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A heteroplasmy level of 83% is observed in position 11,778 of the mitochondrial DNA.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Leber hereditary optic neuropathy (LHON) is a mitochondrial neurodegenerative disease that affects the optic nerve and is characterised by a sudden loss of vision in young adults.<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">1</span></a> Loss of vision has a tendency to stabilise, although there have been reports of spontaneous visual recovery, related to the age of onset of the disease and the type of mutation.<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">2</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">LHON disease is caused by mutations in mitochondrial DNA (mtDNA): more than 90% occur in positions 3460, 11,778 or 14,484, known as primary mutations positions.<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">3</span></a> Unlike other mitochondrial cytopathies, most of these mutations are detected in homoplasmia,<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">4</span></a> i.e. all mtDNA molecules (in a single cell there are hundreds or thousands of mtDNA molecules) are carriers of the deleterious mutation. The existence of normal and mutated mtDNA, known as heteroplasmy, is present only in 10–15% of patients with LHON.</p><p id="par0015" class="elsevierStylePara elsevierViewall">The gold standard technique for the detection of specific mutations in mtDNA is the Sanger sequencing because of its simplicity and availability in most molecular genetic laboratories. However, it does not provide quantitative information about the degree of heteroplasmy and does not detect heteroplasmias of less than 15%.<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">5</span></a> For these cases, using other techniques is useful, such as the PCR quantitative variant based on the amplification-refractory mutation system, and more recently, massive sequencing.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Clinical observation and results</span><p id="par0020" class="elsevierStylePara elsevierViewall">We report the case of a 26-year-old woman who visited the Neurology department because of decreased visual acuity and blurred vision in her right eye. The general neurological examination was normal. She reported no pain with eye movements, impaired colour perception, photophobia or headaches. A nuclear magnetic resonance imaging scan was requested to rule out a possible demyelinating disease, and a lumbar puncture was conducted, which showed no pathological findings. Exploration by the ophthalmology with a slit lamp and of the ocular fundus showed normal findings in both eyes. The intraocular pressure was found within the normal range, and the visual evoked potentials and retinogram were normal. The perimetry detected a peripheral visual field deficit in the right eye and gave a clinical judgement of right retrobulbar optic neuritis.</p><p id="par0025" class="elsevierStylePara elsevierViewall">As the presence of multiple sclerosis was discarded, complementary tests were requested in search of a secondary cause, including the molecular study of LHON, which is the most common inherited cause of optic neuritis.<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">6</span></a> To perform this molecular study, total DNA was first extracted from a blood sample using the automatic extractor MagNA Pure (Roche Diagnostics, West Sussex, UK). Total DNA amplification was performed by PCR with primers specific to mtDNA regions where the most frequent mutations in patients with the disease are found: m.3460G>A, m.11778G>A and m.14484T>C (reference sequence used: Revised Cambridge Reference Sequence, NC_012920). Each PCR reaction had a final volume of 25<span class="elsevierStyleHsp" style=""></span>μl: 100–150<span class="elsevierStyleHsp" style=""></span>ng of total DNA, 12.5<span class="elsevierStyleHsp" style=""></span>μl de PCR Master Mix by Promega<span class="elsevierStyleSup">®</span> (Madison, Wisconsin, USA) 1.5<span class="elsevierStyleHsp" style=""></span>μl MgCl<span class="elsevierStyleInf">2</span>, 25<span class="elsevierStyleHsp" style=""></span>mM and 25<span class="elsevierStyleHsp" style=""></span>pmol from each primer.</p><p id="par0030" class="elsevierStylePara elsevierViewall">An initial denaturation step of 5<span class="elsevierStyleHsp" style=""></span>min was performed at 94<span class="elsevierStyleHsp" style=""></span>°C and 30 cycles of: 1<span class="elsevierStyleHsp" style=""></span>min of denaturation at 94<span class="elsevierStyleHsp" style=""></span>°<span class="elsevierStyleHsp" style=""></span>C, 30<span class="elsevierStyleHsp" style=""></span>s hybridisation at 54<span class="elsevierStyleHsp" style=""></span>°C and 1<span class="elsevierStyleHsp" style=""></span>min extension at 72<span class="elsevierStyleHsp" style=""></span>°<span class="elsevierStyleHsp" style=""></span>C, with a final extension step at 72<span class="elsevierStyleHsp" style=""></span>°C for 7<span class="elsevierStyleHsp" style=""></span>min.