Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Several missplicing events and transcriptional alterations have been described in DM1 patients. A large number of these defects have been reproduced in animal models expressing CTG repeats alone. Recent studies have also reported miRNA dysregulation in DM1 patients. In this work, a Drosophila model was used to investigate miRNA transcriptome alterations in the muscle, specifically triggered by CTG expansions. Twenty miRNAs were differentially expressed in CTG-expressing flies. Of these, 19 were down-regulated, whereas 1 was up-regulated. This trend was confirmed for those miRNAs conserved between Drosophila and humans (miR-1, miR-7 and miR-10) in muscle biopsies from DM1 patients. Consistently, at least seven target transcripts of these miRNAs were up-regulated in DM1 skeletal muscles. The mechanisms involved in dysregulation of miR-7 included a reduction of its primary precursor both in CTG-expressing flies and in DM1 patients. Additionally, a regulatory role for Muscleblind (Mbl) was also suggested for miR-1 and miR-7, as these miRNAs were down-regulated in flies where Mbl had been silenced. Finally, the physiological relevance of miRNA dysregulation was demonstrated for miR-10, since over-expression of this miRNA in Drosophila extended the lifespan of CTG-expressing flies. Taken together, our results contribute to our understanding of the origin and the role of miRNA alterations in DM1.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos.

Novel next-generation sequencing procedures have rapidly emerged into the preimplantation genetic screening framework. This work presents the design and validation of a new low-coverage whole-genome sequencing assay for aneuploidy detection in single blastomeres and trophectodermal samples from preimplantation embryos. The validation ensures analytical sensitivity, specificity, robustness, precision, limit of detection, resolution, and reproducibility. Specific parameters to measure the performance are defined, and the results are compared with a standardized array-based method to stablish the concordance. From the single cell genomics point of view, the main novelties are the length of reads of the libraries (150 nucleotides) together with a paired-end strategy and the design of an original algorithm and copy number viewer. A total of 129 samples were included in six experimental runs using a MiSeq Illumina platform. Samples included: single amniocytes, single blastomeres (cleavage-stage embryos), trophectoderm samples (blastocyst), and diluted DNA. Sensitivity and specificity were calculated per chromosome yielding 96% and 99%, respectively. The percentage of concordant samples was 98.2% and all of the aneuploid samples were confirmed. In conclusion, the validation yields highly reliable and reproducible results, representing an accurate and cost-effective strategy for the routine detection of aneuploidy in human embryos.

Statistical validation of the identification of tuna species: bootstrap analysis of mitochondrial DNA sequences.

Sequencing of the mitochondrial cytochrome b gene has been used to differentiate three tuna species: Thunnus albacares (yellowfin tuna), Thunnus obesus (bigeye tuna), and Katsuwonus pelamis (skipjack). A PCR amplified 528 bp fragment from 30 frozen samples and a 171 bp fragment from 26 canned samples of the three species were analyzed to determine the intraspecific variation and the positions with diagnostic value. Polymorphic sites between the species that did not present intraspecific variation were given a diagnostic value. The genetic distance between the sequences was calculated, and a phylogenetic tree was constructed, showing that the sequences belonging to the same species clustered together. The bootstrap test of confidence was used to determine the statistical validation of the species assignation, allowing for the first time a quantification of the certainty of the species assignation. The bootstrap values obtained from these results indicate that the sequencing of the cytochrome b fragments allows a correct species assignation with a probability > or =95%.

Enfoque diagnóstico molecular utilizando secuenciación exómica en las distrofias musculares cintura-cadera / Molecular diagnosis approach through the use of whole exome sequencing in limb-girdle muscular dystrophies

