymorphisms Polymorphism Heterozygous or Patient ID dHPLC Ampl- homozygous identified icon c.12C>G, V4V heterozygous CG 1, 3, 4, 5, 6, 14, 17, no 1 18, 19, 20, 21, 22, 23, 25, 26, 27, 33, 35 homozygous GG 2, 7, 8, 9, 10, 15, 16, 24 c.158T>C, I53T heterozygous TC 22 yes 1 c.198C>A1, G66G heterozygous CA 4, 23, 33 yes 1 c.312G>A1, R104R heterozygous GA 1, 4, 18, 19, 25, 28 yes 2 homozygous AA 11, 13, 32 Footnote: 1 reported by Petruzzella et al. 2001

3.3.5 NDUFS7 sequencing results Sequencing results for NDUFS7 are listed in Table 30. No mutations were identified in NDUFS7.

75 Table 30: NDUFS7 Sequencing results NDUFS7 dHPLC abnormals Samples Sequencing Comment Amplicon (similar dHPLC sequenced result profiles grouped together) A1 C5, 1, 2, 5, 6, 11, 14, 1-10 c.68T>C, L23P common 17, 18, 19, 21, 22, TC: 1, 2, 5, 6 polymorphism 24, 30, 34, 35, 36 CC: 8, 9 explains dHPLC TT: 3, 4, 7, 10 results A2 nil 1-10 no mutations A3 8 8 c.655C>A polymorphism 3’UTR 1, 2, 3, 4, 5, 6, no mutations / 7, 9, 10, 20 polymorphisms Footnote: Prior to availability of dHPLC Patients 1-10 were sequenced routinely.

NDUFS7 polymorphisms dHPLC identified a common sequence variant (c.68C>T, P23L) in amplicon 1, by the finding of a broad dHPLC curve, rather than a typical heteroduplex (see Discussion). The 16 patients heterozygous for this mutation were identified by dHPLC, though two patients (8 and 9) who were homozygous for this polymorphism were not distinguished by dHPLC as the wild type DNA (C5) was heterozygous for this SNP. This previously reported polymorphism, c.68C>T, P23L (Schuelke et al 1999, Petruzzella et al. 2001), results in an amino acid change in a non conserved area of the gene. The first 38 amino acids of NDUFS7 constitute a mitochondrial import sequence that does not share high homology with other mammals such as the cow or mouse (Hyslop et al. 1996). P23 is not conserved in these species. Of the 20 alleles sequenced, 60% were T and 40% were C at position 68. Considering the lack of conservation and the frequency of this DNA change, c.68C>T (P23L) is considered a polymorphism.

For amplicon 3 only Patient 8 was abnormal by dHPLC. A polymorphism in the untranslated 3’ region, c.655C>A (13bp after stop codon) was identified.

Table 31: NDUFS7 polymorphisms Polymorphism Patient Homozygous or dHPLC Amplicon ID heterozygous identified 1,2 c.68T>C, L23P 1, 2, 5, 6 heterozygous TC yes 1 8, 9 homozygous CC 1 c.655C>A, 3’UTR 8 heterozygous CA yes 3 (13 bp after stop codon) Footnote: 1 reported by Petruzzella et al. 2001, 2 reported by Schuelke et al. 1999

76 3.3.6 NDUFS8 sequencing results NDUFS8 sequencing results are reported in Table 32. No mutations were identified.

Table 32: NDUFS8 sequencing results

NDUFS8 dHPLC Samples Sequencing Comment Amplicon abnormals sequenced result (similar dHPLC profiles grouped together) A1 1 1, 2, 3, 4, 5, 7, no mutations 8, 9 A2 nil 1, 2, 3, 4, 7, 9 no mutations A3 3, 4, 9, 12, 18, 19, 3, 4, 9, 12, 18, het c.647C>T polymorphism 21, 27, 31 27, 31 c.647C>T 16 16 hom c.647C>T explains all the 1, 2, 6, 7 no mutations dHPLC results (hom CC for c.647C>T SNP) Footnote: het: heterozygous, hom: homozygous Prior to availability of dHPLC, some patients (1-10) were sequenced routinely

NDUFS8 polymorphisms A polymorphism was identified in amplicon 3 in the untranslated 3’region, c.647C>T (14 bp after the stop codon). Nine patients were dHPLC heteroduplexes without wild type DNA for amplicon 3 and seven of these were sequenced and found to be heterozygous for the this polymorphism. Patient 16 only became a heteroduplex with addition of wild type DNA, and this patient was homozygous TT for the c.647C>T polymorphism (Table 33).

The control samples (C3 and C5) had a different dHPLC heteroduplex profile compared to all patient samples in amplicon 3. Unfortunately sequencing of the control DNA failed, so the reason for this was not determined. However, this information was not necessary to interpret patient dHPLC results.

Table 33: NDUFS8 polymorphisms

Polymorphis Patient ID Homozygous or dHPLC Amplicon m heterozygous identified c.647C>T1, 3, 4, 9, 12, 18, 27, 31 heterozygous CT yes 3 3’UTR 16 homozygous TT Footnote: 1 reported by Petruzzella et al. 2001

77 4. DISCUSSION

4.1 OVERVIEW

Complex I deficiency is often a devastating neurodegenerative disorder with multi- system involvement that leads to severe disability and death in the first few years of life. Considering the severe phenotype and lack of effective treatment, accurate diagnosis and the availability of prenatal diagnosis is important. However, for the majority of affected families the genetic cause is unknown. In addition to extending our understanding of the genetic basis of complex I deficiency, identification of the causative mutations in a child means that accurate genetic counseling and prenatal diagnosis is available for the family.

In this study, 34 patients with confirmed complex I deficiency were investigated. This is one of the largest groups of complex I deficient patients assembled and studied in this way. Six nuclear encoded complex I subunit genes were screened for mutations (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). Eighteen percent of the complex I deficient patients (6/34) had at least one nuclear encoded complex I subunit mutation identified. Two patients were compound heterozygotes for NDUFV1 mutations, two patients were compound heterozygotes for NDUFS2 mutations and two patients had NDUFS4 mutations identified.

In the following discussion, the methods used in this study are reviewed including their advantages and limitations. The significance of the sequence variants identified is also analyzed in context of what is known about the structure and function of the complex I subunits. For each identified mutation, the phenotype and genotype are examined and compared with previously reported patients with mutations in the same gene. The possible location of mutations in the patients in whom we failed to identify mutations is discussed, in light of what is known about the structure, assembly and regulation of complex I. The approach to the molecular genetic diagnosis of an individual patient with complex I deficiency is examined with particular reference to the different approaches now available.

78 4.2 DISCUSSION OF METHODS

4.2.1 Complementary DNA (cDNA) Complementary DNA (cDNA) was used for mutation screening, rather than genomic DNA (gDNA) because the genes studied contained many small exons (less than 100 base pairs) often separated by large introns. Exon amplification would not have provided suitable DNA fragments for dHPLC analysis, which ideally requires DNA more than 100 base pairs in size. Amplification of each individual exon would have required the amplification of large segments of adjacent intron. As introns are more likely to contain polymorphisms, this would have created numerous false positive dHPLC results, which would then need to be clarified by sequencing. By using cDNA, the amplicon sizes were larger and the number of fragments to screen by dHPLC much smaller. For these reasons cDNA was considered preferable.

However, there are some disadvantages to using a cDNA based method. RNA extraction is more complex than DNA extraction as there is always a risk of RNase mediated RNA degradation. RNA cannot be reliably stored for long periods of time, unlike gDNA. RNA is best stored at -80°C and repeated thawing and refreezing can lead to degradation. In this project, the RNA was converted to cDNA by reverse transcription and the stable cDNA was kept at -4°C for daily use. The RNA was only defrosted briefly once every few months in order to make cDNA. With these precautions RNA degradation was not encountered.

The main disadvantage of the cDNA based method for mutation detection was the loss of mutated alleles in mRNA due to nonsense-mediated mRNA decay (NMD). mRNAs are monitored for errors by a mechanism called RNA surveillance and most mRNAs that cannot be translated along their full length are rapidly degraded by the process of NMD (Culbertson 1999). This avoids the accumulation of truncated proteins that may be deleterious and result in dominant-negative effects (Culbertson 1999). NMD comes into play with nonsense mutations, as well as frame shift mutations that result in premature stop codons. Splicing defects can also result in premature termination codons and mRNAs that are subjected to NMD. Perhaps 25-30% of all mutations causing inherited human disease could be targets for NMD (Culbertson 1999, Wagner and

79 Lykke-Andersen 2002) and so strategies are need to minimize NMD when a cDNA method of mutation detection is used.

For this reason the patient cell fibroblasts were cultured in cycloheximide for a short period prior to harvesting, to minimize NMD (Bateman et al. 1999). However, for patient 17 an apparent homozygous mutation in NDUFS2 (M292T) was in fact, heterozygous when gDNA was studied. The second mutation, located in the donor splice site (IVS9+4A>G), was not identified in cDNA. There was complete loss of this allele in cDNA presumably secondary to destruction of the abnormally spliced mRNA. NMD has been previously reported in patients with premature stop codons in NDUFS4 (Scacco et al. 2003).

In addition, mutation analysis of cDNA is unable to identify splice site sequence variants, as non-coding DNA is not screened. The finding of an abnormally spliced transcript would suggest the possibility of a splicing mutation. However, the abnormally spliced transcript may be subjected to NMD as was the case for Patient 17.

Culture of cells in cycloheximide minimises NMD and this precaution may have allowed identification of the NDUFS2 mutation (R221X) in Patient 25. In this case, dHPLC showed a clear heteroduplex. However, the allele with this stop mutation was in much reduced quantity in cDNA, when compared to the alternate allele that carried the missense mutation (R333Q). This quantification was based on the height of the DNA sequencing peaks (Appendix 3, Figure 29). Fortunately, there was just enough of the allele with the stop mutation, to allow mutation detection by sequencing as well as dHPLC.

A potential advantage of dHPLC is the ability to identify mutations present at low levels and this has been shown previously in patients with mosaicism (Xiao and Oefner 2001). In fact, dHPLC is more likely to detect low percentage mosaicism for a mutation than is direct DNA sequencing. Amplicons with an abnormal dHPLC profile, yet normal sequencing, should not be disregarded (Xiao and Oefner 2001). Mutant alleles present at a proportion of 20% are almost always detected by dHPLC, whereas only 80% will be detected by sequencing (Xiao and Oefner 2001). For Patient 25, the stop mutation

80 would likely have been missed by sequencing of cDNA if the abnormal dHPLC profile had not clearly indicated the presence of a mutation in this amplicon.

While it is possible that mutations subjected to mRNA decay were missed, it is unlikely that this was a common problem. If both mutated alleles were subjected to mRNA decay, very little if any, normal cDNA would be created, and hence the PCR should fail. If only one transcript was subject to mRNA decay, then the second allele and the second mutation should still be identified in cDNA. Therefore NMD is unlikely to explain the large number of patients in whom no mutation was found.