</p><p id="par0035" class="elsevierStylePara elsevierViewall">After verifying the presence of amplification in an agarose gel at 2%, the amplified fragments were sequenced by Sanger sequencing in the ABI 3130 automated sequencer from Applied Biosystems (Life Technologies, Carlsbad, California, USA). Analysis of the sequences obtained by the SeqScape v2.5<span class="elsevierStyleSup">®</span> (Life Technologies) software showed the presence of m.11778G>A mutation in the <span class="elsevierStyleItalic">MT-ND4</span> gene in heteroplasmy (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>). This mutation is present in 70% of patients with LHON, thus confirming the clinical diagnosis of the disease.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0040" class="elsevierStylePara elsevierViewall">Given that mitochondrial diseases are inherited maternally, a test for the mutation in the patient's 53-year-old asymptomatic mother was conducted. It confirmed that she also carried it in heteroplasmy, the amount of mutated mtDNA being greater than normal mtDNA according to the chromatograms obtained with the Sanger sequencing (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>B).</p><p id="par0045" class="elsevierStylePara elsevierViewall">The sister of the index case, who was 23 years old and had no ocular manifestations, came to the clinic for genetic counselling. A test for the mutation m.11778G>A using the same Sanger methodology showed that it was in homoplasmia (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>C). Due to the absence of symptoms and in order to offer suitable genetic counselling, additional studies were requested to verify whether it really was in homoplasmia.</p><p id="par0050" class="elsevierStylePara elsevierViewall">Massive sequencing or next generation sequencing (NGS) represents a valuable tool to quantitatively determine the status of a mutation,<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">7</span></a> and could therefore be useful in this case. To carry out the study, the fragment where the m.11778G>A mutation was found was amplified again using PCR, following the same amplification conditions. Subsequently the library was generated and enriched using the Nextera<span class="elsevierStyleSup">®</span> XT kit (Illumina, San Diego, California, USA). 150<span class="elsevierStyleHsp" style=""></span>bp paired-end sequencing was conducted in the MiSeq<span class="elsevierStyleSup">®</span> (Illumina) mass sequencer, obtaining coverage for that position of 175x, of which 145 corresponded to the alternative allele (A) and 30 to reference allele (G), representing a heteroplasmy of 83% (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). As a primer the BWA was used, and as a variant caller, GATK.</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0055" class="elsevierStylePara elsevierViewall">The subsequent study by NGS of the mutation in question from the index case and in the mother, showed levels of heteroplasmy of 66 and 75%, respectively (the coverage at that position was 958x in the first case, and 909x in the second).</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Discussion</span><p id="par0060" class="elsevierStylePara elsevierViewall">LHON is a disease that does not have a specific treatment, is highly disabling and significantly decreases the quality of life for those who suffer from it, so its diagnosis is vitally important so as to be able establish measures to control risk factors and to give appropriate genetic counselling to patients and families.</p><p id="par0065" class="elsevierStylePara elsevierViewall">This disease is characterised by incomplete penetrance: approximately 50% of men and 90% of women with any of the 3 primary mutations do not develop vision loss.<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">2</span></a> Thus, other genetic or environmental factors that may have an effect on its development must exist. The factors that appear to be most important are age, sex, a difference in the mutated mtDNA in different tissues and the level of heteroplasmy.<a class="elsevierStyleCrossRefs" href="#bib0120"><span class="elsevierStyleSup">8–10</span></a></p><p id="par0070" class="elsevierStylePara elsevierViewall">However, in this family the index case, with less heteroplasmy than her mother, presented optic atrophy at the average age of diagnosis in women who carry the m.11778G>A mutation (24 years old).<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">11</span></a> This could be due to differences in the degree of heteroplasmy in different tissues (blood vs. optic nerve) in the index case and her mother. Because of the low penetrance of the LHON disease, it is common to not observe a history of mitochondrial inheritance and to detect ocular manifestations in one family member which may complicate the diagnostic orientation of these patients.