Antecedentes. La distrofia muscular cintura-cadera tipo 1B es una enfermedad con herencia autosómica dominante y secundaria a una mutación en el gen LMNA. Esta enfermedad se caracteriza por su afectación a nivel neuromuscular y cardiaco. Objetivo. Realizar diagnóstico clínico y confirmatorio molecular en una paciente con debilidad muscular proximal y sintomatología cardíaca a través de secuenciación exómica. Materiales y métodos. Se presenta el caso de una paciente de 57 años de edad con cuadro de debilidad muscular proximal progresiva principalmente en extremidades y posterior afectación cardíaca; adicionalmente, la paciente tiene múltiples familiares con la misma sintomatología. Se realizó estudio de secuenciación exómica con confirmación, por método de Sanger, de la mutación hallada y posteriormente el análisis bioinformático de esta. Resultados. La detección de la mutación R377L en el gen LMNA por secuenciación exómica con confirmación por Sanger, junto con la sintomatología clínica de la paciente y el análisis bioinformático de la mutación hallada, permitió realizar diagnóstico confirmatorio de distrofia muscular cintura-cadera tipo 1B. Conclusión. Es difícil realizar un diagnóstico clínico debido a la heterogeneidad genética del fenotipo de distrofias musculares cintura-cadera. La aproximación diagnóstica es compleja y requiere clasificar las distrofias musculares según el patrón de afectación y el patrón de herencia de la enfermedad. Adicionalmente, debido a los múltiples genes que pueden generar clínica semejante a las diferentes distrofias musculares, se recomienda realizar secuenciación exómica solicitando especial énfasis en los genes candidatos de distrofias musculares cintura-cadera.

Background. Limb-girdle muscular dystrophy type 1B has a dominant autosomal inheritance pattern and is caused by a mutation in the LMNA gene. This disease has a major neuromuscular and cardiac compromise; furthermore, it belongs to the limb-girdle muscular dystrophies. Objective. To make a clinical and molecular confirmatory diagnosis in a patient with proximal muscular weakness and cardiac symptoms using whole exome sequencing. Materials and Methods. This is the case of a 57 year old patient with a slowly progressive proximal muscular weakness and cardiac compromise; furthermore, the patient has many relatives with the same clinical history. Whole exome sequencing with Sanger confirmation and bioinformatics analysis was performed on the found mutation. Results. The detection of mutation R377L in the LMNA gen by whole exome sequencing with Sanger confirmation, the bioinformatic analysis of the mutation and the symptoms exhibited by the patient allowed the confirmatory diagnosis of limb-girdle muscular dystrophy type 1b. Conclusion. Due to genetic heterogeneity in the phenotype of limb-girdle muscular dystrophies it is difficult to make a clinical diagnosis. The diagnostic approach is complex and requires classification of the muscular dystrophies according to the pattern of muscular weakness and to identify the disease inheritance pattern. Additionally, due to the multiple genes that can generate similar symptoms in the different muscular dystrophies, the authors recommend the use of whole exome sequencing with a special emphasis on the candidate genes for limb-girdle muscular dystrophies.

Diagnóstico molecular de enfermedades genéticas: del diagnóstico genético al diagnóstico genómico con la secuenciación masiva / Molecular diagnosis of genetic diseases: from genetic to genomic diagnosis using next generation sequencing

En la actualidad se conocen 8.000 enfermedades genéticas monogénicas. La mayoría de ellas son heterogéneas, por lo que el diagnóstico molecular por técnicas convencionales de secuenciación suele ser largo y costoso debido al gran número de genes implicados. El tiempo estimado para el diagnóstico molecular se encuentra entre 1 y 10 años, y este retraso impide que los pacientes reciban medidas terapéuticas y de rehabilitación específicas, que sus familiares entren en programas preventivos y que reciban asesoramiento genético. La secuenciación masiva está cambiando el modelo de diagnóstico molecular de los afectos, sin embargo, los médicos y profesionales de la salud se enfrentan al dilema de la selección del método más eficiente, con el menor coste sanitario y con la mayor precisión de sus resultados. El objetivo de este trabajo es revisar la tecnología de secuenciación masiva y definir las ventajas y los problemas en su utilización.

Currently 8000 monogenic genetic diseases are known. Most of them are heterogeneous, so their molecular diagnosis by conventional sequencing techniques is labour intensive and time consuming due to the large number of genes involved. The estimated time is between 1 and 10 years for molecular diagnosis and this delay prevents patients from receiving therapy and rehabilitation measures, and their families from entering prevention programs and being given genetic counselling. Next generation sequencing (NGS) is changing the model of molecular diagnosis of patients; however, doctors and health professionals are faced with the dilemma of choosing the most efficient method, with lower health care costs and the most accurate results. The aim of this paper is to review the NGS technology and define the advantages and problems in the use of this technology.