Avances en genética. Utilidad de la NGS y la bioinformática.
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27 Octubre 2014. Presentación de Pablo Lapunzina, Director del Instituto de Medicina Genética Médica y Molecular (INGEMM), de IDIPAZ y de CEBERER, en la "Jornada Avances en Genética y Tecnología Social. La experiencia de la Fundación Síndrome de Dravet ".
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Avances en genética. Utilidad de la NGS y la bioinformática.
- 1. Bilbao 27 de Octubre, 2014 Avances en Genética. Utilidad de la NGS (Next Generation Sequencing) y la Bioinformática. El ejemplo del S. de Dravet. Pablo Lapunzina, M.D., Ph.D On behalf of the Dravet Syndrome Genetic Testing Group and the UBEG group.
- 2. General concepts of epilepsy Epilepsy is a common neurological condition defined by recurrent, unprovoked seizures that affects 1% of the population, including 1/200 children Epilepsy risk: 1% on general population 8-12% on first-degree relatives Clinical classification of epilepsy: Symptomatic Presumed symptomatic Idiopathic Epilepsy genetics Complex disease with strong genetic component Mendelian genetics: autosomal dominant inheritance Genes encoding ion channels and neurotransmitter receptors
- 3. General concepts of epilepsy Known genes involved in epilepsy syndromes Poduri, A., et al., Curr. Op. Genet & Dev., 2011
- 4. Dravet Syndrome clinical features Incidence of 1/20,000 births, approximately Rare congenital disease Genetic epilepsy: Autosomal dominant or de novo 25% with familial history of epilepsy or febrile seizures 1st seizure <1y Fever sensitivity Afebrile seizures Type of seizures: Generalized/partial Tonic/Clonic Myoclonic Status epilepticus Intellectual disability, autism, sleep disturbancies Atypical absences, ataxia, SUDEP No known treatment nor cure Early diagnosis is required
- 5. Genetic diagnosis in DS (I) Clinical diagnosis Medical history of seizures Seizure type Age of onset EEG, CT, … Genetic Diagnosis Familial history Genotype - Phenotype + Personalized treatment and prognosis
- 6. Genetic diagnosis in DS (II) Identification of the molecular cause of DS Genetic counseling Prenatal diagnosis Accurate and personalized treatment Confirmation of clinical diagnosis
- 7. DS is a genetic febrile epilepsy FS: febrile seizures FS+: febrile seizures plus GEFS+: generalized epilepsy with febrile seizures plus SIMFE: severe infantil multifocal epilepsy SMEB: severe myoclonic epilepsy of infancy borderland EMRF: epilepsy with mental retardation limited to female ICE-GTC: intractable childhood epilepsy with generalized tonic clonic seazures SMEI/DS: severe myoclonic epilepsy of infancy www.ice-epilepsy.org
- 8. Genes involved on DS 80% of DS patients have mutations on known genes: SCN1A, PCDH19, GABRG2, SCN2A y SCN1B SCN8A and SCN9A as modifier genes 20% of DS patients with unknown genetic defect
- 9. Sodium channel (I) Depienne, C., et al., J Med. Genet., 2009 333 DS patients
- 10. DS is genetically heterogeneous DS patients with family history of epilepsy: 25% with family history of epilepsy, but 90% with “de novo” mutations DS patients within GEFS+ families DS patients with inherited mutation from asymptomatic or with mild pathological phenotypic parents Mosaicism: May explain a parental clinical history of seizures Percentage of mosaicism detected in lymphocytes is not correlated to other tissues May evaluate recurrence risk Genetic compensation: Protective changes in one ion channel may compensate the damage in another ion channel Modifier genes Other genomic regions that regulate gene expression
- 11. DS Genetic Test of DSF-INGEMM Free DS genetic test Patient has to follow the clinical criteria of DSF Genetic report of SCN1A in ~80 days Genetic test of other DS genes when SCN1A screening is negative Extension to other genes related to genetic epilepsies and phenotype modifier genes in the next future Application of the new high-throughput genomic technology Prenatal screening and genetic testing of relatives OBJECTIVE: 100% of DS patients with genetic diagnosis
