[Leber hereditary optic neuropathy: update on genetics and pathomechanisms]. / La génétique et physiopathologie de la neuropathie optique de Leber.

Leber Hereditary Optic Neuropathy is still a dramatic disease of optic nerve. Origins and mechanisms are extensively studied in the last decades, in link with emergent therapeutic approaches. This article is an update on genetics and pathophysiology of LHON and leber-like inherited optic neuropathies.

Assessing population genetic structure of three New Zealand stream insects using mitochondrial and nuclear DNA markers.

Assessing genetic differentiation among natural populations can aid understanding of dispersal patterns and connectivity among habitats. Several molecular markers have become increasingly popular in determining population genetic structure for this purpose. Here, we compared the resolution of mitochondrial cytochrome c oxidase subunit I (COI) and nuclear single nucleotide polymorphism (SNP) markers for detecting population structure among stream insects at small spatial scales. Individuals of three endemic taxa-Coloburiscus humeralis (Ephemeroptera), Zelandobius confusus (Plecoptera), and Hydropsyche fimbriata (Trichoptera)-were collected from forested streams that flow across open pasture in the North Island of New Zealand. Both COI and SNP data indicated limited population structure across the study area, and small differences observed among these species were likely related to their putative dispersal abilities. For example, fine-scale genetic differentiation between and among neighbouring stream populations for H. fimbriata suggests that gene flow, and hence dispersal, may be more limited for this species relative to the others. Based on the generally similar results provided by both types of markers, we suggest that either COI or SNP markers can provide suitable initial estimates of fine-scale population genetic differentiation in stream insects.

[Usefulness of combined sequencing of the mitochondrial genome and a targeted panel of nuclear genes involved in mitochondrial diseases]. / Intérêt du séquençage combiné du génome mitochondrial et d'un panel ciblé de gènes nucléaires impliqués dans les maladies mitochondriales.

The molecular study of mitochondrial diseases, essential for diagnosis, is special due to the dual genetic origin of these pathologies: mitochondrial DNA and nuclear DNA. Complete mtDNA sequencing still remains the first line diagnostic test followed if negative, by resequencing panels of several hundred mitochondrially-encoded nuclear genes. This strategy, with an initial entire mtDNA sequencing, is currently justified by the presence of nuclear mitochondrial DNA sequences (NUMTs) in the nuclear genome. We designed a resequencing panel combining the mtDNA and 135 nuclear genes which was evaluated compared to the performances of the standard mtDNA sequencing. Method validation was performed on the reading depth and reproducibility of the results. Thirty patients were analyzed by both methods. We were able to demonstrate that NUMTs did not impact the mtDNA sequencing quality, as the identified variants and mutant loads were identical with the reference mtDNA sequencing method. Reading depths were higher than the recommendations of the MitoDiag French diagnostic network, for the entire mtDNA for muscle and for 70% of the mtDNA for blood. These results highlight the usefulness of combining both mtDNA and mitochondrially nuclear-encoded genes and thus obtain more complete results and faster turnaround time for mitochondrial disease patients.

Cytosolic mtDNA released from pneumolysin-damaged mitochondria triggers IFN-ß production in epithelial cells.

Pneumolysin (Ply) is a major virulence factor of Streptococcus pneumoniae. Ply-induced interferon-ß (IFN-ß) expression in host macrophages has been shown to be due to the accumulation of mitochondrial deoxyribonucleic acid (mtDNA) in the cytoplasm during S. pneumoniae infection. Our findings extend this work to show human bronchial epithelial cells that reside at the interface of inflammatory injury, BEAS-2B, adapt to local cues by altering mitochondrial states and releasing excess mtDNA. The results in this research showed that purified Ply induced the expression of IFN-ß in human epithelial cells, which was accompanied by mitochondrial damage both in vivo and in vitro. The observations also were supported by the increased mtDNA concentrations in the bronchial lavage fluid of mice infected with S. pneumoniae. In summary, our study demonstrated that Ply triggered the production of IFN-ß in epithelial cells, and this response was mediated by mtDNA released from Ply-damaged mitochondria. It displayed an impressive modulation of IFN-ß response to S. pneumoniae in epithelial cells.

Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects.

Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to future applications of this approach.

Biodiversity assessments in the 21st century: the potential of insect traps to complement environmental samples for estimating eukaryotic and prokaryotic diversity using high-throughput DNA metabarcoding 1.

The rapid loss of biodiversity, coupled with difficulties in species identification, call for innovative approaches to assess biodiversity. Insects make up a substantial proportion of extant diversity and play fundamental roles in any given ecosystem. To complement morphological species identification, new techniques such as metabarcoding make it possible to quantify insect diversity and insect-ecosystem interactions through DNA sequencing. Here we examine the potential of bulk insect samples (i.e., containing many non-sorted specimens) to assess prokaryote and eukaryote biodiversity and to complement the taxonomic coverage of soil samples. We sampled 25 sites on three continents and in various ecosystems, collecting insects with SLAM traps (Brazil) and Malaise traps (South Africa and Sweden). We then compared our diversity estimates with the results obtained with biodiversity data from soil samples from the same localities. We found a largely different taxonomic composition between the soil and insect samples, testifying to the potential of bulk insect samples to complement soil samples. Finally, we found that non-destructive DNA extraction protocols, which preserve insect specimens for morphological studies, constitute a promising choice for cost-effective biodiversity assessments. We propose that the sampling and sequencing of insect samples should become a standard complement for biodiversity studies based on environmental DNA.

Improving hepatic mitochondrial biogenesis as a postulated mechanism for the antidiabetic effect of Spirulina platensis in comparison with metformin.

Various nutritional and medicinal potencies have been accredited to metabolites from the cyanobacteria, Spirulina platensis (Arthrospira platensis) sp. Hence, our study was designed to examine whether the Spirulina supplementation would possess beneficial effects in type 2 diabetes mellitus (T2DM) in comparison with metformin. High-fat diet/low-dose streptozotocin (HFD/STZ) model was adopted and the diabetic rats were orally treated with metformin (200 mg/kg) or Spirulina (250 or 500 or 750 mg/kg) for 30 days. Spirulina ameliorated the HFD/STZ-induced elevation of fasting blood glucose, insulin, and hepatic enzymes. Moreover, Spirulina successfully rectified disrupted serum lipid profile and exhibited an anti-inflammatory effect via tumor necrosis factor-α and adiponectin modulation. On the molecular level, Spirulina reduced the expression of hepatic sterol regulatory element binding protein-1c (SREBP-1c), confirming its lipotropic effect. Furthermore, Spirulina amended compromised hepatic mitochondrial biogenesis signaling by significantly increasing peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), mitochondrial transcription factor A (Tfam), and mitochondrial DNA (mtDNA) copy number. On almost all parameters, the highest dose of Spirulina showed the best effects, which were comparable to that of metformin. To our knowledge, our study is the first to attribute the various aspects of the effect of Spirulina to the SREBP-1c and PGC-1α/Tfam/mtDNA pathways in liver. The present results clearly proved that Spirulina modulated glucose/lipid profile and exhibited prominent anti-inflammatory properties through SREBP-1c inhibition and hepatic mitochondrial biogenesis enhancement. Thus, Spirulina can be considered as an add-on to conventional antidiabetic agents and might influence the whole dynamics of the therapeutic approaches in T2DM.