4.2.2 Denaturing High Performance Liquid Chromatography (dHPLC)

Sensitivity and specificity dHPLC was chosen as the mutation screening method because of its high sensitivity and specificity, which appear to be consistently better than 96% (Xiao and Oefner 2001). One study evaluated the sensitivity and specificity of dHPLC in detecting mutations in exon H of the Factor IX gene and exon 16 of the Neurofibromatosis type 1 gene (O’Donovan et al. 1998). This study assessed the ability of dHPLC to identify unknown mutations in a blind analysis. Sensitivity and specificity were 100% for a single set of conditions for each exon. Forty-eight unique mutations were correctly identified, as were 55 controls with wild-type alleles. In addition, dHPLC offered high sensitivity mutation detection when using large PCR fragments [438 base pairs (Factor IX) and 549 base pairs (NF1)]. In another study, PCR fragments from the CFTR, TSC1 and TSC2 genes were screened for mutations (Jones et al. 1999). One hundred and three mutations in 42 different gene fragments were identified. dHPLC had a sensitivity of 96%, compared to 85% for single strand conformation polymorphism analysis (SSCA) and 82% for gel based heteroduplex analysis (Jones et al. 1999). In a study of the mutations in the BRCA1 gene, dHPLC had 100% sensitivity while SSCA 94% (Gross et al. 1999). Overall, dHPLC has consistently high sensitivity and specificity. dHPLC compared to other mutation detection methods There are many different mutation detection techniques. These include SSCA, heteroduplex analysis, sequencing, denaturing gradient gel electrophoresis (DGGE),

81 dideoxy fingerprinting and enzymatic or chemical mismatch cleavage methods (O’Donovan et al. 1998). Although the first two methods are simple and require little processing of PCR samples, they lack sensitivity unless conducted under several conditions and temperatures. The other more sensitive methods are correspondingly more labour intensive, time-consuming, expensive or involve toxic reagents (O’Donovan et al. 1998). dHPLC has advantages when high through-put is needed as after the initial temperature optimization, the process is automated and little post-PCR sample processing is needed (O’Donovan et al.1998). Although there is an initial investment to purchase the dHPLC system, running costs are low.

There are several advantages of dHPLC over SSCA. This was demonstrated by screening the fibrillin-1 gene in Marfan syndrome (Liu et al. 1997). dHPLC doubled the number of mutations detected compared to SSCA. Also with SSCA, sensitivity is greatly reduced when the fragment size exceeds 300 base pairs (Xiao and Oefner 2001), unlike dHPLC. The sensitivity of SSCA can be improved by the use of multiple gel running conditions, the use of optimised conditions and the use of digests before analysis (Cotton 1997). However all these factors complicate the process and make it more expensive. Fluorescent single-strand conformation polymorphism analysis (F- SSCA) has been reported to have similar specificity and sensitivity to dHPLC (Dobson- Stone et al. 2000, Ellis et al. 2000). However, F-SSCA has the disadvantage of smaller amplicon size and greater expense (Xiao and Oefner 2001).

Prior to the commencement of this study, the different mutation detection methods were reviewed. Consideration was taken of the specific genes to be studied, the number of patients, the number of amplicons, laboratory experience, sensitivity and specificity of various methods, funds and the technical support available. The main factors determining the decision to use dHPLC were the large number of patients and the large number of genes, which made a high throughput system preferable.

At the time that this project started, the sequencing system we had available was the ABI Prism 310 DNA sequencer. This system was only able to process one sequence per hour and the interpretation of results was manual. The system was expensive and not suitable for the high throughput required for this project. With the recent availability of

82 high throughput sequencing systems, sequencing would have been a better option than dHPLC. Sensitivity and specificity are higher and sequencing costs are reduced with a high through-put system as 96 samples can be analysed in one hour and all these samples can be prepared for sequencing in one plate. The results can be analysed quickly and accurately with computer software alignment and mutation detection programs.

Finally, it needs to be recalled that there are a group of mutations that are poorly detected or characterized by both dHPLC and DNA sequencing. Both techniques only detect what a PCR can amplify, therefore hemizygous mutations are not detectable. Similarly mutations that result in whole exon inversions, insertions, duplications or translocations are not detectable even in the presence of an adequate PCR product and require Southern, pulsed field gel electrophoresis, quantitative PCR strategies or multiprobe/multicolour FISH analysis for detection.

Disadvantages and difficulties with dHPLC At the start of the project, a dHPLC system was purchased and a considerable period of time was needed to learn how to use this technique. The Varian column recommended and used for this project had a short life span and has been superseded. The more advanced columns now available may also have improved mutation detection.

One of the difficulties with dHPLC as a screening method is that its accuracy is very dependent upon selection of the correct dHPLC temperature. The optimal dHPLC temperature was assessed by the Stanford melt algorithm but was also confirmed by individual melt curves for every amplicon, as described in the Methods section. However, as positive controls were not available, the temperature selected may not be optimal for detection of all possible mutations. Moreover the Stanford melt program did not always predict the ideal temperature, especially at very high temperatures. In this situation two temperatures were used.

In this project melt curves were considered more accurate than the Stanford melt program, when determining the optimal temperature for dHPLC. However, determining the melt temperature with melt curves means that some mutations may be missed if

83 there are domains with low melt temperatures. These areas may be completely denatured (for both homoduplexes and heteroduplexes) at the temperature chosen and therefore mutations will not be detected (Xiao and Oefner 2001). For this reason results of the Stanford algorithm were also considered. However, in retrospect, more attention should have been given to the lower temperatures when two temperatures were recommended. An example of this was the NDUFV1 mutation, c.331A>G (K111E) that was not identified by dHPLC in amplicon 1 at the dHPLC temperatures of 64°C and 66°C (Table 34). The mutation was located in a segment of the amplicon with a lower recommended melt temperature of 59°C. The melt curve at 59°C showed a small peak before the main peak, and this is likely to have represented the segment of the amplicon containing the mutation. Fortunately, the mutation was detected in amplicon 2, as it was located in the overlap area.

A number of other amplicons were also screened at a dHPLC temperature different from the Stanford recommended (Table 11). The reasons for this are outlined in Table 34. In most cases this was because of a failure to detect a reduction in retention time until a higher temperature was reached (Appendix 2, Figures 18, 21, 22, 23). The decision to use a different temperature is supported by the identification of sequence variants in many of the amplicons. NDUFS7 and NDUFS8 had very high melt temperatures and as no sequence variations were identified in some of their amplicons, there is the possibility that changes were missed at the temperatures tested.

84 Table 34: Amplicons that were not screened at the Stanford recommended dHPLC temperature Amplicon Stanford Temp Reasons Temp °C Used °C NDUFV1 A1 59, 64 64, 66 The temperature 59°C applied to nucleotides at the very end of the amplicon which were also screened in amplicon 2. The mutation in the 6th last base pair of amplicon 1 (c.331A>G) was not detected at 64°C or 66°C but was identified in amplicon 2. It is possible that an early peak representing these lower melt temp bases was missed at the temperatures chosen. NDUFV1 A2 62 64, 65 No significant reduction in retention time below 64°C. Heteroduplexes were detected at the temperatures chosen. NDUFV1 A5 53, 58, 63 65 The lower temperatures (53°C, 58°C) applied to the first five base pairs of amplicon 5 and these were also screened in amplicon 4. No significant reduction in retention time was seen below 64°C. Heteroduplexes were identified at 65°C. NDUFS4 A1 57, 62 59, 62 No significant reduction in retention time below 59°C. At 57°C the first denatured peak is not evident, but it is by 59°C. A number of heteroduplexes were identified at the temperatures tested. NDUFS7 A1 64 66 No significant reduction in retention time seen below 65°C. NDUFS7 A2 64 66 No significant reduction in retention time seen below 65°C. NDUFS7 A3 64 66.5 No significant reduction in retention time seen below 65°C. NDUFS8 A1 63 64, 66 No significant reduction in retention time seen below 66°C. NDUFS8 A2 58, 63 64, 66.5 The lower temperature (58°C) applied to the first 27 base pairs of amplicon 2 which were also screened in amplicon 1. No significant reduction in retention time seen below 66°C. NDUFS8 A3 53, 58, 63 64, 65 The lower temperatures applied to the last 6 bases which are outside the coding region and the first three bases which are screened in amplicon 2. No significant reduction in retention time was observed below 65°C.

Failure to identify sequence variants with dHPLC A number of sequence variants were not identified by dHPLC and the reasons for this are examined.

The c.1056T>C polymorphism identified in Patient 15 in NDUFV1 amplicon 4, was not identified by dHPLC. Amplicon 4 was screened at the Stanford recommended temperature of 62°C and melt curves supported this decision. The samples were also tested at 2°C above the Stanford recommended and this polymorphism was still not detected. The c.1056 nucleotide is located at the centre of amplicon 4 in exon 7 and is in the middle of a 200 nucleotide segment with the same melt temperature of 62°C. Other sequence variants in amplicon 4 were detected at the temperature tested. The normal splice variant present in NDUFV1 amplicon 4, made dHPLC results complicated as

85 extra peaks were present in many samples, prior to the main peak. The extra peaks from the splice variant may have obscured a heteroduplex peak caused by the polymorphism. Patient 15 had a large amount of splice variant present in both dHPLC and sequencing results (Appendix 4, Figure 31).

The c.12C>G polymorphism in NDUFS4 amplicon 1, was very common and of the 54 alleles sequenced, 20 were C and 34 were G. This polymorphism was not identified by dHPLC. The first 118 base pairs of amplicon 1 have a melting temperature of 62°C and this temperature was used. It is unclear why this polymorphism was not detected by dHPLC when many other sequence variants were detected in amplicon 1 at the temperatures used. Other C>G sequence variants, in other genes, were detected by dHPLC.

The NDUFS2 mutation c.998 G>A (R333Q) was identified in Patient 25 by sequencing of amplicon 4, but was not identified by dHPLC. The optimal temperature by melt curve and algorithm was 60°C and the mutation is located in the central area of the amplicon that was predicted to melt at this temperature. A different mutation (c.875T>C, M292T) and a polymorphism (c.1054C>G, P352A) were detected by dHPLC in this amplicon. Patient 25 showed a homoduplex without wild type DNA. When wild-type DNA was added, which was itself a heteroduplex, all samples became heteroduplexes. One possible explanation for the failure to detect the mutation in Patient 25, is that the allele with the missense mutation (c.998G>A, R333Q) is present in the sample in much greater quantity than the allele with the stop mutation (c.661C>T, R221X) secondary to NMD. Hence in amplicon 4, the missense mutation could almost be interpreted as homozygous when DNA sequencing is assessed (Appendix 3, Figure 29). A homozygous mutation would not be detected until wild-type DNA is added, and as the wild type DNA was already a heteroduplex all samples including Patient 25 became heteroduplexes. Presumably, the missense mutation did not alter the heteroduplex pattern sufficiently to allow distinction from other samples.

Another sequence variant in the same patient (Patient 25) was also missed by dHPLC. This was a silent polymorphism in amplicon 5 of NDUFS2 (c.1290C>T). However, this same polymorphism was detected by dHPLC in Patient 33. If this SNP was present on

86 the same allele as the nonsense mutation (R221X), it may be present in such small quantities that it was not detected by dHPLC. Against this explanation, the R221X mutation was easily detected in amplicon 2 by dHPLC. No other clear explanation is apparent.