- 12. DS Genetic Test procedure (I) www.dravetfoundation.eu Document 1 Document 1 Patient Clinical Info Patient Clinical Info Document 2 Document 2 Informed Consent Informed Consent OK NNeeuurrooppeeddiiaattrriicciiaann Download documents Date Date the the patient patient Blood or gDNA Fill out and signed Dra. Eva Barroso Bloque Quirúrgico, pl-2 Hospital Universitario La Paz Pº La Castellana, 261 28046 MADRID Fill out data and wait for acceptance eevvaa.b.baarrrorossoo@@ddraravveetftofouunnddaatitoionn.e.euu Original signed Genetic report Genetic report Results
- 13. gDNA amplification: PCR PCR: Polymerase Chain Reaction Oligonucleotide designed in intronic regions Oligonucleotides bind specifically to selected regions of genome to the double chain of gDNA Exponential and specific amplification
- 14. Direct sequencing Sanger sequencing: dNTPs + ddNTPs labeled with fluorochromes Capillary electrophoresis + fluorescence detector p.L1670fsX9 p.A486G
- 15. In silico functional analysis by bioinformatic tools Mutation tasting: http://www.mutationtaster.org/ Polyphen2: http://genetics.bwh.harvard.edu/pph2/ SNP&GO: http://snps-and-go.biocomp.unibo.it/snps-and-go/ PANTHER: http://www.pantherdb.org/ MUpro: http://mupro.proteomics.ics.uci.edu/ SIFT Human Coding SNPs: http://sift.jcvi.org/www/SIFT_chr_coords_submit.html Exome Variant Server: http://evs.gs.washington.edu/EVS/ Human Splicing Finder: http://www.umd.be/HSF/
- 16. High-throughput genomic analysis (II) Array-CGH (Comparative Genomic Hybridization): Complex genomic rearrangements analysis Roche-Nimblegen or Agilent aCGH platforms Genetic Epilepsy Panel of ~300 genes
- 17. Inversión del proceso Solicitud de asistencia Recepción del volante Admisión Consulta Clínica Estudio citogenético Microarrays CGH Estudio citogenético Microarrays CGH Estudios moleculares específicos Estudios de ultrasecuenciación (NGS) Evaluación de resultados Emisión del informe Asesoramiento Genético
- 18. The “Genetic” approach Studying “all at the same time” Before Now Gene by gene NGS (exome approach sq or panels)
- 19. High-throughput genomic analysis (I) NGS (Next-Generation Sequencing): High-throughput genomic sequencing Complete sequencing of genome Exome sequencing Sequencing of specific regions accordingly a pre-designed panel Three different NGS platforms on testing DS Panel Genetic Epilepsy Panel of ~300 genes Roche Illumina Ion Torrent
- 20. NGS
- 21. Hospital Universitario La Paz Distribution of Human Genetic Diseases 5 Kb- ? Mb 1 bp- 200 bp No dosage changes GENOMICS GENETICS EPIGENETICS All genetic/genomic or epigenetic diseases with known cause: ~ # 5000 disorders 8-12% 82-87% 2-3% Unknown or No- responsible genes
- 22. The “Genetic Pool” ~ 24,000 MIM genes
- 23. 100% Genetics known gene but not linked to any disorder 100% Genomics 50% Genetics + 50% Genomics 100% Epigenetics 50% Genetics + 50% Unknown The “Genetic Pool”
- 24. Genomas, Exomas y Paneles 1-2 % 100 % 0,05 % 3,000 Millones de pares de bases
- 25. 25 Sequencing the Human Genome 2010: 5K$, 2009: Illumina, 10 days Helicos 40-50K$ 10 2001: Human Genome Project 2.7G$, 11 years price) 8 6 (10Log4 2 Year 2000 2005 2010 2014: 1000$, 36 hrs- Illumina 2008: ABI SOLiD 60K$, 2 weeks 2007: 454 1M$, 3 months 2001: Celera 100M$, 3 years 2015
- 26. Capillary Sequencing (Sanger) vs Next Generation Sequencing (NGS) Before Now Aproximately 300 bp/analysis (exons) Aproximately 75-400 bp per read x millons of reads in one assay.
- 27. Capillary Sequencing (Sanger) vs Next Generation Sequencing (NGS) Antes Ahora 1 Km (1 exon ),conocemos el gen ~ 300 coches/Km. Queremos ver Cual es el coche estropeado. 180,000 Km (exones), varios carriles por Km, No sabemos en que carretera (gen) ni donde está el coche defectuoso. Tenemos muchos coches que parecen defectuosos, pero en realidad NO lo están.