Mitochondrial DNA Analysis of a Sample of Pre-Columbian Acient Remains from Norte de Santander, Colombia (Chitarera Cultural Area) / Análisis de adn mitocondrial en una muestra de restos óseos precolombinos de norte de santander, colombia (área cultural chitarera)

RESUMEN Los análisis de ADN antiguo (ADNa) han incrementado en los últimos años permitiendo conocer la diversidad genética de las poblaciones precolombinas. En Colombia, existen pocos registros arqueológicos de la población prehispánica del Norte de Santander habitada en el siglo XVI por el grupo Chitarero. Por este motivo, nos propusimos analizar la diversidad genética a partir de secuencias de la región HVRI del ADNmt y determinar sus posibles relaciones con otras comunidades tanto antiguas como contemporáneas. Se analizaron siete individuos precolombinos asociados a este grupo prehispánico, recuperados en los municipios de Cácota y Silos en el departamento de Norte de Santander de los Andes Orientales colombianos, siguiendo criterios estrictos de autenticidad para el ADNa. En todos los individuos se logró identificar el haplogrupo B caracterizado por el polimorfismo en la posición 16217C, siendo éste uno de los más frecuentes en comunidades precolombinas y contemporáneas de los Andes Suramericanos. Este hallazgo indica que este grupo poblacional se encuentra estrechamente emparentado por línea materna, con posibles índices de endogamia, con una probable densidad demográfica baja y una baja diversidad genética, similares a lo observado en comunidades pertenecientes a periodos anteriores como el Formativo. Este grupo precolombino exhibe una de las diversidades genéticas más bajas reportadas en las poblaciones pertenecientes a la familia lingüistica Chibcha. Estos resultados genéticos coinciden con los planteamientos sobre el grupo Chitarero de pertenecer a comunidades pequeñas independientes, con asentamientos dispersos, apartados unos de otros.

ABSTRACT In the last few years there has been an increase in ancient DNA (aDNA) analyses that has allowed shedding light on the diversity of pre-Columbian populations. In Colombia, there are few archaeological records belonging to the prehispanic population from Norte de Santander inhabited in the XVI century by the Chitarero. For this reason, we performed a genetic diversity analysis of the HVRI region of mtDNA in order to determine their possible relationships with other communities both ancient and contemporary. We analyzed seven pre-Columbian individuals belonging to this pre-Hispanic group, recovered from the municipalities of Cácota and Silos in the department of Norte de Santander located at the Colombian Andes, following strict authenticity criteria for aDNA. All individuals were identified as belonging to haplogroup B, characterized by the polymorphism found at position 16217C which is one of the most frequent haplogroups in pre-Columbian and contemporary communities of the South American Andes. This finding suggests that this population group was closely related through its maternal lineage, with possible inbreeding indexes, low population density and therefore low genetic diversity, similar to what is observed in communities belonging to previous periods such as the Formative period. This pre-Columbian group exhibits one of the lowest genetic diversities reported in populations belonging to the Chibcha linguistic family. These genetic results coincide with the views on the Chitarero group as belonging to small independent communities, with dispersed settlements separated among them.

IDENTIFICACIÓN MOLECULAR Y DISTRIBUCIÓN POTENCIAL DEL ANFÍPODO TERRESTRE Talitroides topitotum (Crustacea: Amphipoda: Talitridae) EN COSTA RICA / Molecular Identification and Potential Distribution of Terrestrial Amphipod Talitroides topitotum (Crustacea: Amphipoda: Talitridae) in Costa Rica