Taken together, these findings indicate that dHPLC did not have 100% sensitivity for detecting point mutations. The reasons for mutation detection failure were varied. The most likely explanation was that the dHPLC temperature was not ideal to detect that particular sequence change. In order to improve the sensitivity of dHPLC, some studies suggest increasing the column temperature 2°C above the Stanford recommended temperature (Liu et al. 1998, Jones et al. 1999). This was tried for a number of amplicons in this current study (Table 11). Another reason for failure to identify a mutation is that the mutation is located in an extremely high melting domain, surrounded by sequence that melts at significantly lower temperatures (>8°C) (Xiao and Oefner 2001). We did not find evidence of this problem in the current study.

Several of the genes studied in this project had areas with high G-C content, particularly NDUFS7 and NDUFS8, and we wondered whether this may make mutation detection more difficult. However high G-C content alone does not appear to affect dHPLC sensitivity as was shown by analysis of the NOTCH3 gene, which has a mean G-C content of 66% (Escary et al. 2000). dHPLC identified 97% of mutations previously detected by sequencing. However, the dHPLC chromatographic peak was broad rather than multi-peaked for a number of mutations. This was also seen in our study. In amplicon 1 of NDUFS7, the polymorphism c.68T>C (L23P) caused a broad dHPLC peak, rather than a heteroduplex pattern, though the broad curve could still be readily distinguished from homoduplexes. The G-C content of this amplicon is 67%. Again, for amplicon 3 of NDUFS7, with a G-C content of 68%, the c.655C>A 3’UTR polymorphism was identified by a broad dHPLC curve with a prominent left shoulder, but not a heteroduplex pattern. Although high G-C gene content is unlikely to be a major factor affecting mutation detection, it is possible that some mutations were missed for this reason.

87 The use of dHPLC for mutation screening is complicated by the presence of polymorphisms, particularly when the wild type DNA contains single nucleotide polymorphisms (SNPs). This complicated the analysis of the NDUFS1 gene, amplicon 3 where the presence of two unlinked silent polymorphisms (c.966T>G, c.1251A>G) meant that many of the patient samples were heteroduplexes. Although the various combinations of these two polymorphisms explained the complicated dHPLC results, many samples needed to be sequenced. When wild-type DNA also has polymorphisms, identification of a homozygous mutation may be overlooked, if the heteroduplex pattern is similar.

4.2.3 Sequencing

All patients with abnormal dHPLC profiles were considered for sequencing and all mutations were confirmed by sequencing in both directions. Mutations can be missed with cDNA sequencing, due to NMD as discussed above. For some amplicons it was very difficult obtaining good quality sequencing, especially in G-C rich genes.

4.3 DISCUSSION OF RESULTS

4.3.1 NDUFV1

NDUFV1 structure and function NDUFV1 is the 51-kDa subunit of complex I. The gene is located at 11q13 (Ali et al. 1993) and spans about 6kb of genomic DNA (Schuelke et al. 1998). The cDNA transcript is 1566 base pairs and there are 1392 base pairs of coding DNA (Figure 13). There are 10 exons and 9 introns (de Coo et al. 1999), as illustrated in Figure 13. The NDUFV1 protein is 464 amino acids in length, including a 20 amino acid mitochondrial import sequence, which does not appear to be cleaved after import into the mitochondria (Schuelke et al. 1998). The transcriptional start of the NDUFV1 gene is associated with a CpG island (Schuelke et al. 1998). CpG islands are unmethylated and are commonly seen in the 5 prime regions of housekeeping genes which are frequently switched on.

88 There is evolutionary conservation of the NDUFV1 nucleotide sequence and there is high (97%) amino acid sequence homology with the bovine gene (de Coo et al. 1999, Schuelke et al. 1998) (NCBI HomoloGene database). There is also significant homology with bacterial forms of this protein (de Coo et al. 1999).

Figure 13: Organisation of NDUFV1

A. Exons 1 2 3 4 5 6 7 8 9 10

1319 73 729 745 424 195 327 141 106

Introns (base pairs) K111E P252R R386

B. Transcription start ATG Start codon TAG Stop codon

1 2 3 4 5 6 7 8 9 10

1- 73-155 156-326 327-510 511-700 701-913 914-1080 1081- 1163- 1309- 72 1162 1308 1392 Coding 1392 base pairs

Amplicon 1 Amplicon 3 Amplicon 5 PCR and DHPLC Amplicon 2 Amplicon 4 Amplicons

Footnote: A. Genomic structure of NDUFV1 (http://genome.ucsc.edu/cgi refGene_NM_007103). Mutations identified are indicated in boxes. B. cDNA structure, with the amplicons used for PCR and dHPLC analysis indicated

Mitochondrial complex I is the first complex in the mitochondrial respiratory chain. It removes two electrons from NADH and passes them to the electron acceptor, ubiquinone (de Coo et al. 1999). There is simultaneous translocation of four protons

89 into the inter-membranous space. This provides the driving force for the production of ATP by complex V (Schuelke et al.1998). The two electrons are passed initially from NADH to the primary electron acceptor, a non-covalently bound flavin mononucleotide (FMN) and then through the series of iron sulphur clusters to the bound ubiquinone (Benit et al. 2004). NDUFV1 is located in the flavoprotein fraction (FP) of complex I, which consists of three subunits; 51kDa (NDUFV1), 24kDa (NDUFV2) and 10 kDa (NDUFV3). These three subunits mediate the oxidation of NADH (de Coo et al. 1999). NDUFV1 is directly involved in the transfer of two electrons from NADH to ubiquinone.

NDUFV1 protein is ubiquitously expressed and is found in greatest quantities in tissues with the highest energy demands, such as heart, skeletal muscle and areas of the brain (Schuelke 1998).

Schuelke et al. (1998) reported consensus patterns in the NDUFV1 amino acid sequence that indicated the NADH and FMN (flavin mononucleotide) binding sites. A binding site for the tetranuclear iron-sulphur centre was also identified. When NDUFV1 was aligned with the NADH dehydrogenases of a number of prokaryotes, there were three regions of sequence similarity that indicated these binding sites. The locations of these binding sites in the human NDUFV1 amino acid sequence are indicated in Figure 5.

NDUFV1 Mutations Two patients were compound heterozygotes for NDUFV1 mutations; Patient 13 (R386H / K111E) and Patient 27 (R386H / P252R).

R386H: The R386H mutation is located within the consensus motif for the iron sulphur binding site (Figure 5). The signature E-S-C-G-x-C-x-P-C-R-x-G (PROSITE PS00645) (http://www.expasy.org/prosite/) indicates the iron–sulphur binding site of NDUFV1, where the three side chains of the cysteine residues are putative ligands for the tetra iron-sulphur complex (Schuelke et al.1998). The arginine at position 386 is an invariant amino acid in this consensus sequence and is therefore highly conserved across eukaryotic and prokaryotic species (Figure 6) The R386H mutation is predicted to interrupt binding of the iron-sulphur complex.

90 This R386H mutation changes the amino acid from a large polar to one of intermediate polarity. The Grantham chemical difference matrix assesses the chemical relatedness of amino acids based on their composition, polarity and molecular volume (Grantham 1974). Grantham values range from 5-215, with low values indicating chemical similarity and high values indicating radical difference. Values over 120 are likely to indicate a pathogenic change. The Grantham chemical difference values are useful in predicting whether an amino acid substitution is likely to be deleterious (Miller and Kumar 2001). The Grantham chemical difference for arginine and histidine is low (29), placing greater importance on conservation data for the R386H mutation.

This mutation was not present in 50 controls. Based on conservation data and the involvement of a conserved motif, the R386H missense mutation is believed to be of pathogenic significance. It was identified in two apparently unrelated patients, 13 and 27.

K111E: The K111E mutation does not involve one of the known binding sites in NDUFV1, though it is very close to the putative NADH binding site (Figure 5). Lysine and glutamate are both large polar amino acids and the Grantham chemical difference is not high (56). However, this mutation was not identified in 50 controls and it is located in a conserved area of the gene (Figure 6). For these reasons, the mutation is thought to be of pathogenic significance in Patient 13, who also has the R386H mutation.

P252R: The P252R mutation does not involve the known NDUFV1 binding sites, but again it is in a highly conserved area of the gene (Figure 6). The proline is changed to an arginine, and so there is a change in both the charge and size of the amino acid and the Grantham chemical difference is also high at 103 (Grantham 1974). The mutation is not seen in 50 controls. For all these reasons the mutation is believed to be pathogenic in Patient 27, who also has the R386H mutation.

Patient 13 and 27 are compound heterozygotes for NDUFV1 mutations and they show a similar abnormal complex I assembly profile by BN-PAGE analysis (personal communication D. Thorburn, see below for description of BN-PAGE analysis). This adds further support to the pathogenicity of the identified mutations Instead of the

91 normal 900kDa complex, they have a smaller complex of ~750kDa. BN-PAGE analysis has not been reported for patients with NDUFV1 mutations, but a similar profile has been shown for a patient with NDUFS4 mutations (Scacco et al. 2003). Both NDUFV1 and NDUFS4 are located in the complex I hydrophilic arm that protrudes into the mitochondrial matrix. The mutations identified are likely to prevent proper assembly of the matrix arm.

Genotype /Phenotype Correlations in patients with NDUFV1 Mutations Patients 13 and 27 are compound heterozygotes for NDUFV1 mutations. They share one mutation R386H. Patient 13 had postmortem confirmation of Leigh syndrome. Patient 27 had suspected Leigh syndrome based on MRI findings and clinical features. Both patients presented at 5 months of age and had a severe, progressive course with neurological deterioration and death. The patients had similar clinical features, though the Leigh phenotype is not specific to patients with NDUFV1 mutations.

There have been six cases reported of complex I deficiency and NDUFV1 mutations (Table 4). Four had Leigh-like syndromes, one had a progressive macrocystic leukodystrophy and one had lethal neonatal mitochondrial disease. Two of these cases had myoclonic epilepsy (Schuelke et al. 1999). There is a predominance of ocular features in the reported cases (strabismus, ptosis, ophthalmoplegia, retinal depigmentation, blindness) (Table 4) and Patient 26 from this study had ophthalmoplegia and ptosis. All these ocular features can be seen in Leigh syndrome of any cause and are not specific to NDUFV1 mutations. However, ocular features appear to be common in this group. None of the patients reported to date with NDUFV1 mutations have had cardiomyopathy.

4.3.2 NDUFS2

NDUFS2 structure and function The NDUFS2 gene (Genbank Accession No. AF050640) is located at chromosome 1q23 (Procaccio et al. 1998) and codes for the 49kDa subunit of human complex I. The NDUFS2 gene has an open reading frame of 2061 base pairs, which codes for a 463 amino acid protein. The gene has 15 exons (Figure 14).

92 The NDUFS2 gene is highly conserved in prokaryotes and eukaryotes indicating its functional importance (Loeffen et al.1998C). There is 96% homology with the bovine amino acid sequence (Loeffen et al.1998C), 62% homology with N. crassa and 40% homology with E. coli (Loeffen et al. 2001) (NCBI website, Homologene).

Figure 14: Organisation of NDUFS2

A. Exons 1 2 3 4 5 6 7 8 9 10 11 12131415

R221X R333Q

M292T

B.

