Development and validation of a novel PCR-RFLP based method for the detection of 3 primary mitochondrial mutations in Leber's hereditary optic neuropathy patients
© Eustace Ryan et al. 2015
Received: 22 July 2015
Accepted: 6 October 2015
Published: 25 October 2015
Leber’s Hereditary Optic Neuropathy (LHON; MIM 535000) is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset between the ages of 15–30. The worldwide incidence of LHON is approximately 1 in 31,000. 95 % of LHON patients will have one of 3 primary mitochondrial mutations, G3460A (A52T of ND1), G11778A (R340H of ND4) and T14484C (M64V of ND6). There is incomplete penetrance and a marked gender bias in the development of visual morbidity with approximately 50 % of male carriers and 10 % of female carriers developing optic neuropathy. Visual recovery can occur but is dependent on the mutation present with the highest level of visual recovery seen in patients who have the T14484C mutation. The 3 primary mutations are typically identified by individual end-point PCR-restriction fragment length polymorphism (RFLP) or individual targeted bi-directional Sanger sequencing reactions. The purpose of this study was to design a simple multiplex PCR-RFLP that could detect these 3 primary LHON mutations in one assay.
PCR primers were designed to incorporate a MaeIII restriction site in the presence of 3460A and 14484C mutations with the 11778A mutation naturally incorporating a MaeIII site. A multiplex PCR-RFLP assay was developed to detect the 3 common mutations in a single assay. Synthetic LHON controls based on the mitochondrial genome harbouring the 3 common mutations were synthesized and cloned into plasmids to act as reliable assay controls. DNA from previously tested patients and the synthetic LHON controls were subjected to the multiplex PCR-RFLP assay. The RFLP products were detected by agarose gel electrophoresis.
The novel PCR-RFLP assay accurately detects the 3 primary mutations both in patient DNA and in synthesized DNA control samples with a simple visual mutation detection procedure. The synthesized DNA was demonstrated to be a robust control for the detection of LHON Mutations.
In this paper, we describe a novel, robust and simple PCR-RFLP based method for the detection of mutations causing LHON, and report the generation of a series of LHON DNA controls suitable for all currently published assays.
Leber’s hereditary optic neuropathy (LHON; MIM 535000) is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults [1, 2]. The disorder is the result of mitochondrial dysfunction wherein primary mitochondrial DNA (mtDNA) mutations affect complex I subunits of the respiratory chain . LHON is the most common among primary mitochondrial diseases with a prevalence of 1 in 31,000 in the North of England, 1 in 39,000 in the Netherlands and 1 in 50,000 in Finland [4–6]. In some countries, approximately 95 % of individuals with LHON have one of 3 primary mtDNA mutations; G3460A (A52T of ND1), G11778A (R340H of ND4) and T14484C (M64V of ND6) [3, 7, 8] while other rare mutations (such as G13730A, G14459A, C14482G, A14495G, C14498T, C14568Tand T14596A) account for the final 5 % [9–16]. LHON demonstrates marked gender bias and an incomplete penetrance, with approximately 50 % of males and 10 % of females who harbour one of the above mutations actually developing optic neuropathy [17–19]. This indicates that environmental or other genetic factors must play a role in penetrance. Alcohol consumption, smoking, certain prescription medications, stress and critical illness have been implicated in the onset of symptoms in LHON mutation carriers [17, 18, 20, 21].
The peak age of LHON onset is between 15–30 years and 95 % of carriers who will experience visual failure will do so before the age of 50 years . However, visual deterioration can occur any time during the first to the seventh decade of life . Clinically, there tends to be an acute loss of vision in one eye generally followed by loss of vision in the other eye within 8 weeks. In the majority of cases, LHON pathology is limited to a highly specialized group of cells within the eye known as retinal ganglion cells (RGCs). Histological analysis of the optic nerve in LHON patients reveals minimal evidence of any inflammation, but shows general axonal depletion centrally and fibrocytic scarring. Any residual axons were limited to superior and temporal peripheral clusters .
Visual recovery can occur in some LHON patients, but the extent of which is influenced by the kind of mutation involved in the development of a particular patient’s LHON. The highest level of visual recovery is seen with patients who have the T14484C mutation (up to 58 %), followed by those with the G3460A mutation (up to 25 %). Patients who harbour the G11778A mutation have the lowest level of visual recovery [25–28]. Thus, an accurate mutation detection strategy can have a significant prognostic value to the LHON patient.
Current diagnostic strategies for the 3 most common mutations causing LHON include individual endpoint PCR-RFLP , allele specific PCR , real time PCR  and PCR followed by Sanger / pyrosequencing . In this study, we successfully designed a simple multiplex PCR-RFLP to detect the 3 primary mitochondrial LHON mutations and also describe the synthesis of a series of LHON control sequences that act as a robust and patient-free resource for LHON test controls and assay development.
Patient DNAs were obtained from the Centre for Medical Genetics, Our Lady’s Hospital for Sick Children, Dublin, Ireland and Oxford Medical Genetics Laboratories, Oxford, UK. DNA was extracted from peripheral blood using the Centra Puregene Blood Kit (Qiagen, Manchester, UK) or the EZ1 Blood Kit on the EZ1 advanced XL instrument (Qiagen, Manchester, UK) according to the manufacturers’ instructions. All samples used in this study were previously tested for LHON mutations using PCR amplification and DNA sequencing. To maintain patient confidentiality during this study, aliquots of residual DNA material from the diagnostic test were labelled with the LHON mutation detected and irreversibly anonymised. The use of patient DNA in this study has received ethical approval from the Dublin Institute of Technology Research Ethics Committee (RN: 14–06).