RESUMEN El anfípodo terrestre, Talitroides topitotum, es un talítrido distribuido mundialmente en regiones subtropicales y templadas, con un amplio rango de distribución altitudinal, temperatura y humedad. Se colectaron y procesaron especímenes desde el año 2012 al 2016, mediante remoción-filtración de sustratos húmedos. Se identificaron taxonómicamente por características fenotípicas diagnósticas, se determinó su estado de desarrollo y se separaron por sexo. Se extrajo ADN de anfípodos completos, seguido de una PCR de los genes citocromo oxidasa subunidad 1 y del ARN ribosomal de la subunidad 16S. Se obtuvo un árbol filogenético por máxima verosimilitud con un modelo GTR-GAMMA. El análisis de la distribución potencial de T. topitotum se estimó utilizando 19 variables bioclimáticas. En este estudio, se amplía la distribución previamente reportada y en altitudes entre los 1900 a 595 m s.n.m. Se analizaron 39 localidades, en las cuales: 1) Hay presencia de T. topitotum, 2) no hubo presencia de anfípodos terrestres, 3) no hubo presencia de Talitroides sp., pero sí de un anfípodo nativo. La abundancia proporcional de T. topitotum se inclina hacia las hembras adultas, una proporción alta de juveniles y no se detectaron individuos machos. El análisis bioinformático determinó el posicionamiento taxonómico de la especie T. topitotum dentro del agrupamiento de anfípodos terrestres, además, la especie exógena diverge de Cerrorchestia hyloraina demostrando una separación filogenética entre especies, las cuales pueden estar compartiendo hábitats. T. topitotum, según el modelo de máxima entropía, posee una alta capacidad de dispersión y estaría siendo favorecida, en cuanto a su asentamiento y propagación, por elementos climáticos como temperatura, precipitación y humedad, y factores como la altitud. Nuestros hallazgos son relevantes para la toma de decisiones de manejo y monitoreo del desplazamiento de especies nativas de anfípodos terrestres en la región.

ABSTRACT The land-hopper, Talitroides topitotum, is a talitrid amphipod distributed worldwide in subtropical and template regions, with a wide range of altitudinal distribution, temperature and humidity. Specimens were collected and processed since 2012 until 2016, by collection-filtration of wet substrates. Specimens were taxonomically identified using diagnostic phenotypic characteristics, and the developmental stage and sex were recorded. DNA was extracted from whole amphipods, followed by PCR of cytochrome oxidase subunit 1 and ribosomal RNA subunit 16S genes. Partial genetic sequences were obtained and a maximum-likelihood phylogenetic tree was calculated based on a GTR-GAMMA model. The analysis of potential distribution of T. topitotum was estimated using 19 bioclimatic variables. This study extends the previously reported distribution and elevations between 1900 and 595 m a.s.l. Thirty-nine localities were analyzed, where the following categories were registered: 1) T. topitotum is present, 2) terrestrial amphipods are not present, 3) T. topitotum is not present, but the native amphipod is present. The relative abundance of T. topitotum corresponds to adult females, a high proportion ofjuveniles and no males were collected. The bioinformatic analysis established the taxonomic position of T. topitotum within a group of terrestrial amphipods; moreover, the invasive species diverges of Cerrorchestia hyloraina, demonstrating the phylogenetic separation between these species that could be sharing habitats. Based on the model of maximum entropy, T. topitotum shows a high dispersion capacity and its establishment and propagation are been improved by climatic elements such as temperature, precipitation, humidity, and elevation. Our findings are relevant for management policies and monitoring the distribution of native species of terrestrial amphipods in the region.

Lower mitochondrial dysfunction in survivor septic patients with mitochondrial DNA haplogroup JT.

OBJECTIVE: The comparison on mitochondrial function between severe septic patients and healthy control subjects according to mitochondrial deoxyribonucleic acid (mtDNA) haplogroup has not been previously reported; and this was the objective of the current study. METHODS: Prospective, multicenter, observational study. We obtained blood samples from 198 severe septic patients at days 1, 4 and 8 of severe sepsis diagnosis and from 96 sex- and age-matched healthy controls to determine mtDNA haplogroup and platelet respiratory complex IV (CIV) specific activity. The endpoint of the study was 30-day mortality. RESULTS: We included 198 severe septic patients (38 with mtDNA haplogroup JT and 160 with mtDNA haplogroup non-JT) and 96 healthy control subjects (16 with mtDNA haplogroup JT and 80 with mtDNA haplogroup non-JT). We have no found statistically significant differences in platelet CIV specific activity between healthy controls and survivor severe septic patients with mtDNA haplogroup JT at days 1, 4 and 8 of severe sepsis diagnosis; and the remaining severe septic patients showed lower platelet CIV specific activity than healthy controls with the same mtDNA haplogroup. CONCLUSIONS: The new finding of our study was that survivor severe septic patients and healthy controls with mtDNA haplogroup JT showed no different platelet Civ specific activity.