- 28. El Proceso Actual Antes Ahora - Extracción de ADN en cada Lab - Extracción de ADN centralizada - Amplificación x PCR amplification - NGS experiments (panels or exomes) - Screening de amplicones - Análisis de Bioinformática Ej..(HRM, etc) o Sanger Seq - Send to “experts” in the field - Análisis y reporte - Análisis, validación y reporte.
- 29. Platforms Illumina HiSeq X Ten and NextSeq 500 Ion Proton
- 30. El Proceso de Dx Genético
- 32. NGS (Next Generation Sequencing)
- 33. Interfaz de inicio www.dravetfoundation.eu/bbdd-mutaciones-geneticas.
- 34. Lista de variables– Descripción de la mutación *Mutation_ID *ID *Gene *Variant Pathogenicity *Exon *cDNA change *Protein change Chr coordinate change (hg19) dbSNP ClinVar *Type of variant *Mutation effect *Protein domain affected *RNA change Reference Template Technique Lista de variables – Descripción del fenotipo clínico Clinical phenotype description Answer General features Seizure onset (y) Number *Patient Phenotype/Epilepsy Classification Description Seizure type Generalized tonic clonic seizure Y/N/DK Myoclonic seizure Y/N/DK Atypical absence Y/N/DK Partial seizure Y/N/DK Hemiconvulsion Y/N/DK Secondary generalization Y/N/DK Status epilepticus (N) Y/N/DK (Number) Number of seizures Number Seizures provoked Y/N/DK Afebrile seizures Y/N/DK Photosensitivity Y/N/DK Other stimulus (e.g. vaccination, heat bath, excitation, period) Description Neurological abnormalitie s Mental retardation Y/N/DK Motor delay Y/N/DK Ataxia Y/N/DK Autism Y/N/DK Sleep disturbancies Y/N/DK Others Description IQ Number Complement ary diagnosis tests Neurophysiology EEG Description Neuroimage MRI CT Scan Description Hattori score Number Genetic inheritance *Inheritance Type if known Family history Description Treatment Current treatment Description Ethnic origin Ethnic origin Description Remarks/Oth
- 35. Aproximación diagnóstica actual Paneles de genes personalizados para epilepsias genéticas. Exomas (23,000 genes) para investigación.
- 36. Genes MIM en un solo tubo Para Encefalopatías epilépticas: • (Casi) todos los genes de relevancia clínica • Sólo un estudio para todos los genes juntos (más de 300). • Tiempo de respuesta (aproximadamente 2-3 meses)
- 37. Genes implicados en Epilepsias La vida en Blanco y Negro de la Genética La vida en Colores de la Genética
- 38. ¿Hacia donde vamos? 5 Kb- ? Mb 1 bp- 200 bp No dosage changes GENOMICS CGH-array SNP-aray Karyotype FISH GENETICS NGS panel screening Exome seq Sanger Seq Exome Seq EPIGENETICS MS-arrays Meth- PCR confirmations First Step Further
- 39. Agradecimientos Todos los integrantes de la Fund. S. de Dravet BBK-KutxaBank Microsoft & Microsoft Azure INGEMM
- 40. H o s p i t a l U n i v e r s i t a r i o L a P a z INGEMM – Instituto de Genética Médica y Molecular Instituto de Genética Médica y Molecular
Editor's Notes
- Epilepsy is a common neurological condition defined by recurrent, unprovoked seizures that affects 1% of the population, including 1/200 children. The epileptic risk on general population is approximately 1%, but this risk raises up to 8-12% among first-degree relatives of individuals with idiopathic generalized epilepsy. This illustrates the complexity of this group of pathologies and their strong genetic component, but a complex one that does not always show a pattern consistent with genetic Mendelian inheritance. The clinical classification of epilepsy includes symptomatic epilepsy, presumed symptomatic or sub-clinical epilepsy and idiopathic epilepsy. The latter one is caused, at least in part, by genetics, influenced by genetic susceptibility. Epilepsy genetics has been based on Mendelian genetics, since many of the familial epilepsy syndromes exhibit an autosomal dominant inheritance, which identified mutations that are located in many of the genes encoding subunits of ion channels or neurotransmitter receptors, important in epileptogenesis.