Exons 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 3 5 PCR and dHPLC Amplicons 2 4

Coding cDNA

Footnote: A. Genomic structure of NDUFS2. Mutations identified are indicated. B. cDNA structure of NDUFS2 with amplicons used in this study indicated

The NDUFS2 subunit is located in the extra membranous matrix arm of complex I near the membrane domain (Loeffen et al. 2001). It is part of the IP fraction. NDUFS2 contains a highly conserved SLR protein kinase C phosphorylation site at amino acids 340-342 (Figure 15). NDUFS2 is also thought to be involved in proton pumping and ubiquinone binding (Loeffen et al. 2001).

93 Figure 15: The amino acid sequence of NDUFS2.

MAALRALCGFRGVAAQVLRPGAGVRLPIQPSRGVRQWQPDVEWAQQFGGA 50

VMYPSKETAHWKPPPWNDVDPPKDTIVKNITLNFGPQHPAAHGVLRLVME 100

LSGEMVRKCDPHIGLLHRGTEKLIEYKTYLQALPYFDRLDYVSMMCNEQA 150

YSLAVEKLLNIRPPPRAQWIRVLFGEITRLLNHIMAVTTHALDLGAMTPF 200 R221X FWLFEEREKMFEFYERVSGARMHAAYIRPGGVHQDLPLGLMDDIYQFSKN 250 M292T FSLRLDELEELLTNNRIWRNRTIDIGVVTAEEALNYGFSGVMLRGSGIQW 300 R333Q DLRKTQPYDVYDQVEFDVPVGSRGDCYDRYLCRVEEMRQSLRIIAQCLNK 350

MPPGEIKVDDAKVSPPKRAEMKTSMESLIHHFKLYTEGYQVPPGATYTAI 400

EAPKGEFGVYLVSDGSSRPYRCKIKAPGFAHLAGLDKMSKGHMLADVVAI 450

IGTQDIVFGEVDR 463

Footnote: The putative mitochondrial leader sequence is underlined (Procaccio et al. 1998). The SLR consensus protein kinase C phosphorylation site is boxed. Mutations identified in this study shown in bold face.

NDUFS2 mutations Patient 17 has an apparent homozygous missense mutation (M292T) in exon 10 of NDUFS2 cDNA. The M292T missense mutation leads to a change from the highly conserved large amino acid methionine to the small amino acid threonine and the Grantham chemical difference is 81 (Grantham 1974). The mutation was not identified in 50 controls. Sequencing of genomic DNA indicated that the mutation was heterozygous. There was loss of the second allele in cDNA presumably due to destruction of an abnormal mRNA transcript by NMD.

This phenomenon has been reported before for the NDUFV1 mutation c.1,268C>T, which appeared to be homozygous in cDNA, but was heterozygous in genomic DNA due to the loss of the paternal allele in cDNA due to a premature stop mutation (R59X) (Schuelke1999). Benit et al. (2001) report four apparent homozygous mutations, two in NDUFV1 and two in NDUFS1 cDNA, which were heterozygous in genomic DNA. In

94 three of these cases the second mutations lead to a prematurely short or unstable mRNA. In the fourth, there was de novo deletion of the paternal allele.

Study of genomic DNA in Patient 17 identified the second mutation as a splice site mutation in intron 9, c.780+4 A>G (or IVS9+4A>G). This mutation would result in the loss of the donor splice site and hence the production of an abnormally spliced transcript which is very likely to be unstable and subject to mRNA decay.

Patient 25 has a premature stop mutation (R221X) in exon 7 which would result in a truncated NDUFS2 protein. He also has a missense mutation in exon 11 (R333Q). Arginine and glutamine are both large polar amino acids and the Grantham chemical difference is only 43 (Grantham 1974). However, this arginine is highly conserved and the mutation was not identified in 50 controls. The entire gene was sequenced in cDNA and no other mutation identified.

The two NDUFS2 missense mutations (M292T, R333Q) do involve the consensus protein kinase C phosphorylation site. However, both the R221X and c.780+4 A>G mutations would result in loss of the phosphorylation site through failure of expression of these unstable alleles.

Genotype /Phenotype Correlations in patients with NDUFS2 Mutations Patients 17 and 25 are both compound heterozygotes for different NDUFS2 mutations. The clinical features were different. Patient 17 had neurological symptoms and lactic acidosis and was still alive at 6 years of age. Patient 25 had a more severe course with probable Leigh syndrome, lactic acidosis and death at 8 months of age.

Hypertrophic cardiomyopathy has been associated with NDUFS2 mutations, being reported in two of three previous cases (Loeffen et al. 2001) (Table 4). However, neither patient in this study with NDUFS2 mutations had documented cardiomyopathy. Also, NDUFS2 mutations were not identified in the 5 patients (Patients 16, 30, 33, 34, 37) with hypertrophic cardiomyopathy or one patient with congestive cardiac failure (Patient 35). Cardiomyopathy is a common clinical finding in mitochondrial encephalomyopathies (Loeffen et al.2001). Hypertrophic cardiomyopathy has also been

95 described in two patients with NDUFS4 mutations (Table 4). In summary, hypertrophic cardiomyopathy is not specific to NDUFS2 mutations, and most patients with cardiomyopathy do not have NDUFS2 mutations.

Optic atrophy was present in one of our patients with NDUFS2 mutations, Patient 17. Optic atrophy was noted in two of three families with previously reported NDUFS2 mutations, and could not be assessed in the third child due to early demise (Loeffen et al. 2001). However, again optic atrophy is common in complex I deficiency and was reported in at least six of 34 of our patient group. Therefore, although optic atrophy appears to be a common feature of NDUFS2 mutations, it is clearly not specific to mutations in this gene.

4.3.3 NDUFS4

NDUFS4 structure and function The NDUFS4 gene is located at 5q11.1 (Emahazion et al.1998). This 18-kDa subunit of complex I has 5 exons and 525 base pairs of coding DNA (Figure 16). The gene is large and spans over 120 kb of genomic DNA, due to the large size of the first two introns that each span over 42 kb (Figure 16). The NDUFS4 protein consists of 175 amino acids including a 42 amino acid mitochondrial leader sequence that is removed after import into the mitochondria (Figure 17).

96 Figure 16: NDUFS4 organization.

A.

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Intron Intron Intron Intron 42, 691 bp 42, 702 bp 12145 24,493

B. Splice variant: 172bp of intron inserted between exons 2 and 3 c.221del C c.198C>A, G66G c.12C>G, c.312G>A, R104R V4V I53T

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5

Amplicon 1

Amplicon 2 Coding DNA

Footnote: A. NDUFS4 genomic structure B. NDUFS4 cDNA structure with location of the amplicons used for dHPLC and sequencing indicated. The mutations, splice variant and polymorphisms are indicated.

97 Figure 17: Human NDUFS4 cDNA and amino acid sequences.

-24 atcctggcgtttgcctgcagcaag -1

atggcggcggtctcaatgtcagtggtactgaggcagacgttgtggcggagaagggcagtg 60 M A A V S M S V V L R Q T L W R R R A V 20

gctgtagctgccctttccgtttccagggttccgaccaggtcgttgaggacttccacatgg 120 A V A A L S V S R V P T R S L R T S T W 40

c.158T>C (I53T) agattggcacaggaccagactcaagacacacaactcaTaacagttgatgaaaaattggat 180 R L A Q D Q T Q D T Q L I T V D E K L D 60 Mature protein c.221delC atcactactttaactggcgttccagaagagcatataaaaaCtagaaaagtcaggatcttt 240 I T T L T G V P E E H I K T R K V R I F 80

gttcctgctcgcaataacatgcagtctggagtaaacaacacaaagaaatggaagatggag 300 V P A R N N M Q S G V N N T K K W K M E 100

tttgataccagggagcgatgggaaaatcctttgatgggttgggcatcaacggctgatccc 360 F D T R E R W E N P L M G W A S T A D P 120

ttatccaacatggttctaaccttcagtactaaagaagatgcagtttcctttgcagaaaaa 420 L S N M V L T F S T K E D A V S F A E K 140

aatggatggagctatgacattgaagagaggaaggttccaaaacccaagtccaagtcttat 480 N G W S Y D I E E R K V P K P K S K S Y 160

ggtgcaaacttttcttggaacaaaagaacaagagtatccacaaaataggttggcactgac 540 G A N F S W N K R T R V S T K - 175

tatatctctgcttgactgtgaataaagtcagctatgcagtatttatagtccatgtataat 600

aaatacatctcttaatctcctaataaattggacctttaaactacaaaaaaaaaaaaaaaa 660

Footnote: The cDNA sequence was obtained from GENBANK (Accession number NM_002495). The predicted amino acid sequence was translated by the Expasy nucleotide/protein translation program (http://www.expasy.org/tools/dna.html). The mitochondrial leader sequence (amino acids 1-42) is italicized. Exon/exon boundaries are indicated by the arrows. Mutations and polymorphisms are in boxes. The phosphorylation consensus sites in the mitochondrial leader sequence (RTS, amino acids 36-38) and in the mature protein (RVS, amino acids 171-173) are underlined. This figure uses information from Papa et al. (1996), Petruzzella et al. (2001) and Scacco et al. (2003).

The NDUFS4 gene is not as highly evolutionarily conserved as the other subunits studied in this project and NDUFS4 does not have a counterpart in E. coli. However NDUFS4 is highly conserved in mammals and there is around 90% amino acid homology with M. musculus and B. taurus. There is about 45% homology with C. elegans and 25% homology with N. crassa (Scacco et al. 2000, Papa et al. 2002).

98 Human NDUFS4 has at least two important roles. These are: i) cAMP-dependent phosphorylation of the NDUFS4 subunit activates complex I (Papa et al. 1996, Papa et al. 2001) and this is thought to play a critical role in the regulation of complex I activity (Petruzzella et al. 2002). The NDUFS4 subunit is phosphorylated by the cAMP-dependent protein kinase (PKA) (Papa et al. 1996) which is located on the matrix side of the inner mitochondrial membrane, as well as in the cytosol (Papa et al. 2002). Elevation of cellular cAMP stimulates respiratory chain activity via phosphorylation of NDUFS4 (Scacco et al. 2000). The cAMP cascade mediates many hormone and neurotransmitter signals that regulate cell processes such as steroidogenesis, neuronal activity, cell proliferation, cell differentiation, cell death, utilisation of lipid stores as well as mitochondrial respiration (Papa et al. 2002).

Phosphorylation / de-phosphorylation sites are frequently associated with regulation of biological processes (van den Heuvel et al.1998). There are two consensus phosphorylation sites in human NDUFS4; one in the mitochondrial leader sequence (RTS) and one close to the end of the mature protein (RVS) (Figure 17). The S residue of the RVS site is phosphorylated (Scacco et al. 2000). Phosphorylation of NDUFS4 is associated with stimulation of activity of complex 1 and overall respiratory chain activity (Scacco et al. 2000). The presence of two phosphorylation sites, one in the mitochondrial leader sequence and one in the mature protein suggests that phosphorylation occurs in both cytosolic and mitochondrial compartments (Papa et al. 1996). This could possibly exert a positive effect on the import of NDUFS4 into the mitochondria or on the assembly of complex I (Papa et al. 1996, Scacco et al. 2000).

