Osteogenesis imperfecta (OI) is a heterogeneous disease of connective tissue, the cardinal symptom being fractures and severity ranging from mild to lethal. Dominant mutations in collagen I, encoded by COL1A1 and COL1A2, cause >90% of cases. To delineate genotype-phenotype correlations and pharmaco-genetic response, collagen I was sequenced in 150 unrelated Swedish families and clinical data were collected in Paper I. Mutation type, gene affected, and N- to C-terminal location correlated with phenotype and severity. Bisphosphonate response assessed by calculated yearly change in lumbar spine bone mineral density (BMD) was inversely related to age and BMD at treatment initiation. Mutations associated with a more severe phenotype exhibited an increased response after 2 years; however, all types of OI responded well. To investigate the effect of naturally occurring variations in collagen I, the only common coding single nucleotide polymorphism (rs42524 in COL1A2) was genotyped in 2004 healthy men in Paper II. Heterozygous genotype was associated with decreased BMD and an increased risk of stroke. An adolescent with repeated fractures despite a markedly high BMD harbored a unique C-terminal procollagen cleavage-site mutation in COL1A1, which motivated extensive investigations in concert with a similar COL1A2 case in Paper III. The probands were found to have impaired procollagen processing, incorporation of collagen with retained C-propeptide in matrix and increased mineral to matrix ratio, which demonstrates that C-propeptide cleavage is crucial to normal bone mineralization and structure. Bisphosphonate therapy has insufficient effect in OI, and as classical OI is a dominant disorder severe cases would benefit from silencing of the mutated allele. In Paper IV and V small interfering RNAs (siRNAs) were used to allele-specifically target primary human bone cells heterozygous for I) a coding polymorphism in COL1A2 and II) insertion/deletions in the 3’UTR of COL1A1 and COL1A2. Results were promising with altered allele ratios and decreased mRNA levels in the predicted fashion. To summarize, this thesis found that collagen I is crucial to bone and connective tissue and that collagen I mutations create markedly diverse phenotypes. Age, BMD and pharmaco-genetic effects influence the response to bisphosphonate therapy in individuals with OI; however, novel approaches are needed. Utilizing allele-specific siRNAs may be a way forward in the treatment of severe OI.