Three new active members of the I-OnuI family of homing endonucleases.

In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.

Differential effect of hypoxia and acidity on lung cancer cell and fibroblast metabolism.

This study examined the metabolic response of lung cancer cells and normal lung fibroblasts to hypoxia and acidity. GLUT1 and HXKII mRNA/protein expression was up-regulated under hypoxia in the MRC5 fibroblasts and in the A549 and H1299 lung cancer cell lines, indicating intensified glucose absorption and glycolysis. Under hypoxia, the LDHA mRNA and LDH5 protein levels increased in the cancer cells but not in the fibroblasts. Acidity suppressed the above-mentioned hypoxia effect. PDH-kinase-1 (PDK1 mRNA and protein) and inactive phosphorylated-PDH protein levels were induced under hypoxia in the cancer cells, whereas these were reduced in the MRC5 lung fibroblasts. In human tissue sections, the prevalent expression patterns supported the contrasting metabolic behavior of cancer cells vs. tumor fibroblasts. The monocarboxylate/lactate transporter 1 (MCT1) was up-regulated in all the cell lines under hypoxic conditions, but it was suppressed under acidic conditions. The mitochondrial DNA (mtDNA) content per cell decreased significantly in the A549 cancer cell line under hypoxia, but it increased in the MRC5 fibroblasts. Taking into account these findings, we suggest that, under hypoxia, cancer cells intensify the anaerobic direction in glycolysis, while normal fibroblasts prefer to seek energy by intensifying the aerobic use of the available oxygen.

Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels.

Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species' presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.

IMPLEMENTACIÓN DE LA METODOLOGÍA DE ANÁLISIS DE ADN MITOCONDRIAL EN Rhinoclemmys nasuta (TESTUDINES:GEOEMYDIDAE) / Implementation of DNA Mitochondrial Analysis in Rhinoclemmys nasuta (Testudines: Geoemydidae)

Rhinoclemmys nasuta (Testudines: Geoemydidae) es considerada una especie casi endémica de Colombia y la más primitiva del género, sin embargo, se encuentra clasificada por la IUCN como deficiente de datos, ya que la información disponible no es suficiente para hacer una evaluación directa o indirecta de su riesgo de extinción. En este trabajo se describe la implementación del método para realizar la secuenciación de la región control del ADN mitocondrial de R. nasuta, con el propósito de generar herramientas técnicas para futuros estudios de evolutivos y de conservación. Se utilizó el método de desalamiento (Salting-out) para extraer ADN a muestras sanguíneas procedentes de Isla Palma y Playa Chucheros (Bahía Málaga-Pacífico Colombiano) y se utilizó una pareja de cebadores degenerados (reportada para Chrysemys picta Testudines: Emydidae) para realizar la amplificación. Se obtuvieron fragmentos de 800pb siendo exitosa la reacción de secuenciación de los amplificados, y se estableció un porcentaje de homología mayor al 92 % entre las secuencias obtenidas y las secuencias de ADNmt de Sacalia quadriocellata (Testudines:Geoemydidae) y Cuora aurocapitata (Testudines:Geoemydidae), depositadas en el GeneBank. Este resultado demuestra que el método descrito puede ser una herramienta útil para el estudio de las poblaciones de R. nasuta del Pacífico colombiano, lográndose una efectiva secuenciación de la región control del ADNmt de esta especie.