- Here I show you a table that comprises most of the known genes involved in epilepsy syndromes, but the genes discovered thus far do not account for the majority of idiopathic genetic epilepsy.
- DS is a rare disease, since it is present in 1 in 20,000 births, approximately, so that it is considered a rare congenital disease, and also a genetic epilepsy that follows an autosomal dominant or de novo inheritances. It shows a high genetic component, because approximately 25% of patients with DS have a familial history of epilepsy or febrile seizures. First seizure comes up at age under 1 year, firstly associated with a febrile process or vaccination, but after that these seizures become recurrent and afebrile, or responding to other stimulus such as photosensivity, stress, changes in temperature, etc. Seizures can be partial or unilateral, or generalized, tonic/clonic, myoclonic, and sometimes it results in a status epilepticus. Mental retardation is shown from 2 years old and it ussually get worse in the next years, together with other alterations like autism, hyperexcitability, sleep disturbancies. It may also manifestate not typical seizures but atypical absences and other motor alterations such as ataxia and cumpsy walk. There is also a high mortality among DS patients, leaded by SUDEP or suden unexpected death in epilepsy patient. Additionally to these severe clinical features, this disease has not cure nor appropriate treatment. In deed, traditional antiepileptic drugs, such as carbamazepine, lamotrigine, phenytoin and others may aggravate symptoms of DS patients and this wrong treatment must be stopped as soon as possible. Thus, early diagnosis is a key step in the searching for seizure control of DS patients and the minimization of other symptoms.
- So that, we are trying to merge both clinical and genetic diagnosis. Clinical diagnosis details the medical history of seizures, like type of seizure, age of onset, anticonvulsant treatment, etc, together with other diagnostic tests, such as electroencephalography and computed tomography. But the genetic diagnosis gives us additional information in order to get a final accurate diagnosis. We can suspect that a disease has a genetic component when there is an associated familial history of similar symptoms, so there is a genetic marker inherited along generations. It makes also possible to determine a genetic profile of genotype that correlates with a specific clinical profile or phenotype, that can be applied in a correct and personalized treatment and prognosis of disease. Then, the implementation of genetic diagnosis in the clinical practice guidelines of hospitals, in combination with clinical diagnosis provided by neurologists, in the case of DS, will allow to establish a diagnostic routine of quality that will benefit patient.
- Genetic diagnosis for DS identifies the molecular cause of the disease, that can be applied in an appropriate family genetic counseling, and the possibility of access to a prenatal diagnosis. Otherwise, this information may help to confirm the previous clinical diagnosis, helping also in the establishment of pharmacological patterns according to patient genetic background.
- Furthermore, they share also common molecular factors, because mutations in the same genes may trigger different type of diseases, with more or less severity.
- Approximately 80% of patients with DS present one mutation in one of these known genes associated with this disease: SCN1A, PCHD19, GABRG2, SCN2A, SCN1B, SCN8A y SCN9A. Of these, 75% are due by mutations in SCN1A, and the remaining 5% are the mutations in the other genes. 20% of DS patients do not have a genetic diagnosis and the causative mutation remain unknown.
- Depienne and cols. carried out a genetic analysis of 333 DS patients in 2009, and they detected mutations in SCN1A in 242 (73%) patients. 228 mutations were missense mutations (42%), but other were nonsense, slipe-site mutations and indels, and 14 microrearrangments, most of them complete or partial deletions of gene. Besides, this figure shows the location of mutation in protein, and there is not a hot-spot domain for being mutated, and there is not any correlation between location of mutation and clinical feature profile of DS patient. If only DS patients with an accurate clinical feature profile of DS are considered, the mutation rate of SCN1A rises up to 78%. However, in this study such as later studies, do not identify other genes of genomic regions responsible of the remaining percentage of DS patients without genetic diagnosis.