Anticiper l’impact de fluctuations environnementales de nature climatique ou alimentaire est un enjeu crucial dans les systèmes de productions animales, et plus particulièrement sur la volaille. Cette influence de l’environnement sur les phénotypes passe en partie par des phénomènes épigénétiques, notamment la méthylation de l’ADN, et qui peuvent intervenir dans la régulation de l'expression des gènes. Ce sont des mécanismes qui n'affectent pas la séquence d'ADN mais qui peuvent être transmis par la mitose ou la méiose. Ces interactions entre épigénomes et expression des gènes sont de plus en plus étudiées dans les modèles animaux et chez les plantes. Cependant, les mécanismes de régulation de l'expression du génome par la méthylation de l’ADN sont assez peu connus chez les oiseaux. Ce travail de thèse repose sur deux dispositifs expérimentaux réalisés chez la poule, le but étant de caractériser le méthylome par séquençage haut-débit. Les profils de méthylation le long du génome, et le lien avec l’expression, sont établis d’abord par un séquençage tout-génome (WGBS) au sein d’embryons entiers, puis par un séquençage d'une sous-représentation du génome (RRBS) au sein d’hypothalamus d’individus adultes. À ce jour, aucune étude d'analyses de méthylome par RRBS chez la poule n'a été publiée. Ces deux analyses sont réalisées grâce au développement d'un pipeline bioinformatique, optimisé, disponible à la communauté scientifique. Globalement, le profil de méthylation chez la poule est similaire à ce qui est connu chez les mammifères : les îlots CpG - régions riches en dinucléotides CG, souvent peu méthylées, qui ponctuent le génome principalement dans les régions promotrices des gènes - sont globalement peu méthylés dans les promoteurs sur les données WGBS et RRBS. Les analyses du méthylome des embryons ont confirmé l'absence d'un phénomène de compensation de dose sur les chromosomes sexuels, ou la présence sur le chromosome Z d'une région hyperméthylée. Les analyses des données RRBS révèlent une hyperméthylation globale des CG sur le génome, suggérant une réponse de la méthylation à un stress environnemental. Sur les données WGBS, le niveau de méthylation dans le promoteur est négativement corrélé à l'expression du gène associé. Une méthylation allèle spécifique est également détectée entre les lignées, phénomène mis en évidence pour la première fois chez la poule et dont la fréquence est comparable à ce qui a été observé chez l'Homme. Sur les données RRBS, des résultats préliminaires de la réponse du méthylome aux stress environnementaux montrent le caractère complexe de cette relation. L’utilisation d’aliments moins énergétiques entraînerait une plus grande mobilisation des réserves lipidiques, tandis que les individus soumis à un stress à la chaleur ont un poids corporel plus léger. Une intégration de ces données à des mesures phénotypiques permettrait de faire le lien entre méthylation et environnement. Au-delà de l'aspect fondamental de cette thèse, l'application plus concrète de ces connaissances peut s'appliquer aux systèmes d'élevage pour obtenir des animaux mieux adaptés à l’environnement, en améliorant les caractères de production / Anticipating the impact of environmental changes (on climate and feed) is a crucial issue for livestock production systems, including poultry. The influence of the environment on phenotypes is partly mediated by epigenetic phenomena, including DNA methylation, which may be involved in the regulation of gene expression. These mechanisms do not affect the DNA sequence but can be inherited by mitosis or meiosis. The interactions between epigenomes and gene expression are increasingly being studied in animal models and in plants. However, the mechanisms of regulation of genome expression through DNA methylation are relatively unknown in birds. This thesis work is based on two experimental devices realized in chicken aiming to characterize the methylome by high-throughput sequencing. The methylation patterns across the genome, and their link with expression, were first established by whole-genome bisulfite sequencing (WGBS) in whole embryos, following a reduced representation bisulfite sequencing (RRBS) from hypothalamus of adults. To date, no specific chicken RRBS study has been published. These two analyses were carried out by developing an optimized bioinformatics pipeline, available for scientific community. Overall, the pattern of methylation in chicken is like those in mammals: CpG islands - dinucleotides CG-rich regions which are often poorly methylated, and which are found mainly in the promoter regions of the genome - are generally poorly methylated in promoters on WGBS and RRBS data. Embryo methylome analyses confirmed the absence of a dose-compensation phenomenon on sex chromosomes, or the presence of a hypermethylated region on the Z chromosome. The analyses of RRBS data revealed an overall hypermethylation of CGs across the genome, suggesting a methylation response to environmental stress. From the analysis of WGBS data, we found that the level of methylation in promoters was negatively correlated with the expression of the associated gene. For the first time, a specific allele methylation was also detected between chicken lines whose frequency is comparable to that observed in humans. On the RRBS data, preliminary results of the methylome response to environmental stresses showed the complex nature of this relationship. The use of a low-energy diet would led to greater mobilization of body fat, while individuals with heat stress had a lighter body weight. Integrating these data with phenotypic measurements would allow to link methylation and environment. Beyond the fundamental aspect of this thesis, the method developed in this work could be applied to livestock systems to breed animals better adapted to a changing environment, by improving production traits.

L’édition du génome utilisant CRISPR/Cas9 est récemment apparue comme une stratégie thérapeutique potentielle des maladies génétiques. Pour les mutations dominantes de type gain de fonction, la correction allèle-spécifique pourrait être l'approche la plus appropriée. Ici, nous avons testé l'inactivation ou la correction d'une mutation hétérozygote du gène de la dynamine 2 (DNM2) causant la forme autosomique dominante de la myopathie centronucléaire (CNM). Des ARN-guides tronqués ciblant spécifiquement l'allèle muté ont été testés sur des cellules de patients et des myoblastes d'un modèle murin. L'allèle muté a été ciblé avec succès et des clones ont été obtenus avec inactivation ou correction précise du génome. Les myoblastes Dnm2R465W/+ ont montré une altération de l'endocytose et de l'autophagie. L'inactivation ou la correction allèle-spécifique a normalisé ces phénotypes. L'allèle muté a également été ciblé avec succès dans les muscles de la souris Dnm2R465W/+. Ces résultats illustrent le potentiel de CRISPR/Cas9 à cibler et corriger de manière allèle-spécifique les mutations ponctuelles hétérozygotes de type de gain de fonction. / Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. Here we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene causing the autosomal dominant form of centronuclear myopathies (CNM). Truncated single guide RNAs targeting specifically the mutated allele were tested on cells derived from a mouse model and patients. The mutated allele was successfully targeted in patient fibroblasts and Dnm2R465W/+ mouse myoblasts, and clones were obtained with both precise genome correction or inactivation. Dnm2R465W/+ myoblasts showed an alteration in transferrin uptake and autophagy. Specific inactivation or correction of the mutated allele rescued these phenotypes. The mutated allele was also successfully targeted in Dnm2R465W/+ mouse muscles. These findings illustrate the potential of CRISPR/Cas9 to target and correct heterozygous point mutations leading to a gain-of-function effect in an allele-specific manner.