Rhinoclemmys nasuta (Testudines:Geoemydidae) is considered an almost endemic specie to Colombia and the most primitive species of Rhynoclemmys. However, it is classified data deficient by IUCN because the available information is not enough to make a direct or indirect assessment of its extinction risk. Here, we describe the implementation of the method to analyze the mitochondrial DNA control sequence (mtDNA) of R. nasuta in order to generate tools for future studies in systematics and population conservation. Genomic mtDNA was extracted by salting-out from blood samples from Isla Palma and Playa Chucheros (Bahía Málaga-Colombian Pacific Coast) and we used a pair of degenerate primers (reported for Chrysemys picta, Testudines:Emydidae) to perform amplification. Fragments of 800pb were obtained and the sequencing reaction was effective. A homology percentage above of 92 % was established between the obtained sequences and mtDNA sequences from Sacalia quadriocellata (Testudines:Geoemydidae) ,and Cuora aurocapitata (Testudines:Geoemydidae) reported in the GenBank. This result shows that the described method can be a useful tool for the study of R. nasuta populations in the Colombian Pacific region, achieving an effective sequencing of the mtDNA control region of this species.

Filogeografía de Holothuria (Halodeima) inornata Semper, 1868 (Echinodermata: Holothuroidea)

Se evaluó la estructura genética de las poblaciones de Holothuria (Halodeima) inornata Semper, 1868, y se investigó cuáles podrían ser las barreras para el flujo de genes y procesos históricos. Se recolectaron muestras tratando de abarcar su ámbito de distribución, desde México hasta el norte de Perú. Con base en secuencias del gen COI se detectaron 118 haplotipos en 220 individuos y las diferencias entre dichos haplotipos fueron debidas a 97 sitios variables (21.41%) en los 453 pb secuenciados. Se observó una alta diversidad de haplotipos (h=0.979) y moderada diversidad nucleotídica (π=0.017). Para analizar la diferenciación genética, se utilizaron los valores de Fst, el test exacto de diferenciación poblacional y los análisis de varianza molecular (AMOVA). Estos análisis sugieren que existen dos poblaciones: las del norte, frente a las costas de: Sinaloa, Jalisco, Michoacán, Guerrero y Oaxaca; y las del sur, frente a las costas de: Chiapas, El Salvador, Panamá y Perú. Los acontecimientos históricos y los patrones oceanográficos podrían ser los principales factores que determinaron la dispersión y estructura de la población de H. inornata, es probable que la población original se haya extendido inicialmente en el sur y luego hacia el norte. Además, la separación entre estas dos poblaciones podría deberse al Golfo de Tehuantepec, el cual está constituido por una serie de eventos tectónicos y oceanográficos que constituyen una barrera para el asentamiento de H. inornata.

Genetic structure of the populations of H. inornata was evaluated and the barriers for genetic flux and historic processes were investigated. Samples were collected trying to cover the distribution range of the species, from Mexico to northern Perú. Based on COI sequences, 118 haplotypes from 220 specimens were detected; the differences between such haplotypes were due to 97 variable sites (21.41%) of the 453 bp sequenced. A high haplotype diversity (h=0.979) and a moderate nucleotidic diversity were observed. The values of Fst, the exact test of population differentiation, and the molecular variance analysis (AMOVA) were used in order to analyze the genetic differentiation. These analyses suggest the existence of two populations: northern, off the coasts of Sinaloa, Jalisco, Michoacán, Guerrero, and Oaxaca, and southern, off the coasts of Chiapas, El Salvador, Panamá and Perú. Historic events and oceanographic patterns may be the main factors determining dispersion and structure of Hi populations. It seems probable that the original population have extended first in the south and then northern. Besides, the split between these two populations may be due to several tectonic and oceanographic events constituting a barrier for H. inornata settling.

An overview of neurological and neuromuscular signs in mitochondrial diseases.