- En definitiva, el SD es genéticamente heterogéneo, tanto por los genes que pueden verse afectados, los tipos de mutación que se han identificado hasta la fecha, que están repartidas a todo lo largo de los genes afectados, sin existir un hot-spot específico, haciendo muy difícil la correlación genotipo-fenotipo para esta enfermedad. Se trata este último punto de un reto que debemos conseguir por el bien de los pacientes y sus familias. Hasta ahora se ha podido comprobar que, si bien SD comparte características clínicas e incluso causa genética con una suerte de patologías congénitas que van desde FS, pasando por GEFS+ hasta DS en su máxima expresión, se considera que existe un 95% de pacientes SD con mutaciones “de novo”, pero un 25% de los pacientes SD presentan una historia familiar de epilepsia, crisis febriles convulsivas, etc., que sugiere un componente genético familiar que se escapa. Así, por ejemplo, se ha visto que pacientes SD están en familias que tienen miembros con GEFS+, donde SD se debe a haploinsuficiencia del gen SCN1A, mientras que GEFS+ se debe a una pérdida parcial de función del gen que no llega a ser haploinsuficiencia. Además, pacientes con una mutación claramente familiar, tienen padres asintomáticos o con un fenotipo muy leve. Todo ello indica que existen otros mecanismos o elementos que intervienen en definir un fenotipo leve o grave, que existe todo un espectro de expresión de la enfermedad que debe ser evaluado, tanto desde un punto de vista genético como ambiental. Así por ejemplo, el mosaicismo germinal y/o somático podría explicar la presencia de una historia parental asociada a epilepsias sólo una generación atrás. La detección de mosaicismo parental puede ser de vital importancia en aquellos casos en los que padres de un niño enfermo desean tener más descendencia, dado que se controlaría el riesgo de recurrencia al hacer estudios prenatales. Sin embargo, la evaluación del nivel de mosaicismo en un tejido como los linfocitos, no es extrapolable al tejido nervioso o al tejido germinal. Otro mecanismo podría ser la compensación genética y funcional a través de genes modificadores del fenotipo. En este caso, cambios en un canal iónico podrían compensar el daño en otro canal, y revertir o minimizar el fenotipo patológico. Estos cambios podrían ser no sólo cambios codificantes, sino también cambios que afecten a elementos reguladores de la expresión génica, como elementos reguladores del splicing, regiones promotoras, miRNAs, etc.
- The genetic test developed by DSF and INGEMM has the following characteristics.
- Workflow
- PCR
- Sanger sequencing
- Bioinformatic tools
- Otra tecnología que vamos a utilizar y que permite identificar grandes reordenamientos cromosómicos, deleciones, duplicaciones, etc., es el array CGH, para el que se dispone de dos plataformas plenamente funcionales en el INGEMM: Roche-Niblegen y Agilent. El diseño que se utilizará para montar el panel de genes de epilepsia y SD es el mismo que para NGS. Aquí pueden ver una imagen de ejemplo de lo que podremos detectar: 3 deleciones de diferente tamaño, que probablemente no se podrían haber visto con un cariotipo tradicional, ni con un MLPA.
- La implementación de las tecnologías de última generación que están ya presentes en el INGEMM y que serán el futuro próximo de la Genética a un nivel global, es clave para la obtención de los mejores resultados posibles en este proyecto. Así, la denominada NGS o secuenciación masiva del genoma es ya una realidad. El 1990 se puso en marcha el Proyecto Genoma Humano con el fin de cartografiar el genoma humano, con un plazo de 15 años de ejecución. La secuenciación completa del genoma se publicó en 2003, dos años antes de lo previsto, gracias al esfuerzo internacional a nivel técnico y académico. Sin embargo, tras sólo 9 años, estos datos resultan casi ridículos cuando el avance tecnológico de los últimos años permite secuenciar el genoma completo de una persona con un equipo de poyata y en tan sólo 1 día, aunque hay que añadirle al menos un mes de trabajo de análisis de datos. En cualquier caso, maximizamos los beneficios y el rendimiento de estas pruebas utilizando estas nuevas tecnologías, reduciendo tiempos de espera y costes. En nuestro caso, la secuenciación completa del genoma o del exoma no son útiles, así que estamos diseñando un panel de genes o regiones enriquecidas específicas para SD o epilepsias en general. Por ahora hay tres plataformas en período de prueba: Roche, Illumina y Ion Torrent.
- I would like to begin with an overview of the history of human genome sequencing. Despite significant improvements … it was clear that Sanger sequencing would not make massive DNA sequencing at a low cost and high speed feasible. Several technologies were developed at the time, of which the 454 Life Sciences sequencer was the first to become commercial in 2005. 2 years later it was used for … Whether …, but the direction is clear: in a few years from now very fast and cheap sequencing technologies will be available for commercial and research purposes