Efficienza diagnostica dei pannelli NGS per la

diagnosi genetica delle malattie mitocondriali:

risultati di 3 anni di attività presso IRCCS BestaAndrea Legati, Daniele Ghezzi (UO Neurogenetica Molecolare)

Prospettive e strategie di ricerca

studies of the molecular mechanisms causing the disease

studies of therapeutic approaches in cellular models

studies of therapeutic approaches in animal models

therapeutic clinical trials

Genetic studies to identify the genetic cause(s) of the disease

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Road to Road to successful successful researchresearchon diseaseon disease

Elevata eterogeneità genetica delle malattie mitocondriali

Difetti del DNA mitocondriale

• Geni correlati alla sintesi proteica mitocondriale (rRNAs, tRNAs)• Geni codificanti proteine strutturali MRC• Grosse delezioni

Mutazioni DNA nucleare

• Geni codificanti proteine strutturali MRC• Geni codificanti fattori d’assemblaggio MRC• Geni codificanti enzimi per biosintesi di lipidi o cofattori• Geni codificanti per fattori importanti per mantenimento mtDNA• Geni codificanti per fattori per la sintesi proteica mitocondriale• Geni coinvolti nella biogenesi/dinamica mitocondriale

DNA mitocondriale

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2001 Shoudbridge2004 Zeviani&Didonato2006 Zeviani&Carelli2011 Ghezzi&Zeviani2014 Rotig2015 Turnbull&Rustin

Heterogeneous genetic disorder

Targeted resequencing

(panels)

Candidate 1

Candidate 2

Candidate 3

Candidate n

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Strategie combinate basate su NGS

diagnostic

Exome sequencing All Exons !!!

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I I I

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new disease gene research

• Requires DNA at 50-250 ng/µl (15 µl)• Can allow a maximum of ~1500 amplicons• Coverage profile similar to blocks

• Requires 5 ng/µl (10 µl)• Can allow a maximum of ~100.000 amplicons• Coverage profile similar to a Gaussian distribution

Pochi geni per tanti pazienti

Tanti geni per pochi pazienti1 run = 4 - 7.500.000.000 bp

Pannello MitoisoB(difetti isolati, PDH, MMDS, CoQ10)

119 geni (705 esoni; 1231 ampliconi)

Pannello MitomixB(difetti multipli, alterazioni mtDNA)

94 geni (825 esoni; 1526 ampliconi)

Pannello MitONE230 geni (2111 targets; 3504 sonde)

Dettagli pannelli

TruSeq Custom Amplicon

Nextera rapid capture

Filtraggio varianti NGS

MITOCHONDRIAL GENES PANEL

(230 genes)

TOTAL VARIANTS

EXOME SEQUENCING(~20.000 genes)

30035.000

QUALITY Q > 30COVERAGE > 10X 28030.000

MAF < 1% (1000g Project, EVS) 5-7600

MISSENSE, FRAMESHIFT, STOP,

SPLICING8010.000

CANDIDATE VARIANTS

COVERAGE ANALYSIS

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Sanger/qPCR validation + SegregationFunctional studies

VARIANTSVARIANTSFILTERINGFILTERING

GENOTYPE/PHENOTYPEGENOTYPE/PHENOTYPECONCORDANCECONCORDANCE

NGSNGSPANEL / WESPANEL / WES

Recessive hypothesis

One One HomozygousHomozygous

Compoundheterozygous

Only one Heterozygous

No Variants

Sporadic / Single cases

COVERAGE COVERAGE ANALYSISANALYSIS

TRANSCRIPTTRANSCRIPTANALYSIS ANALYSIS

(mRNA)(mRNA)

Heterozygous deletion

HomozygousDeletion

mRNA splicing alteration / sequencing for variants

confirmation

Heterozygous deletion

mRNA splicing alteration / sequencing for variants

confirmation

if negative

if negative

Unsolved

Unsolved

if negative Dominant hypothesis

One Heterozygous

No Variants

Defined dominant cases

Schema dell’analisi dati NGS

Copertura NGS – Analisi trascritti (se identificata una sola variante eterozigote in un gene «interessante»)

1) Looking for exons not covered by NGS

MTFMT: - heterozygous mutation c.626C>T p.Ser209Leu

(Pathogenic)

- exon1 not sequenced by NGS; Sanger-seq revealed the second

heterozygous mutation c.73C>T p.Gln25*.