</p><p id="par0075" class="elsevierStylePara elsevierViewall">For the mother, the risk of developing visual impairment is low; the vast majority of patients who have this mutation develop symptoms before the age of 50.<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">12</span></a> Because of the index case's sister's age and because she has a high percentage of heteroplasmy, she has been advised to visit the ophthalmologist every 6 months for regular check-ups. The fact that the index case has a higher percentage of mutated mtDNA than her mother is explained by the “bottleneck” phenomenon.<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">13</span></a> LHON, and in general all mitochondrial diseases caused by mutations in mtDNA, is maternally inherited: mothers pass the mutation to all their children. However, the percentage of mutated mtDNA in the ova of a woman carrying a mutation varies by this phenomenon, which is simply a restriction and subsequent amplification process of mtDNA molecules produced in the oocyte during embryogenesis.</p><p id="par0080" class="elsevierStylePara elsevierViewall">NGS, unlike the Sanger sequencing, facilitates reading each base genome numerous times, so this feature can be used in the case of mtDNA (present in multiple potentially different copies) to read different sequences and detect low levels of heteroplasmy and quantify heteroplasmic mutations. The number of times each base genome is present in the readings that the mass sequencer carried out is called coverage.<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">14</span></a> For example, a 30x coverage for a position indicates that 30 readings of that nucleotide have been made. Traditionally, analytical sensitivity is defined as the proportion of biological samples that have a positive outcome and are correctly classified as positive. In Sanger sequencing and NGS techniques, this term refers to the probability that a variant is detected when present in the analysed region.<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">15</span></a> The analytical sensitivity of the NGS to detect heteroplasmias depends on the coverage; the greater coverage, the greater its sensitivity. A minimum of 40 reads to detect low degrees of heteroplasmy is recommended.<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">16</span></a></p><p id="par0085" class="elsevierStylePara elsevierViewall">We have seen that NGS is useful not only in detecting low levels of heteroplasmy, but also distinguishing between high levels of heteroplasmy and true homoplasmy. Confirming that a mutation is in homoplasmia in a patient is important for the purposes of genetic counselling. If a woman is homoplasmic for a mutation in mtDNA, all her children will also inherit them in homoplasmia; however, if it is heteroplasmic, she may transmit a low level of mutated mtDNA to her offspring, with a lower risk of developing the disease.<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">9</span></a></p><p id="par0090" class="elsevierStylePara elsevierViewall">In the LHON disease, the reported percentage of patients with mutations in heteroplasmy is low. However, this is based on studies using other methodologies, such as the primer extension assay.<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">4</span></a> It would therefore be advisable to confirm, using new, more sensitive technologies, such as NGS, if the percentage of homoplasmia in this disease is really so high.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Conflict of interest</span><p id="par0095" class="elsevierStylePara elsevierViewall">The authors declare no conflict of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:9 [ 0 => array:3 [ "identificador" => "xres672928" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background and objective" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Patients and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec679274" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres672927" "titulo" => "Resumen" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Fundamento y objetivo" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Pacientes y métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusiones" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec679273" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:2 [ "identificador" => "sec0010" "titulo" => "Clinical observation and results" ] 6 => array:2 [ "identificador" => "sec0015" "titulo" => "Discussion" ] 7 => array:2 [ "identificador" => "sec0020" "titulo" => "Conflict of interest" ] 8 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-07-09" "fechaAceptado" => "2015-10-01" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec679274" "palabras" => array:4 [ 0 => "Mitochondrial deoxyribonucleic acid" 1 => "Leber hereditary optic atrophy" 2 => "Next-generation sequencing" 3 => "Homoplasmy" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec679273" "palabras" => array:4 [ 0 => "Ácido desoxirribonucleico mitocondrial" 1 => "Neuropatía óptica hereditaria de Leber" 2 => "Secuenciación de última generación" 3 => "Homoplasmia" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background and objective</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Leber hereditary optic neuropathy is characterised by acute and subacute visual loss, produced by mitochondrial DNA mutations.