Nucleic acid technology has assumed an essential role invarious areas ofin vitrodiagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis. The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products. Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection. Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy&gt;99%is required. <b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.

Developments within the field of genetic analysis have during the last decade become enormous. Advances in DNA sequencing technology have increased throughput from a thousand bases to over a billion bases in a day and decreased the cost thousandfold per base. Nevertheless, to sequence complex genomes like the human is still very expensive and efforts to attain even higher throughputs for less money are undertaken by researchers and companies. Genotyping systems for single nucleotide polymorphism (SNP) analysis with whole genome coverage have also been developed, with low cost per SNP. There is, however, a need for genotyping assays that are more cost efficient per sample with considerably higher accuracy. This thesis is focusing on a technology, based on competitive allele-specific extension and microarray detection, for genetic analysis. To increase specificity in allele-specific extension (ASE), a nucleotide degrading enzyme, apyrase, was introduced to compete with the polymerase, only allowing the fast, perfect matched primer extension to occur. The aim was to develop a method for analysis of around twenty loci in hundreds of samples in a high-throughput microarray format. A genotyping method for human papillomavirus has been developed, based on a combination of multiplex competitive hybridization (MUCH) and apyrase-mediated allele-specific extension (AMASE). Human papillomavirus (HPV), which is the causative agent in cervical cancer, exists in over a hundred different types. These types need to be determined in clinical samples. The developed assay can detect the twenty-three most common high risk types, as well as semi-quantifying multiple infections, which was demonstrated by analysis of ninety-two HPV-positive clinical samples. More stringent conditions can be obtained by increased reaction temperature. To further improve the genotyping assay, a thermostable enzyme, protease, was introduced into the allele-specific extension reaction, denoted PrASE. Increased sensitivity was achieved with an automated magnetic system that facilitates washing. The PrASE genotyping of thirteen SNPs yielded higher conversion rates, as well as more robust genotype scoring, compared to ASE. Furthermore, a comparison with pyrosequencing, where 99.8 % of the 4,420 analyzed genotypes were in concordance, indicates high accuracy and robustness of the PrASE technology. Single cells have also been analyzed by the PrASE assay to investigate loss of alleles during skin differentiation. Single cell analysis is very demanding due to the limited amounts of DNA. The multiplex PCR and the PrASE assay were optimized for single cell analysis. Twenty-four SNPs were genotyped and an increased loss of genetic material was seen in cells from the more differentiated suprabasal layers compared to the basal layer. / QC 20100714

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

<p>Nucleic acid technology has assumed an essential role invarious areas of<i>in vitro</i>diagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis.</p><p>The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products.</p><p>Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection.</p><p>Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy>99%is required.</p><p><b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.</p>

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

Magister Scientiae - MSc / The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South Africa; a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot® and Multiplex AS-PCR genotyping assays, and also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the Cape Metropolitan area. Two SNaPshot® Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific – PCR (MAS-PCR) genotyping system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan large numbers of samples for novel genetic variations. / South Africa

Leber’s hereditary optic neuropathy (LHON) is a genetic disease that causes the patients to become blind, first in one eye and then the other, around the ages of 10-75 years. The disease is caused by mutations in the mitochondrial DNA, which disturbs the respiratory chain leading to the deterioration of the retinal ganglion cells. This study’s aim is to optimize a multiplex amplification-refractory mutation system PCR for detection of three primary mutations causing LHON. This was done through a series of PCRs, including PCR aimed at the ß-globin gene, conventional simplex PCR and a simplex ARMS-PCR aimed at the three primary mutations causing LHON. This study was, however, terminated prematurely due the Covid-19 outbreak and the optimization of the ARMS-PCR could therefore not be done. This study’s aim was adapted to the new circumstances to instead provide guidance on how to perform the optimization using the results from the PCRs that were done before the termination. The results found that for the ARMS-PCR 2 mM of magnesium would suffice as a start point overall and the need to solve the problems with the two 14484 plasmids was evident. The ARMS-PCR is one of many methods that can be used to the detect single nucleotide polymorphism, but its availability and robustness makes this a method worth optimizing. To continue with the optimization of the ARMS-PCR several factors would have to be tested, including annealing temperature, primer concentrations and magnesium concentration.