MIM614947: Combined oxidative phosphorylation deficiency 15

DNA

cDNA

2) Looking for heterozygous exonic deletion

DARS2: - one heterozygous variant c.29C>T p.Leu10Pro,

all exons covered

- Heterozygous deletion of exons 15-16

-Sanger-seq on cDNA dispayed the c.29C>T as homozygous;

-WB showed strongly reduced protein

MIM611105: Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation

Ct Pt+ P

WB α-DARS2

gDNA cDNA

3) Looking for alterations in the cDNA

NDUFAF6: - one heterozygous variant c.136G>C p.Glu46Gln ,

all exons covered

- no exonic deletions

- Sanger on cDNA showed the c.136G>C as homozygous

MIM256000: Leigh syndrome due to mitochondrial complex I deficiency

4) if no additional finding and the phenotype is consistent, dominant

inheritance?

ISCU: - one heterozygous mutation p.Gly96Val

- all exons covered, no exonic deletion, cDNA hetero

- absent in parents = de novo

-Dominant effect?

MIM255125: Myopathy with lactic acidosis, hereditary (AR)

DNA cDNA

∆∆/ISU1/isu1G97V clone1

∆∆/ISU1/pFL39

∆∆/ISU1/ISU1

∆∆/ISU1/isu1G97V clone2

∆∆/ISU1/isu1G97V clone3

∆∆/isu1G97V /pFL39 clone1

∆∆/isu1G97V /pFL39 clone2

105 104 103 102 101

Glucose 2%

105 104 103 102 101

Lactate 2%

Copertura NGS – Analisi trascritti (se identificata una sola variante eterozigote in un gene «interessante»)

TRUSEQ CUSTOM AMPLICON

Genes sequenced: 132

Target regions: CDS + UTR

Samples sequenced: 125

Statistiche mutation discovery con NGS

Statistiche mutation discovery con NGS

TRUSEQ CUSTOM AMPLICON

Genes sequenced: 210

Target regions: CDS

Samples sequenced: 133 (21)

TRUSEQ CUSTOM AMPLICON

Genes sequenced: 210

Target regions: CDS

Samples sequenced: 58 (15)

Subgroup of patients (58) more recently collected (≤ 1 gene by Sanger)

TSCA24 pz con difetto biochimico cI

38%Mutations in:

- 6 genes encoding for structural subunits of complex I: NDUFA1, NDUFA10, NDUFS2, NDUFS3, NDUFS4 (2 samples), NDUFV1;

- 1 gene encodes for one of the assembly factors of complex I: ACAD9;

- 1 gene previously associated with multiple complex deficiencies: ELAC2.

Whole exome seq22 pz con difetto biochimico cI

45%

18%Mutations in:

- 5 genes that encode for structural subunits of complex I: NDUFV1, NDUFV2, NDUFS4, NDUFS6, NDUFA12;

- 2 genes previously associated with multiple complex deficiencies: TMEM70 and ELAC2;

- 3 new candidate genes never associated before with mitochondrial dysfunctions: FBXL4 (mitochondrial F-box protein), VARS2 (mitochondrial valyl-tRNA synthetase), COQ4 (component of the multisubunit complex for CoQ10 biosynthesis).

Eterogeneità genetica: Complex I

The proband (P) presented with:

•Bilateral ptosis•Muscle biopsy: few COX negative fibers•SB: multiple deletions

Mother, grandmother, 2 aunts affected

Uno, l’altro o entrambi: casi digenici?

P

C10orf2#609286 - PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA WITH MITOCHONDRIAL DNA DELETIONS, AUTOSOMAL DOMINANT 3; PEOA3hetero c.1001G>A p.Arg334Gln (rs28937887 pathogenic)

POLG#157640 - PROGRESSIVE EXTERNAL OPHTHALMOPLEGIA WITH MITOCHONDRIAL DNA DELETIONS, AUTOSOMAL DOMINANT 1; PEOA1c.1685G>A p.Arg562Gln (Exac=0)

Mitochondrial panel

POLGor

c10orf2?