Mitochondrial disorders have a broad clinical spectrum and are genetically heterogeneous, involving two genomes. These disorders may be develop at any age, with isolated or multiple system involvement, and any pattern of inheritance. Neurological involvement is the most frequent, and concerns muscular, peripheral and central nervous system. Among these diverse signs, some are suggestive of mitochondrial disease, such as progressive external ophthalmoplegia, exercise intolerance, psychomotor regression, stroke-like episodes, refractory epilepsy and Epilepsia Partialis Continua. Others are less specific and mitochondrial hypothesis may be evocated because of either association of different neuromuscular signs or a multisystemic involvement. This review describes the wealth of this neurological and neuromuscular symptomatology through different syndromes reported in the literature, according to preponderant signs and to modes of inheritance, as key elements to guide genetics testing.

A mitochondrial implication in a Tunisian patient with Friedreich's ataxia-like.

Genes encoding the DNA helicase TWINKLE (C10orf2) or the two subunits of mtDNA polymerase γ (POLγ) (POLG1 and POLG2) have a direct effect on the mitochondrial DNA replication machinery and were reported in many mitochondrial disorders. Friedreich's ataxia (FRDA) is the common cause of ataxia often associated with the expansion of a GAA repeat in intron 1 of the frataxin gene (FXN). Mitochondrial DNA could be considered as a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. We screened the FXN, POLG1 and C10orf2 genes in a Tunisian patient with clinical features of Friedreich's ataxia-like. The results showed the absence of the expansion of a GAA triplet repeat in intron 1 of the FXN gene. Besides, the sequencing of all the exons and their flanking regions of the FXN, POLG1 and C10orf2 genes revealed the presence of intronic polymorphisms. In addition, screening of the mtDNA revealed the presence of several mitochondrial known variations and the absence of mitochondrial deletions in this patient. The detected m.16187C>T and the m.16189T>C change the order of the homopolymeric tract of cytosines between 16184 and 16193 in the mitochondrial D-loop and could lead to a mitochondrial dysfunction by inhibiting replication and affecting protein involved in the replication process of the mtDNA which could be responsible for the clinical features of Friedreich ataxia observed in the studied patient.

Uso de la técnica SSCP para detectar mutaciones puntuales del ADN mitocondrial humano

En el presente trabajo se evalúa la técnica de SSCP (polimorfismo de conformación de cadena individual de ADN) para detectar mutaciones puntuales, tanto por su sensibilidad en la detección (alrededor 80% en condiciones ideales), como por su implementación fácil y económica. Se utilizaron como controles positivos y negativos, ADN de voluntarios caracterizados previamente para las mutaciones puntuales de 5 RFLPs mitocondriales. Para la optimización de la prueba fueron ensayadas concentraciones variables del tampón TBE (1X y 0,5X) y del glicerol (10%, 5% y 0) en geles de poliacrilamida. Cuatro de los 5 RFLPs fueron detectados en las condiciones utilizadas y pueden ser usados en estudios de rutina sin usar enzimas de restricción. Además, la técnica SSCP permitió determinar mutaciones desconocidas en un segmento de 394 nucleótidos de la región hipervariable (HVI) del ADNmt. Diferencias en correspondencia a los distintos haplotipos fueron detectados e incluso permitió discernir grupos dentro del mismo subtipo. La secuenciación de dos muestras del subtipo B1 con migración diferencial en SSCP, corroboró la existencia de siete nucleótidos distintos.

We evaluate the use of SSCP (single strand conformational polymorphism), a relatively easy and inexpensive technique for the detection of point mutations with a sensibility around 80% under ideal conditions. To test the technique, we used samples of volunteers whose DNA had been previously characterized for the presence or absence of 5 mitochondrial RFLPs. Optimization of the tests included variations in TBE (1X and 0,5X) and of glycerol concentration (10%, 5% and no glycerol) in polyacrylamide gels. Four out of five RFLPs were detected under the conditions used and could be applied routinely without using restriction enzymes. In addition, the SSCP technique allowed detection of unknown mutations in a 394 bp nucleotide segment of the hypervariable (HVI) region of mtDNA. Differences corresponding to different haplotypes were detected, helping to distinguish groups within the same subtype. Sequencing of two samples of subtype B1 with differential migration on SSCP gels, proved the existence in seven different nucleotides.