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.

Bilbao27 de Octubre, 2014

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 Mendelian genetics: autosomal dominant inheritance Genes encoding ion channels and neurotransmitter receptors

Complex disease with strong genetic component

General concepts of epilepsy

Poduri, A., et al., Curr. Op. Genet & Dev., 2011

Known genes involved in epilepsy syndromes

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

Dravet Syndrome clinical features

No known treatment nor cure

Early diagnosis is required

Personalized treatment and prognosis

Clinical diagnosis

Medical history of seizures Seizure type Age of onset

EEG, CT, …

Genetic Diagnosis

Familial history Genotype - Phenotype

+

Genetic diagnosis in DS (I)

Identification of the molecular cause of DS

Genetic counseling

Prenatal diagnosis

Accurate and personalized treatment

Confirmation of clinical diagnosis

Genetic diagnosis in DS (II)

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

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

Depienne, C., et al., J Med. Genet., 2009

Sodium channel (I)

333 DS patients

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

DS is genetically heterogeneous

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

Prenatal screening and genetic testing of relatives

Application of the new high-throughput genomic technology

OBJECTIVE: 100% of DS patients with genetic diagnosis

DS Genetic Test procedure (I)

Document 1Patient Clinical Info

Document 1Patient Clinical Info

Document 2Informed Consent

Document 2Informed Consent

OK

NeuropediatricianNeuropediatrician

Download documents

www.dravetfoundation.eu

Dra. Eva BarrosoBloque Quirúrgico, pl-2Hospital Universitario La PazPº La Castellana, 26128046 MADRID

Date the patient

Date the patient

Blood or gDNA

eva.barroso@dravetfoundation.eueva.barroso@dravetfoundation.eu

Fill out data and wait for acceptance

Original signed

Fill out and signed

Genetic report

Genetic report

Results

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

Direct sequencing

Sanger sequencing:

dNTPs + ddNTPs labeled with fluorochromes

Capillary electrophoresis + fluorescence detectorp.L1670fsX9

p.A486G

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/

Array-CGH (Comparative Genomic Hybridization): Complex genomic rearrangements analysis Roche-Nimblegen or Agilent aCGH platforms Genetic Epilepsy Panel of ~300 genes

High-throughput genomic analysis (II)

Solicitud de asistenciaRecepción del volante

Admisión

Consulta Clínica

Evaluación de resultados

Emisión del informe

Asesoramiento Genético

Estudios de ultrasecuenciación (NGS)

Inversión del proceso

Estudio citogenético

Microarrays CGH

Microarrays CGH

Estudio citogenético

Estudios moleculares específicos

The “Genetic” approachStudying “all at the same time”

Before Now

Gene by gene NGS (exomeapproach sq or panels)

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

High-throughput genomic analysis (I)

NGS

Hospital Universitario La Paz

GENOMICS GENETICS EPIGENETICS

All genetic/genomic or epigenetic diseases with knowncause: ~ # 5000 disorders

8-12% 82-87% 2-3%

5 Kb- ? Mb 1 bp- 200 bp No dosage changes

Unknown or No- responsible genes

Distribution of Human Genetic Diseases

The “Genetic Pool”

~ 24,000 MIM genes

100% Genetics

100% Genomics

100% Epigenetics

50% Genetics + 50% Genomics

50% Genetics + 50% Unknown

known gene but not linked to any disorder

The “Genetic Pool”

Genomas, Exomas y Paneles

100 %

1-2 %0,05 %

3,000 Millones de pares de bases

25

2010: 5K$, 10 days2009: Illumina,

Helicos40-50K$

Sequencing the Human Genome

Year

Log10(price)

201020052000

10

8

6

4

22014: 1000$, 36 hrs- Illumina

2008: ABI SOLiD60K$, 2 weeks

2007: 4541M$, 3 months

2001: Celera100M$, 3 years

2001: Human Genome Project2.7G$, 11 years

2015

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.

Antes Ahora

1 Km (1 exon ),conocemos el gen~ 300 coches/Km. Queremos verCual 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.

Capillary Sequencing (Sanger) vs Next Generation Sequencing (NGS)

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.

PlatformsIllumina HiSeq X Ten and NextSeq 500

Ion Proton

El Proceso de Dx Genético

Bioinformática- La UBEG

NGS (Next Generation Sequencing)

www.dravetfoundation.eu/bbdd-mutaciones-geneticas. 

Interfaz de inicio

*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 de la mutación

Lista de variables – Descripción del fenotipo clínicoClinical 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/DKOther stimulus (e.g. vaccination, heat bath, excitation, period) Description

Neurological abnormalities

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 NumberComplementary diagnosis tests

Neurophysiology EEG Description

Neuroimage MRI CT Scan Description

Hattori score Number

Genetic inheritance

*Inheritance Type if known

Family history DescriptionTreatment Current treatment DescriptionEthnic origin Ethnic origin DescriptionRemarks/Other comments Remarks/Other comments Description

Paneles de genes personalizados para epilepsias genéticas.

Exomas (23,000 genes) para investigación.

Aproximación diagnóstica actual

Genes MIM en un solo tubo

• (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)

Para Encefalopatías epilépticas:

Genes implicados en Epilepsias

La vida en Blanco y Negro de la Genética

La vida en Colores de la Genética

¿Hacia donde vamos?

5 Kb- ? Mb 1 bp- 200 bp No dosage changes

GENOMICS

CGH-arraySNP-aray

KaryotypeFISH

GENETICS

NGS panel screening

Exome seq

Sanger Seq

Exome Seq

EPIGENETICS

MS-arrays

Meth- PCR confirmations

FirstStep

Further

Agradecimientos

Todos los integrantesde la Fund. S. de Dravet

BBK-KutxaBank

Microsoft & Microsoft Azure

INGEMM

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Instituto de Genética Médica y Molecular