</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Patients and methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">The molecular study of a family with only one affected member is presented.</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">In the index case and in her mother, the mitochondrial mutation m.11778G>A in the <span class="elsevierStyleItalic">MT-ND4</span> was detected in the heteroplasmic state. The index case's sister, without ocular manifestations, asked for genetic counselling. The study of the mentioned mutation by Sanger sequencing identified it in an apparent homoplasmic state. However, by means of next-generation sequencing (NGS), the mutation was actually in a heteroplasmic state.</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Regarding genetic counselling, verifying a mutation in homoplasmic state is really important. We have observed that NGS allows us to discriminate between high levels of heteroplasmy and homoplasmy, meaning that it is a useful technique for the analysis of apparent homoplasmic results obtained with less sensitive technique, as Sanger sequencing.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Background and objective" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Patients and methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusions" ] ] ] "es" => array:3 [ "titulo" => "Resumen" "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Fundamento y objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">La neuropatía óptica hereditaria de Leber se caracteriza por una pérdida de visión aguda o subaguda, producida por mutaciones en el ADN mitocondrial.</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Pacientes y métodos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Presentamos el estudio molecular realizado en una familia en la que solo uno de sus miembros presentaba signos de la enfermedad.</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">En el caso índice y en la madre se detectó la mutación m.11778G>A en el gen <span class="elsevierStyleItalic">MT-ND4</span> en heteroplasmia. La hermana del caso índice, sin manifestaciones oculares, acudió a consulta para consejo genético. El estudio de dicha mutación por secuenciación Sanger mostró que la portaba en homoplasmia. Sin embargo, mediante secuenciación de última generación (NGS) se pudo comprobar que se encontraba realmente en heteroplasmia.</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusiones</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Confirmar que una mutación se encuentra en homoplasmia es importante cara al consejo genético. Hemos observado que la NGS permite distinguir entre grados elevados de heteroplasmia y verdaderas homoplasmias y, por tanto, es de utilidad en el estudio de pacientes en los que se detectan mutaciones en homoplasmia con metodologías de menor sensibilidad analítica.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Fundamento y objetivo" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Pacientes y métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusiones" ] ] ] ] "NotaPie" => array:1 [ 0 => array:2 [ "etiqueta" => "☆" "nota" => "<p class="elsevierStyleNotepara" id="npar0005">Please cite this article as: Carrasco Salas P, Palma Milla C, López Montiel J, Benito C, Franco Freire S, López Siles J. Neuropatía óptica de Leber: utilidad de la secuenciación masiva en el estudio de mutaciones mitocondriales en aparente homoplasmia. Med Clin (Barc). 2016;146:163–166.</p>" ] ] "multimedia" => array:2 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Fig. 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 3108 "Ancho" => 2988 "Tamanyo" => 438459 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Electropherograms in which the mutation m.11778G>A in heteroplasmia shown in the index case (A), in her mother (B), and in homoplasmia, in the index case's sister (C).</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Fig. 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1618 "Ancho" => 2927 "Tamanyo" => 408769 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Display of the mass sequencing data in the index case's sister through the Integrative Genomics Viewer. 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Clinical report
Leber hereditary optic neuropathy: Usefulness of next generation sequencing to study mitochondrial mutations on apparent homoplasmy
Neuropatía óptica de Leber: utilidad de la secuenciación masiva en el estudio de mutaciones mitocondriales en aparente homoplasmia