The proband (P, II-4) presented with:

•Psychomotor development referred normal.•At 15 months, after a febrile illness, she presented acute psychomotor regression, losing previously acquired psychomotor skills in about a week. •Generalized hypotonia, hyperreflexia, no postural control, poor voluntary movements, marked irritability with frequent crying. •No seizures. •Lactate and pyruvate were elevated in plasma.•Brain MRI showed diffuse hyperintensity of the hemispheric white matter and corpus callosum.•MRC: cII deficiency in muscle and fibros

All her siblings were reported in good health

In the proband homozygous variant c.143A>T p.Asp48Val in SDHBBoth parents are heterozygous for the variantOne sister (II-1) homozygous One sister (II-2) heterozygousOne sister (II-3) wild type homozygous

SDHB Succinate Dehydrogenase Complex, Subunit B (cII)

Segregazione: necessaria e/o sufficiente?

Sanger validation / segregationFunctional studies

Genes coding for protein with mitochondrial

functions

Whole exome sequencing

TOTAL VARIANTS

QUALITY Q > 30COVERAGE > 10X

MAF < 1% (1000g Project, EVS)

MISSENSE, FRAMESHIFT, STOP, SPLICING

EXOME SEQUENCING(~20.000 genes)

35.000

30.000

10.000

700

TYPE OF INHERITANCE(recessive / dominant) 30 / 670

CodeBiochemical defect

Gene MutationsExonic Deletions

Trait NotesExAC frequency

Pathogenic variants

NGSP67 multi PC p.N647D homoz / ARConsanguineous parents; 1 homozygous affected sister

Ø

NGSP73 cIV "COA-X" compound heteroz / ARConsanguineous parents: heterozygous

Ø

NGSP65 PDH CYP2U1 c.1283_1288+8del / ARConsanguineous parents: heterozygous

Ø

NGSP66 cIV PREPL / ex.6-14 AR Consanguineous parents

NGSP110 multi RANBP2 p.T585M / AD "Affected" mother: heterozygous <0,01%

Probably pathogenic variants

NGSP91 PDH E4F1 p.K144Q / ARParents: heterozygous; 1 homozygous affected brother

<0,01%

Unsolved WES cases

NGSP47mtDNA depl TRMT1 p.N70S+p.A171V / AR

1 affected sibling. Variants on the same allele

0,1%; Ø

NGSP49Neg TENM4 p.N1799H+p.Q2527K / AR

2 affected sibling. Variants on the same allele

0,2%; 0,2%

NGSP89 PDH GNAO1 p.D134N+p.A165V / AR Different phenotype Ø

NGSP116 multi MRS2 p.R446H homoz / AR Consanguineous parents 0,33%

Whole exome sequencing (BBA cohort)

- Inclusion criteria based on clinical evaluation affect the disease mutation discovery rate

Phenotype heterogeneity

RIASSUNTO

- Different strategies for disease mutation discovery:differences in detection power, output size and data interpretation

Sanger: 1-5 genes, needs high phenotipe/genotype concordance

NGS panel: 200 genes, more power, disease mutation easy to find if sequenced

NGS Exome: 20,000 genes, very likely to sequence the disease mutation but hard to

find

Homogeneous cohorts, increased rate of successful discovery

Heterogenous cohorts, progressive lower rates

CollaborazioniMassimo Zeviani, Aurelio Reyes, Alan Robinson (MRC Cambridge, UK)Holger Prokisch, Tobias Haack, Thomas Meitinger (Helmoltz Center, Munich, Germany)E. Baruffini, C. Dallabona, P. Goffrini, I. Ferrero (Università di Parma)R. Costa, C. DePittà, F. Argenton (Università di Padova) I. Moroni, A. Ardissone, G. Uziel, G. Piscosquito, D. Pareyson, E. Salsano, L. Farina, C. Pantaleoni, T. Granata, C. Mariotti… (Istituto Neurologico “Besta”)E. Bertini, D. Diodato (Osp. Pediatrico Bambin Gesù, Roma), A. Burlina (Università di

Padova), M.A. Donati (Osp. Meyer, Firenze), R. Parini (Osp. San Gerardo, Monza)M. Van der Knaap (University Medical Center, Amsterdam), R. Taylor (University of Newcastle, UK)

www.mitopedia.org

Grazie! Ministero della Salute

Exome seq

Lievito

Clinici

Drosophila

Confronto costi strategie NGS

vanNimwegen C., ESHG Congress 2016

Targeted resequencing

Aspetti positivi:- Possibilità analisi di molti geni contemporaneamente- Buona copertura- Applicabile ad un numero elevato di pazienti

Exome sequencing

Aspetti negativi:- Analisi solo dei geni presenti- Copertura geni presenti non completa- Strumentazione (hardware/software) dedicata

Per la diagnostica…

Per la ricerca di nuovi geni malattia…

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