Scacca et al. (2003) examined the effect of three homozygous NDUFS4 mutations on the function of NDUFS4. The mutations were 1. AAGTC duplication at position 466-470 (van den Heuvel et al. 1998) 2. c.290delG (W96X) (Budde et al. 2000) 3. c.44G>A (W15X) (Petruzzella et al. 2001).

99 All three mutations led to loss of the carboxy terminal phosphorylation consensus site and the activity of complex I could not be stimulated by cAMP-dependent phosphorylation in these patients. ii) Mutations in NDUFS4 result in failure of normal complex I assembly (Petruzzella et al. 2002, Scacco et al. 2003). All three mutations assessed by Scacca et al. (2003), resulted in impairment of assembly of complex I, as assessed by two dimensional gel analysis (blue native PAGE/SDS-PAGE). There was absence of normally assembled functional complex I in all patients and a non-functional lower molecular weight subcomplex of about 800kDa. As the NDUFS4 subunit is normally located at the junction of the peripheral matrix arm and the membrane arm (Petruzella and Papa 2002), it is not surprising that the loss of the NDUFS4 subunit would lead to failure of assembly of normal complex I.

NDUFS4 mutations Patient 26 is homozygous for the NDUFS4 mutation, c.221delC. The c.221delC would be predicted to result in a translational frame shift and a stop codon 18 codons downstream (91X) (see Results). This mutation was not found in 50 anonymous controls or 40 Lebanese controls. There is little doubt that this mutation would be pathogenic as it would lead to a truncated protein and loss of the NDUFS4 phosphorylation consensus site. In addition, two previously reported patients with premature stop codons in NDUFS4, both failed to assemble normal complex I (Scacco et al. 2003).

Patient 26 was also found to have an NDUFS4 splice variant that resulted in the insertion of 172 base pairs of intronic sequence between exon 2 and exon 3. This 172bp of intronic sequence is usually found in intron 2 (IVS2 nt +1194 to +1365). Subsequent work done by D. Thorburn has shown that the abnormally spliced NDUFS4 variant seen in Patient 26 is a normal splice variant that occurs at low levels in many controls in the presence of cycloheximide. This splice variant is unstable without cycloheximide. It is an alternatively spliced NDUFS4 that generates an unstable mRNA, due to a translational frame shift and a premature stop codon 73 amino acids after the start of the

100 insertion (132X). This instability, as well as the low levels of expression, are likely to explain why this variant was not identified in other patients in our study.

The c.221C mutation leads to the stabilization of the NDUFS4 splice variant in Patient 26, so that it is present in greater quantities in this patient. The c.221delC mutation stabilizes the mRNA splice variant by correcting the translational frame shift, as described in the Results section.

Patient 19 is heterozygous for the same NDUFS4 mutation, c.221delC. Patient 19 has a sister who died with Leigh syndrome and she is also heterozygous for the c.221delC mutation. Analysis of microsatellite markers show they share the same other allele (D. Thorburn). Although the NDUFS4 splice variant was not identified in Patient 19 in this study, it has been demonstrated subsequently in this patient (D Thorburn).

Although the nature of the c.221delC mutation makes pathogenicity almost certain, there are a number of complicating factors in Patient 19. All reported patients with NDUFS4 mutations have autosomal recessive mutations. However, a second mutation has not been identified in this patient. This second mutation may be located in non coding areas of the gene. This patient’s parents are first cousins and yet the c.221delC mutation is heterozygous. However, consanguinity does not exclude the possibility that both parents have different mutations. Alternatively, it is possible that this is a dominant mutation causing complex I deficiency. This is less likely, as this mutation is seen in homozygous form in Patient 26 whose unaffected parents must be heterozygous for this mutation.

In addition, reported patients with NDUFS4 mutations express complex I deficiency in fibroblasts as well as muscle. This was not the case for Patient 19 who does not express complex I deficiency in fibroblasts (personal communication D. Thorburn). Also BN- PAGE immunoblotting in Patient 19 did not demonstrate a smaller complex I assembly intermediate, unlike Patient 26 and other published NDUFS4 patients (D. Thorburn). BN-PAGE on 24hr postmortem muscle from Patient 19 is being undertaken by D. Thorburn to see if complex I assembly is abnormal in different tissues. There may be

101 tissue-specific silencing of the "wildtype" allele of NDUFS4, leading to an NDUFS4 defect in muscle and brain, but not fibroblasts.

Genotype /Phenotype Correlations in patients with NDUFS4 Mutations Patient 26 had a progressive neurological Leigh-like illness with death at 7 years. The patients reported in the literature with NDUFS4 mutations all had very early lethality (Table 4). The slightly less severe phenotype in Patient 26 is difficult to explain as he has a homozygous truncating mutation. However, this mutation leads to a stabilized splice variant which may provide some functional NDUFS4 as the RVS phosphorylation site would be present in this stabilized splice variant in Patient 26.

Patients 26 and 19 have NDUFS4 mutations and both had Leigh-like syndromes. There were no unusual or specific features, except that Patient 19 had hyperkeratotic skin lesions. In the literature, NDUFS4 mutations have been described in five patients. The phenotypes associated with these mutations are summarised in the Table 4. All these patients had Leigh syndrome or Leigh like syndrome. One patient had hypertrophic cardiomyopathy and another concentric hypertrophy of the left ventricle with hyercontractility. The Leigh syndrome phenotype, as well as hypertrophic cardiomyopathy, are not specific to patients with NDUFS4 mutations. Leigh syndrome is seen in other complex I subunit mutations, both mitochondrial encoded as well as nuclear encoded, as well as in other respiratory chain deficiencies. Hypertrophic cardiomyopathy has also been described in patients with NDUFS2 mutations ( Loeffen et al. 2001) and NDUFV2 mutations (Bénit et al. 2003A).

No specific genotype and phenotype correlations which differentiate NDUFS4 mutations from other subunit mutations have emerged.

4.3.4 NDUFS1, NDUFS8 and NDUFS7

Mutations were not identified in the genes NDUFS1, NDUFS7 and NDUFS8. In the literature only six patients have had mutations in these genes identified (Table 4). Therefore it is not surprising that we did not identify mutations in these genes. However, it is possible that mutations in these genes were missed with the mutation

102 screening method chosen, as discussed above. Both NDUFS7 and NDUFS8 are G-C rich and high dHPLC temperatures were used. Although benign sequence variants were identified in some amplicons, mutation detection may have been less than ideal at these high temperatures. For NDUFS1, recommended temperatures were used and several benign sequence variants were identified. It is less likely that a mutation was missed in this gene.

4.3.5 Assessment of pathogenicity of the identified mutations

The evidence that a particular mutation is responsible for the phenotype observed was assessed. Nonsense mutations, frame shift mutations resulting in a downstream premature stop codon, large deletions or insertions or splice mutations can be assumed to be deleterious. The issue of pathogenicity becomes important with missense mutations. Several missense mutations were identified in this study. Some of the criteria considered optimal for determination that a mutation is “disease causing” are summarised from Cotton and Scriver (1998) and Goldgar et al. (2004): • the mutation needs to be confirmed on two separate PCR samples to exclude PCR artifact • the entire gene should be analyzed and mutation detection should not stop after the first mutation is identified as there may be allelism in cis. The region of DNA analyzed and the efficiency of mutation detection should be indicated. • expression studies are ideally needed to confirm the pathogenicity of a missense mutation. If these are not possible the other criteria must be accepted. • segregation analysis to determine whether the mutation segregates with the disease. • evolutionarily conserved amino acids are more likely to be functionally important • replacement of an amino acid with one with a different physical character is more likely to be significant (Miller and Kumar 2001). • one hundred control chromosomes should be studied, as a mutation is more likely to found in less than 1% of alleles.

103 Golgar et al. (2004) developed a model whereby the clinical significance of a missense mutation can be evaluated based on the above factors, as well as additional specific information relevant to the disease of interest. The likelihood ratios for each component of the analysis, such as evolutionary conservation, were calculated. This model allowed classification of several BRCA1 and BRCA2 sequence variants as likely disease causing or benign and will have application for other diseases.

The missense mutations identified in this study mostly fulfill the above criteria. Three missense mutations were identified in NDUFV1, c.1157G>A (R386H), c.331A>G (K111E) and c.755C>G (P252R). In NDUFS2 two missense mutations were identified c.875T>C (M292T) and c.998G>A (R333Q). All mutations were confirmed in both cDNA and gDNA, excluding PCR artifact. The entire gene was always screened by dHPLC to identify any other sequence variants. For NDUFS4 and NDUFS2 the entire gene was also sequenced in patients with identified mutations. One limitation of this study was the failure to sequence the entire NDUFV1 gene in Patients 13 and 27 who were compound heterozygotes for NDUFV1 mutations. All amplicons were screened by dHPLC and all abnormal amplicons sequenced. As the sensitivity of dHPLC is less than 100% it is possible that one of the mutations identified in these patients is not the disease causing mutation, which may be located elsewhere in the gene. The K111E mutation in Patient 13 causes a conservative amino acid change, and ideally the entire gene should have been sequenced, in addition to screening by dHPLC, to ensure that all sequence variants were detected. Considering the high sensitivity of dHPLC the chance that another mutation was missed is low.

None of the mutations were identified in 100 control chromosomes. The amino acids affected were all highly conserved. Not all mutations resulted in the substitution of an amino acid with very different physical characteristics eg. R386H, R333Q, K111E. For these mutations other evidence was strong, such as evolutionary conservation of the amino acid. Also the finding of a clearly pathogenic second mutation suggested that the missense mutation was significant. Parental samples were available for some patients, allowing segregation analysis (see Results). As described above, studies such as BN- PAGE have been subsequently performed to assess complex I assembly in some of our

104 patients. Evidence of a complex I assembly defect supported the conclusion that these mutations were pathogenic.

The three missense changes (R147W in Patient 15 in NDUFV1, I53T in Patient 22 in NDUFS4 and the NDUFS2 missense change P352A in Patients 3, 14, 18, 22 and Control 5) did not have sufficient evidence of pathogenicity, based on the criteria above. The analysis of evidence for each of these missense changes is presented in the Results. These sequence variants are of uncertain, but unlikely, pathogenic significance.

4.4 WHERE ARE THE MUTATIONS IN THE OTHER PATIENTS WITH COMPLEX I DEFICIENCY?

4.4.1 Summary of results on our cohort of 34 complex I deficient patients

Thirty four patients with confirmed complex I deficiency were studied and mutations in nuclear encoded subunits were identified in only 6 of these 34 patients (Table 35). Before commencement of this study, known mitochondrial DNA complex I subunit mutations were excluded in this patient group. However, further mitochondrial DNA complex I subunit mutations have since been identified, including the novel ND3 mutation 10158T>C in Patients 6 and 7 (McFarland et al. 2004) and the heteroplasmic mtDNA mutation 12706T>C in Patient 15 (D. Thorburn). Homozygous NDUFS6 subunit mutations were recently identified in Patients 2 and 3 who presented with primary lactic acidosis (Kirby et al. 2004). This still leaves 23/34 (68%) patients in whom the genetic basis for complex I deficiency remains unknown. The possible aetiological basis of complex I deficiency in these patients is discussed below.