Synthetic control DNA
Synthetic LHON control plasmids and mixes
3275 – 4272
3275 – 4272
11580 – 12118
11580 – 12118
14484 T (N)
14449 – 15022
14449 – 15022
Primer design and PCR
Primer sequences for multiplex PCR-RFLP
PCR product and restriction product sizes expected in diagnostic test
Uncut PCR products
This study aimed to develop a novel PCR-RFLP based multiplex assay for the detection of the 3 common primary mutations leading to Leber hereditary optic neuropathy (LHON). Approximately 95 % of LHON patients will have one of these 3 mutations, G3460A (13 %), G11778A (69 %) and T14484C (14 %). In approximately 110 diagnostic tests conducted in Ireland, only the 11778A mutation has been detected (data not shown). The PCR-RFLP approach was chosen because it provides a simple, cost effective, robust and easy to read output with minimal requirement for advanced technology. It also allows the detection of all three primary mutations in one multiplex analysis and allows the detection of heteroplasmy to a level of approximately 10 %. This is a significant advantage over individual endpoint PCR-RFLP  that requires 3 simplex PCRs followed by digestion with 3 separate restriction enzymes and electrophoresis. Allele-specific PCR , either simplex that requires 3 separate PCRs, or multiplex that requires only one PCR are efficient, but will not detect heteroplasmy in currently published formats. Individual real-time PCRs have been reported , but require more advanced technologies and are more costly. Individual endpoint PCR followed by bidirectional Sanger/pyrosequencing require significantly more hands-on time, and despite the decreasing costs of sequencing, are still significantly more expensive than the test reported here. As can been seen in Figs. 2b and 3, mutation identification is clear and robust, both with patient DNA and with the synthesised controls described above.
The genetic testing registry (http://www.ncbi.nlm.nih.gov/gtr/) (accessed July 2015) shows that the vast majority of LHON testing involves uni- or bi-directional Sanger sequencing targeted to the 3 primary mutations (n = 20), targeted simplex PCR-RFLP (n = 8), and other testing methods (including real-time mutation detection, PCR followed by hybridisation and pyrosequencing) (n = 4) with 2 laboratories offering a targeted 37 mitochondrial gene Next generation sequencing (NGS) panel (mtSEEK®). PCR followed by Sanger/Pyrosequencing can be used for LHON mutation detection, but requires individual PCRs followed by individual sequencing reactions, and despite decreasing costs for sequencing, still costs significantly more than the test described in this study. NGS with appropriate panels, will detect the 3 primary mutations  but requires significantly more hands-on time for both set up and bioinformatics analysis and, at present, is unlikely to be used for the detection of known mutations due to the costs involved and the simplicity of alternative tests. NGS would, however, be invaluable in the detection of rarer / unknown mutations post initial screening for the 3 primary mutations and could be used for suspected LHON patients negative for the primary mutation screen. The assay reported in this study will allow diagnostic laboratories to avoid costly NGS assays for the vast majority of LHON patients and allow resources to be focussed on patients with unknown mutations requiring further analysis. We suggest that the test described in this study will allow detection of the 3 primary LHON mutations in a single test format at minimal cost, with a rapid turnaround time and without the need for advanced technology.
It is currently estimated that 35,000 individuals worldwide are vision-impaired due to LHON. With the addition of extended family members to this figure, there is a significant requirement for a simple cost-effective and robust diagnostic strategy for LHON mutation detection. The steps required to obtain approval for this diagnostic test for clinical applications include validation of the strategy in larger scale trials in multiple laboratories to ensure reproducibility and sensitivity/specificity followed by applications to the national and international competent authorities such as FDA and EU medical devices sections.
We also describe the generation of a series of controls for LHON applicable for all currently described LHON testing algorithms and demonstrate their applicability in this novel test. The generated controls have also been tested in simplex PCR-RFLP (as described in Marotta et al. 2004) and ARMS PCR (as described in Bi et al. 2010) – data not shown. The controls will provide an unlimited, reliable, and patient-free resource for LHON testing across all current testing platforms. This resource may allow the development of further tests in the future.
We developed a novel, cost-effective multiplex PCR-RFLP based assay for the detection of the 3 most common mutations causing LHON and demonstrated the robustness of the assay in patient and synthetic controls. The assay provides a significant advantage over simplex PCR-RFLP and Sanger/pyrosequencing approaches to mutation detection in terms of costs and hands-on time required. A series of cloned LHON and normal controls were developed and their uses in this and other testing strategies confirmed. This will be a useful resource for future test development and diagnostic laboratories as it provides an unlimited and patient-free source of control material.
We thank the Oxford Medical Genetics Laboratories, Oxford, UK for LHON patient samples. We thank Mr. B. Jennings FAOI from Fight for Sight Ireland and Mr. G. Meynet from the National Council for the Blind Ireland for helpful discussions on LHON. This study was supported by a grant from Fight for Sight to SER. SER is in receipt of a fee waiver from the Dublin Institute of Technology.
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