4.4.2 Complex I assembly and activation

Mutations in the nuclear encoded structural subunits have been shown to cause complex I deficiency. This is different to the situation with Complex IV deficiency, in which no mutations have been identified in the 10 nuclear encoded subunits (Tiranti et al 1998). In complex IV deficiency mutations in SURF1, which is important in the biogenesis of the whole complex have been found (Zhu et al. 1998, Tiranti et al. 1998) and have been

105 associated with a specific Leigh syndrome phenotype with relative sparing of cognitive function (Thorburn et al. 1999). Mutations in other complex IV assembly factors (SCO2, SCO1, COX10) can also cause complex IV deficiency (Shoubridge 2001). The clinical features in complex I deficiency are more variable than those in complex IV deficiency and this may be explained in part by the even greater number of genes involved in complex I deficiency.

It is likely that there are also complex I assembly and regulation factors involved in complex I deficiency. Mutations in these genes may account for complex I deficiency in our undiagnosed group. Two novel chaperones for complex I membrane arm assembly (CIA30 and CIA84) were identified in N. crassa (Küffner et al. 1998). The human homologue of CIA30 has been identified, proving that complex I assembly factors do exist (Ugalde et al. 2004). Mutations causing human disease have not yet been identified.

Ugalde et al. (2004) studied a number of patients with complex I deficiency and showed that those with nuclear subunit mutations had abnormal assembly of complex I. They also studied nine complex I deficient patients where the gene defect was unknown. Complex I subunit genes (45/46) had been studied and no mutations identified. Blue native electrophoresis identified two separate groups of patients- those with assembly defects (2 patients) and those with catalytic defects (7 patients). The two patients with assembly defects had very low levels of fully assembled complex I and very low levels of complex I activity. This suggests an assembly defect and the two patients are good candidates for mutations in a complex I subunit or in a complex I assembly factor. The seven patients in the second group have normal levels of fully assembled complex I, but reduced levels of complex I activity, suggesting a catalytic defect. Mutations would be expected to be found in nuclear genes that regulate complex I activation.

On the basis of this, it is likely that some of our patients without identified subunit mutations, will have mutations in either nuclear encoded assembly genes or in a gene involved in complex I activation or regulation.

106 Table 35: Genetic defects identified in our patients with complex I deficiency.

Patient Complex ID No. Clinical Diagnosis Deficiency Genetic Defect 1 Leigh syndrome I - 2 Primary Lactic acidosis I NDUFS6 1 3 Primary Lactic acidosis I NDUFS6 1 4 Primary Lactic acidosis I - 5 ?Leigh-like syndrome I - 6 Leigh syndrome I ND3 T10158C 2 7 Leigh-like syndrome I ND3 T10158C 2 8 affected pregnancy I - 9 LIMD4 I (III) - 10 mitochondrial myopathy I - 13 Leigh syndrome I NDUFV1 14 Leigh syndrome I - 15 Leigh syndrome I ND5 T12706C 3 16 Leigh syndrome I - 17 Lactic acidosis I NDUFS2 18 Leigh syndrome I - 19 Leigh-like syndrome I NDUFS4 20 Leigh syndrome I - 21 Leigh syndrome I - 22 Leigh syndrome I - 23 ?Leigh syndrome I - 24 Leigh syndrome I - 25 Leigh-like syndrome I NDUFS2 26 Leigh syndrome I NDUFS4 27 Leigh-like syndrome I NDUFV1 29 Leigh disease I - 30 Cardiomyopathy I - 31 Leigh syndrome I (,IV?) - 32 Primary Lactic acidosis I - 33 Cardiomyopathy I - 34 Cardiomyopathy I - 35 mitochondrial disease I - 36 Leigh-like syndrome I - 37 Cardiomyopathy I - Footnote: 1 Patients 2 and 3 subsequently identified as having homozygous NDUFS6 mutations (Kirby et al. 2004). 2Patients 6 and 7 identified as having a mitochondrial DNA point mutation (T10158C) in the structural complex I subunit ND3 (McFarland et al. 2004) 3A heteroplasmic pathogenic mutation in ND5 (12706T>C) subsequently identified (personal communication Dr D. Thorburn). 4LIMD Lethal Infantile Mitochondrial Disease

107 4.5 CLINICAL AND LABORATORY APPLICATIONS OF SUBUNIT MUTATION ANALYSIS

4.5.1 Approach to molecular genetic diagnosis of complex I deficiency

Mammalian mitochondrial complex I is composed of at least 46 subunits- seven encoded for by mitochondrial DNA and the remainder encoded for by genomic DNA (Carroll et al. 2003). Although mutations in complex I subunits can cause complex I deficiency these mutations account for only a proportion of complex I deficiency. It is highly likely that genes involved in complex I assembly, stability, activation or regulation will also be involved in human disease. Hence, when a child is diagnosed with complex I deficiency, the genetic diagnosis is complicated by the large number of genes that are potentially involved. It is not practical, nor possible, in most cases to screen this large number of genes using currently available technology.

For this reason, alternative techniques to identify and characterize the complex I defect prior to mutation analysis are being developed. Characterization of the complex I defect means that mutation detection can be targeted towards a particular gene or group of genes. For example, if complex I deficiency results from impaired catalytic activity or from failure of correct assembly of complex I, the genes likely to be involved are different (Antonicka et al 2003). Impaired complex I assembly is seen in subunit mutations and so these genes should be examined first. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a technique that can identify assembly defects and so can guide mutation detection efforts.

4.5.2 Blue native gel Electrophoresis and Complex I assembly

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a useful technique for the diagnosis of respiratory chain disorders. Van Coster et al. (2001), describes this technique in detail, as summarised below. The oxidative phosphorylation complexes (I, II, III, IV, V) can be separated by BN-PAGE. The complexes remain in their native state during separation and hence the enzymatic activity of complexes I, II, IV and V is maintained and can be demonstrated by catalytic staining. Patients with deficiency of a

108 particular respiratory chain complex will have reduction in the corresponding enzyme band on the gel. In addition, the quantity of complex I (and V and III) can be assessed in samples from heart and muscle by BN-PAGE followed by Coomassie or silver staining. In fibroblasts and liver, the complex protein quantity can be assessed by immunoblotting after BN-PAGE. Following the first BN-PAGE, a second Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) can be undertaken to separate the individual subunits of each respiratory chain complex. The subunits can be visualized by protein staining or by immunoblotting using specific antibodies. The subunit profile may indicate the likely defect. Distinct patterns of both fully and partially assembled complexes are associated with specific defects. These techniques do not replace the spectrophotometric diagnostic assays of complex activity but do provide additional information as to the aetiology of the complex deficiency.

Triepels et al. (2001) used monoclonal antibodies to characterize distinct subunit assembly patterns in patients with complex I deficiency. They used a group of monoclonal antibodies that react with the 39, 30, 20, 18, 15 and 8kDa subunits of complex I. Western blotting with these antibodies, combined with sucrose gradient studies and enzymatic activity measurements, was able to differentiate catalytic defects from assembly defects. For patients with assembly defects, the assembly profile could distinguish between mutations in different subunits as different mutations in the same gene showed very similar subunit profiles.

Antonicka et al. (2003) used two-dimensional Blue-Native /SDS gel electrophoresis and a panel of 11 antibodies directed against subunits of complex I to investigate complex I assembly. They studied muscle mitochondria from four patients with complex I deficiency. Two patients had mtDNA mutation and two had nuclear gene mutation as the cause for the complex I deficiency. All patients had reduced levels of complex I activity on BN-PAGE. Western blot analysis showed that all patients had markedly reduced levels of fully assembled complex I. Antibodies against two nuclear (39-kDa, 49-kDa) and one mitochondrial (ND1) subunit, identified five common subcomplexes with molecular masses of 250, 310, 380, 480 and 650kDa. Eleven antibodies directed against complex 1 subunits were then used to determine the composition of the subcomplexes, in combination with two dimensional gel electrophoresis. Two further

109 subcomplexes, 200 and 230kDa were identified in this way. In total, seven different complex I subcomplexes were identified in second dimension denaturing gels in all complex I patients. They all had the same constellation of subcomplexes, regardless of the underlying cause of their complex I deficiency, although the relative amount of each of the subcomplexes varied between patients. Antonicka et al. (2003) suggest a complex I assembly model based on the concept that these seven subcomplexes are intermediates in complex I assembly. This model of complex I assembly is quite different for that proposed for N. crassa (Schulte 2001) as many more assembly intermediates are observed and results suggest that the membrane arm and the peripheral arm are not assembled in separate independent pathways.

Ugalde et al. (2004) used blue native electrophoresis to study 15 patients with complex 1 deficiency- six with known nuclear-encoded subunit mutations (NDUFS2, NDUFS4, NDUFS7 and NDUFS8) and nine with unknown nuclear defects. Patients with known mutations in nuclear-encoded subunits, all had decreased levels of fully assembled complex I indicating that either complex I stability or assembly had been compromised. Decreased levels of fully assembled complex I correlated with the reduced complex I activity levels, suggesting that the assembly/stability defect is the primary pathogenic mechanism. NDUFS2, NDUFS4, NDUFS7 and NDUFS8 are all located in the peripheral arm, and mutations in these genes would be predicted to affect assembly or activity of this section. Depending on the gene involved (NDUFS2, NDUFS4, NDUFS7 or NDUFS8) different patterns of low molecular weight subcomplexes were identified, suggesting that the assembly of the peripheral arm is disrupted at an early stage. There is an accumulation of these smaller subcomplexes 100-450kDa. However, these subcomplexes are also seen in all patients and controls and are likely to be normal assembly intermediates as described in the proposed complex I assembly pathway (Antonicka et al. 2003). They accumulate in patients with subunit mutations, who fail to complete the correct assembly process.

All the reported patients with NDUFS4 mutations have very low levels of fully assembled complex I together with high levels of a 800kDa subcomplex. This subcomplex represents the complex I peripheral arm with subunits that are normally found in the boundary between the peripheral and membrane arm (NDUFS5,

110 NDUFA9). The membrane arm and peripheral arm have not joined together in NDUFS4 mutation patients. Also the NDUFS4 subunit is not found in the 800kDa subcomplex, indicating that NDUFS4 would normally be incorporated at a late stage of the assembly process, as suggested in the proposed assembly model (Antonicka et al. 2003). It is noted that rather than a failure of assembly, it is possible that assembly occurs but the complex I is unstable and therefore disrupted or that the rate of assembly is affected by the NDUFS4 mutations.

Ugalde et al (2004) also observed that complex I subunit mutations can also affect the stability of other respiratory chain complexes. There was a decrease in fully assembled complex III in patients with NDUFS2 and NDUFS4 mutations (but not NDUFS7 or NDUFS8 mutations or the group of patients with unknown mutations). The decreased stability of complex III seems to cause only a slight if any reduction in complex III activity. The reduced complex III stability fits with the finding of combined complex I and III deficiency in three patients with NDUFS4 mutations (Budde et al. 2000, Budde et al. 2003). Also combined complex I and III deficiency has been identified in a patient with a cytochrome b gene mutation (Lamentea et al. 2002). These findings suggest a physical interaction between complex I and complex III that is perhaps mediated through the subunits NDUFS2 and NDUFS4. This physical interaction is supported by the known joining of OXPHOS complexes into supercomplexes (or respirasomes) (Schägger 2004). Complex I and complex III combine to form a stable core respirasome in the inner mitochondrial membrane and Complex IV can also bind to this complex (Schägger 2004). This finding may also explain why patients with NDUFS2 and NDUFS4 mutations have similar phenotypes.

In our study, BN-PAGE electrophoresis was performed by D Thorburn in the patients with identified mutations. The two patients (13, 27) compound heterozygous for NDUFV1 mutations both had similar abnormal assembly profiles for complex I by BN- PAGE analysis. Instead of the normal 900kDa complex, they had predominantly a smaller complex of ~750 kDa. Abnormal complex I assembly has not been previously reported for NDUFV1 mutations, but a similar abnormal assembly profile has been described for NDUFS4 mutations (Scacco et al. 2003). Both NDUFV1 and NDUFS4 subunits are in the complex I hydrophilic arm that protrudes into the mitochondrial

111 matrix. The mutations identified may prevent proper assembly of this matrix arm. BN- PAGE in Patient 25 who has NDUFS2 mutations showed a decreased amount of full size complex I with no abnormal smaller assembly intermediates, consistent with previous reports of patients with NDUFS2 mutations (Ugalde et al. 2004). BN-PAGE analysis was also undertaken in the two patients with NDUFS4 mutations. BN-PAGE immunoblotting using Patient 19 fibroblasts was normal and did not demonstrate a smaller assembly intermediate of 750-800kDa, unlike Patient 26 and all other published NDUFS4 patients (Ugalde et al. 2004). As there is evidence that the c.221 delC mutation is pathogenic in Patient 19, this finding was unexpected. BN-PAGE, using postmortem muscle from Patient 19, is being undertaken by D.Thorburn to see if complex I assembly is abnormal in other tissues.

4.6 CONCLUSIONS AND SIGNIFICANCE OF FINDINGS

There have been previous reports of mutations in nine nuclear-encoded complex I genes, in a total of 25 families worldwide and so there is still only limited information about the spectrum of genes and mutations involved in complex I deficiency. Complex I subunit mutations account for an unknown proportion of complex I deficiency – somewhere less than 50% (Antonicka et al. 2003).

In one research cohort of 24 of complex I deficient patients, 10/24 (42%) had nuclear encoded subunit mutations identified when all nuclear encoded subunits were sequenced (Triepels et al. 2001, Ugalde et al. 2004) (see Introduction). Benit et al. (2001) identified complex I nuclear subunit mutations in 6/36 (17%) complex I patients, though not all complex I subunit were screened. This group has subsequently identified mutations in three further patients in NDUFV2, NDUFS3 and NDUFS4. (Bénit et al. 2003A, 2003B, 2004) This would mean around 20-25% of the patients have nuclear encoded complex I subunit mutations identified (see Introduction).

In our study, 18% of complex I patients (6/34) had nuclear complex I subunit mutations identified and subsequently two further patients have had NDUFS6 mutations identified (Kirby et al. 2004). This makes a total of eight patients (8/34, 24%) with identified

112 nuclear encoded subunit gene mutations in our patient group - not significantly different from previous studies. Patients with known mitochondrial DNA mutations were excluded prior to study entry, making the proportion of complex I patients with nuclear subunit mutations a little lower than the figure suggests. However the other major study (Triepels et al 2001) also excluded patients with known mitochondrial mutations.

There are several factors that influence the mutation detection rate in different studies. Firstly, there are likely to be differences in the ethnic background of the different study groups and this may result in different mutation spectrums, with different genes being involved. Secondly, and most importantly, it is possible that nuclear mutations are located in genes that were not screened in our study, particularly the core complex I subunit genes NDUFV2 and NDUFS3. The accuracy of the mutation screening method also affects mutation detection. The study by Triepels et al. (2001) and colleagues used direct DNA sequencing, whereas our study utilized dHPLC which has a slightly lower sensitivity (Xiao and Oeffner 2001). Finally it is also possible that the category of mutation present is not easily detectable by PCR technologies, and other systems that can analyze genomic position and/or the quantitation of exon number may be required.

By identifying gene mutations in the Australian group of patients, further valuable knowledge about the incidence and spectrum of nuclear encoded complex I subunit mutations has been achieved. NDUFS4, NDUFS2, NDUFV1 and NDUFS1 are the nuclear encoded complex I subunit genes that are the most commonly involved in complex I deficiency. NDUFS4 is the subunit with the most identified mutations to date (Table 4).

The majority of nuclear encoded complex I subunit mutations are novel and specific to an individual family (Table 4). There are only three mutations that have been identified in more than one complex I patient. The NDUFS4 mutation 316C>T, R106X, has been identified in two unrelated patients (Budde et al. 2000, Budde et al. 2003). In our study, the NDUFV1 mutation R386H was identified in two apparently unrelated individuals and the NDUFS4 mutation c.221delC was also identified in two Lebanese patients. Although these patients were not known to be related, it is possible that they share a distant ancestor.

113

Although there were some similarities in phenotype between individuals with the same mutation, there were also differences. In our study, there were no specific or characteristic clinical features to implicate a particular gene or mutation. NDUFV1 mutations were seen in two patients with Leigh or Leigh-like syndrome. Ocular features appear to be common in patients with NDUFV1 mutations and cardiomyopathy has not been reported in this group. Further information is needed to assess the significance of these observations. Our two patients with NDUFS4 mutations both had Leigh-like syndrome, though there were no clinical features that distinguished them from other patients, other than Lebanese ancestry. The phenotype of our two NDUFS4 patients was less severe than the previously reported patients, though all NDUFS4 patients have had Leigh syndrome or Leigh–like syndrome (Table 4).

Our two patients with NDUFS2 mutations had different mutations and different clinical features. Patient 17 had neurological symptoms and lactic acidosis and was still alive at six years of age. Patient 25 had a more severe course with probable Leigh syndrome, lactic acidosis and death at eight months of age. Cardiomyopathy has been reported to be associated with NDUFS2 mutations (Loeffen et al. 2001). Our NDUFS2 patients did not have cardiomyopathy, and our six patients with cardiomyopathy did not have NDUFS2 mutations.

This information contributes to our understanding of genotype and phenotype in complex I deficiency. It was hoped that specific correlations could be identified so that patients suspected of this disorder could undergo gene mutation analysis of blood, potentially avoiding the need for invasive diagnostic muscle and liver biopsies. The attempt to correlate clinical or biochemical phenotype with genotype is particularly important because of the large number of genes potentially involved. At this point however, specific genotype/phenotype correlations have not emerged. Even the previously reported association of cardiomyopathy and NDUFS2 mutation is not specific. What is likely to be more practical is the correlation between complex I assembly patterns and nuclear encoded subunit involvement, as described above.

114 Screening of six nuclear encoded complex I subunits has identified a small but significant number of causative mutations in complex I deficient patients (18%). For our six families with identified mutations in complex I genes, prenatal diagnosis, carrier testing and molecular genetic diagnosis in affected family members can now be offered. Of utmost importance, accurate genetic counseling can be provided for the family allowing them to make informed reproductive decisions. Complex I deficiency is a rare disorder and there is limited information available on the molecular genetic basis of this disorder. This study contributes further important information on the spectrum of mutations in complex I deficiency, as well as adding to the accumulating data on clinical correlates in patients with complex I subunit mutations.

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131 ELECTRONIC DATABASE INFORMATION

1. Online Mendelian Inheritance in Man (OMIM) http://www.ncbi.nlm.nih.gov/Omim/

2. NCBI HomoloGene databasehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene

3. Expasy nucleotide/protein translation program http://www.expasy.org/tools/dna.html

4. NCBI Conserved Domain Database (CDD) Marchler-Bauer A, Anderson JB, DeWeese-Scott C et al (2003), "CDD: a curated Entrez database of conserved domain alignments", Nucleic Acids Research 31:383-387

5. Enzyme EC reference numbers from http://www.expasy.org/cgi-bin/nicezyme

6. Stanford dHPLC melt programme (http://insertion.stanford.edu/melt.html)

7. Genomic structure of NDUFV1 (http://genome.ucsc.edu/cgi refGene_NM_007103

132 Appendix 1: RNA concentrations

Patient Total RNA Conc No. Disease Status RNA/volume ug/ml OD260/OD280 1 Affected 40 µg/500 µl 80 1.568 2 Affected 82 µg/500 µl 164 1.556 3 Affected 27 µg/300 µl 90 1.732 4 Affected 81 µg/300 µl 270 1.68

5 Affected 76 µg/300 µl 253 1.617

Affected 6 130 µg/300 µl 433 not done 7 Affected 124 µg/300 µl 413 not done 8 Affected 74 µg/300 µl 246 not done 9 Affected 156 µg/300 µl 520 not done 10 Affected 24 µg/300 µl 80 not done 11 Not Confirmed 45 µg/300 µl 150 not done

12 Not Confirmed 45 µg/300 µl 150 not done

Affected not done 13 64 µg/300 µl 213 14 Affected 184 µg/300 µl 613 not done 15 Affected 69 µg/300 µl 230 not done 16 Affected 64 µg/300 µl 213 not done 17 Affected 82 µg/300 µl 273 not done 18 Affected 69 µg/300 µl 230 not done

19 Affected 126 µg/300 µl 430 not done

Affected not done 20 130 µg/300 µl 433 21 Affected 144 µg/300 µl 480 not done 22 Affected 48 µg/ 300µl 160 not done 23 Affected 108 µg/300 µl 360 not done 24 Affected 69 µg/300 µl 230 not done 25 Affected 75 µg/300 µl 250 not done

26 Affected 87 µg/300 µl 290 not done

not done 27 Affected 100 µg/300 µl 333 28 Not Confirmed 57 µg/300 µl 190 not done 29 Affected 94 µg/300 µl 315 not done 30 Affected 117-180 µg/ 300 µl 390-600 not done 31 Affected 102 µg/300 µl 340 not done 32 Affected 93 µg/300 µl 310 not done

33 Affected 57 µg/300 µl 180 not done

Affected not done 34 202 µg/300 µl 673 35 Affected 165 µg/300 µl 550 not done 36 Affected 84 µg/300 µl 280 not done 37 Affected 58 µg/300 µl 193 not done

133 Appendix 2: Figure 18 – NDUFV1 melt curves

18A. NDUFV1 amplicon 1 dHPLC melt profile- selected temperatures 64°C and 66°C

58.5°C

63°C

62°C

61°C

50°C

18B. NDUFV1 amplicon 1 dHPLC melt profile- selected temperatures 64°C and 66°C

50°C

62°C 62°C 59°C 57°C 60°C 63°C 64°C 65°C

63°C

134 Appendix 2: Figure 18 – NDUFV1 melt curves

18C. NDUFV1 amplicon 2 dHPLC melt profile- selected temperatures 64°C and 65°C

50°C 62°C

63°C

64°C

65°C

50°C

18D. NDUFV1 amplicon 3 dHPLC melt profile- selected temperatures 63°C and 64°C

50°C 50°C 63°C 62°C 62°C 64°C59°C 57°C 60°C 65°C 64°C63°C

63°C

66°C 65°C

135 Appendix 2: Figure 18 – NDUFV1 melt curves

18E. NDUFV1 amplicon 4 dHPLC melt profile - selected temperatures 62°C and 64°C

64°C 50°C50°C 63°C 65°C 63°C 62°C 64°C 62°C61°C

66°C 65°C

18F. NDUFV1 amplicon 5 dHPLC melt profile- selected temperature 65°C

50°C 61°C

65°C 63°C 64°C 62°C

136 Appendix 2: Figure 19 – NDUFS1 melt curves

19A. NDUFS1 Amplicon 1 dHPLC melt profile - selected temperature 60°C .

50°C

59°C

60°C

62°C 61°C

19B. NDUFS1 Amplicon 2 dHPLC melt profile - selected temperatures 58.5°C and 59°C

50°C

58°C

59°C 58.5°C 60°C

137 Appendix 2: Figure 19 – NDUFS1 melt curves

19C. NDUFS1 Amplicon 3 melt profile - selected temperatures 59°C and 60°C. Control 5 was a heteroduplex.

50°C

58°C 59°C 61°C 60°C

19D. NDUFS1 Amplicon 4 melt profile - selected temperature 59°C (Control 2 is a homoduplex)

50°C

58°C

58.5°C

59°C 59.5°C 60°C

138 Appendix 2: Figure 19 – NDUFS1 melt curves

19E. NDUFS1 Amplicon 4 melt profile - selected temperature 59°C (Control 5 is a heteroduplex)

50°C

58°C

59°C 59.5°C

60°C

19F. NDUFS1 Amplicon 5 melt profile - selected temperatures 58.5°C and 59°C)

50°C

58°C

58.5°C 60°C 59°C

139 Appendix 2: Figure 20 – NDUFS2 melt curves

20A. NDUFS2 Amplicon 1 dHPLC melt profile - selected temperature 63°C

65°C 63°C 64°C 66°C 50°C 62°C

20B. NDUFS2 Amplicon 2 dHPLC melt profile - selected temperature 61°C

50°C

60°C

61°C

62°C

63°C

140 Appendix 2: Figure 20 – NDUFS2 melt curves

20C. NDUFS2 Amplicon 3 dHPLC melt profile - selected temperature 61°C

50°C

60°C

61°C

63°C 62°C

20D. NDUFS2 Amplicon 4 dHPLC melt profile - selected temperature 60°C

50°C

63°C

60°C 59°C

62°C 61°C

141 Appendix 2: Figure 20 – NDUFS2 melt curves

20E. NDUFS2 Amplicon 4 dHPLC melt profile - selected temperature 60°C

50°C

59°C 60°C

61°C

62°C

142 Appendix 2: Figure 21 - NDUFS4 melt curves

21A. NDUFS4 amplicon 1 dHPLC melt profile (50°C - 61°C) - selected temperatures 59°C and 62°C

61°C

60°C

59°C 58.5°C 58.5°C

58°C

57°C

50°C

21B. NDUFS4 amplicon 1 dHPLC melt profile (57°C- 63°C)- selected temperatures 59°C and 62°C

61°C50°C

59°C 62°C 57°C 60°C

63°C

143 Appendix 2: Figure 21 NDUFS4 melt curve

21C.NDUFS4 amplicon 2 dHPLC melt profile- selected temperature 58°C

50°C

57°C

57..5°C

58°C 59°C

21D. NDUFS4 amplicon 1 dHPLC melt profile- selected temperatures 59°C and 62°C

61°C50°C

59°C 62°C 60°C 57°C

63°C

144 Appendix 2: Figure 22 – NDUFS7 melt curves

22A. NDUFS7 amplicon 1 dHPLC melt profile- selected temperatures 66°C and 66.5°C

50°C 65°C 66°C 64°C

66.5°C

67°C

22B. NDUFS7 amplicon 2 dHPLC melt profile- selected temperature 66°C

61°C 65°C 66°C 63°C 50°C

67°C

145 Appendix 2: Figure 22 – NDUFS7 melt curves

22C. NDUFS7 amplicon 2B dHPLC melt profile- selected temperature 66°C

50°C

63°C 64°C 65°C 66°C

67°C

22D. NDUFS7 amplicon 3 dHPLC melt profile- selected temperature 66.5°C

64°C 50°C 65°C

66°C 66.5°C

67°C

146 Appendix 2: Figure 23 – NDUFS8 melt curves

23A. NDUFS8 amplicon 1 dHPLC melt profile- selected temperatures 64°C and 66°C (continued in next figure)

61°C 50°C 64°C

63°C

23B. NDUFS8 amplicon 1 dHPLC melt profile- selected temperatures 64°C and 66°C (continued)

66°C

67°C 65°C

64°C

147 Appendix 2: Figure 23 – NDUFS8 melt curves

23C. NDUFS8 amplicon 2 dHPLC melt profile- selected temperatures 64°C and 66.5°C (continued in next figure)

64°C 63°C 62°C 61°C 50°C

23D. NDUFS8 amplicon 2 dHPLC melt profile- selected temperatures 64°C and 66.5°C (continued)

67°C 66°C 65°C 64°C

148 Appendix 2: Figure 23 – NDUFS8 melt curves

23E. NDUFS8 amplicon 3 dHPLC melt profile- selected temperature 64°C and 65°C (continued in next figure)

62°C 61°C 50°C 63°C

64°C

23F. NDUFS8 amplicon 3 dHPLC melt profile- selected temperatures 64°C and 65°C (continued)

65°C 66°C

64°C

149 Appendix 3, Figure 24: Sequencing of Patients 13 and 27, NDUFV1 mutation R386H

Patient 13: primer NDUFV1 F5M c.1157G>A, R386H

Patient 27: primer NDUFV1 F5M c.1157G>A, R386H

Normal for comparison

150

Appendix 3, Figure 25: Sequencing of Patient 13, NDUFV1 mutation K111E

Patient 13: primer NDUFV1 F2M c.331A>G, K111E

Normal for comparison

151

Appendix 3, Figure 26: Sequencing of Patient 27, NDUFV1 mutation P252R

Patient 27: Primer NDUFV1 F3 c.755C>G, P252R

Normal for comparison

152

Appendix 3, Figure 27: Sequencing of Patient 17, NDUFS2 mutation M292T

Patient 17: primer NDUFS2 F4 (cDNA sequencing) apparent homozygous c.875T>C, M292T

Normal for comparison

153 Appendix 3, Figure 28: Sequencing of Patient 25, NDUFS2 mutation R221X

Patient 25: primer NDUFS2 F3 c.661C>T, R221X

Normal for comparison

Patient 25: primer NDUFS2 R3 c.661C>T, R221X

Normal for comparison

154

Appendix 3, Figure 29: Sequencing of Patient 25, NDUFS2 mutation R333Q

Patient 25: primer NDUFS2 F4 c.998G>A, R333Q

Normal for comparison

Patient 25: primer NDUFS2 R4 c.998G>A, R333Q

Normal for comparison

155

Appendix 3, Figure 30: Sequencing of Patients 19 and 26, NDUFS4 mutation c.221delC

Patient 19: NDUFS4 heterozygous c.221delC, seen best with primer NDUFS4 R1

Normal sequence for comparison

Patient 26: homozygous NDUFS4 c.221delC is indicated by the arrow. The sequencing primer is NDUFS4 R1, so the deletion is of a G. The deletion could not be seen with the forward primer due to the insertion. The beginning of the 172 base pair insertion (splice variant) between exon 2 and 3 is indicated.

homozygous c.221delC Insertion

Normal sequence for comparison

156 Appendix 4, Figure 31: dHPLC chromatograms for NDUFV1 amplicon 4

Patient 15: NDUFV1 Amplicon 4, 62°C Patient 29: NDUFV1 Amplicon 4, 62°C

Patient 24: NDUFV1 Amplicon 4, 62°C Patient 14: NDUFV1 Amplicon 4, 62°C

Patient 27: NDUFV1 amplicon 4, 62°C Patient 13: NDUFV1 Amplicon 4, 62°C c.1157G>A, R386H mutation c.1157G>A, R386H mutation

Footnote: Patient 15 and 29 were considered to have a large amount of normal splice variant present as indicated by the height of the two extra peaks preceding the main peak. The extra peaks in Patient 24 and 14 are not as prominent indicating that less splice variant is present. These conclusions were supported by sequencing data. The R386H mutation in patients 13 and 27 gave a distinct heteroduplex pattern.

157

Appendix 4

Figure 32: dHPLC chromatograms for NDUFV1 amplicon 2

Patient 13: NDUFV1 Amplicon 2, 64°C Patient 15: NDUFV1 Amplicon 2, 64°C c.331A>G, K111E c.439C>T, R147W and c.549 C>G, G183G

Patient 20: NDUFV1 Amplicon 2, 64°C Normal homoduplex to compare

Footnote: Patients 13 and 15 are both heteroduplexes for this amplicon- and the heteroduplex pattern is different, indicating the presence of different mutations.

158

Appendix 4

Figure 33: dHPLC chromatograms for NDUFV1 amplicon 3

Patient 27: NDUFV1 Amplicon 3, 64°C Patient 18: NDUFV1 Amplicon 3, 64°C c.755C>G, P252R Normal homoduplex to compare

Footnote: Patient 27 is a heteroduplex due to the presence of the P252R mutation

Figure 34: dHPLC chromatograms for NDUFS2 amplicon 3

Patient 25: NDUFS2 Amplicon 3, 61°C Patients 14 and 31: NDUFS2 A3, 61°C c.661C>T, R221X homoduplexes

159 Appendix 4

Figure 35: dHPLC chromatograms for NDUFS2 amplicon 4

Patient 17: NDUFS2 Amplicon 4, 60°C Patient 22: NDUFS2 Amplicon 4, 60°C c.875T>C, M292T c.1054C>G, P352A

Patient 30: NDUFS2 Amplicon 4, 60°C Normal homoduplex to compare

Footnote:. Patient 17 has a distinct heteroduplex when wild type DNA was added and the mutation c.875T>C, M292T was identified. Patients 3, 14 and 22 had the same heteroduplex pattern and they shared the same polymorphism c.1054C>G, P352A.

160

Appendix 4

Figure 36: dHPLC chromatograms for NDUFS4 amplicon 1

Patient 26: NDUFS4 Amplicon 1 59°C Patient19: NDUFS4 Amplicon 1 59°C homozygousc.221delC and splice variant heterozygous c.221delC

Patient 22: NDUFS4 Amplicon 1 59°C Patient 23: NDUFS4 Amplicon 1 59°C c.158T>C, I53T c.198C>A, G66G

Patients 14, 15 and 18: NDUFS4 A1 59°C Normals to compare Footnote: Patients 26, 19, 22 and 23 were all heteroduplexes for NDUFS4 Amplicon1, but they all had different heteroduplex patterns as they all had different sequence variants. Even though Patients 26 and 19 share the c.221delC mutation, Patient 26 also had large amounts of a splice variant present and this has resulted in a different heteroduplex pattern.

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