Next Generation Sequencing mapping mutations in congenital heart

Heart Failure Research Center

Next Generation Sequencing

mapping mutations in

congenital heart disease

AV Postma PhD


Academic Medical Center

Amsterdam, the Netherlands


Overview talk

• Congenital heart disease and genetics

• Next generation sequencing

• HeartRepair NGS

• Ebstein’s anomaly and MYH7 mutations

• Discussion


Congenital Heart Disease

Incidence: ~ 8 per 1000 live births

~ 20 per 100 spontaneous abortions

Netherlands: ~ 1600 children with CHD born per year

~ 25,000 children with CHD

~ 25,000 adult CHD patients


• Familial and twin studies suggest major genetic

component

• High mortality -> low number of large CHD families

• Large amount of families with accumulation of defects

• (Few) causative genes identified by linkage/candidate

gene approaches (GATA4, NKX2.5, TBX5, NOTCH1,

MYH6, ACTC1 etc)

Mutations represent <1% CHD cases, so what is the cause

in the remaining patients?

Next generation sequencing; hypothesis free approach

Genetics and Congenital Heart Disease


Next Generation Sequencing

Whole Genome/Exome Sequencing

Mega Sequencing

2nd Generation Sequencing

Massive parallel Sequencing

High Throughput Sequencing

Deep Sequencing

General characteristics include:

- Amplification of genetic material by PCR

- Ligation of amplified material to a solid surface

- Short reads applications; sequence and then

use computers to assemble the small pieces


Exome sequencing

The human "exome" is 1 percent of the human genome; 180,000 exons (~30 Mb of DNA)

• Proof of principle:

• Freeman-Sheldon syndrome; 4 probands, 8 HapMap control

individuals => MYH3 Ng et al, Nature 2009

• Bartter syndrome; 5 probands => one patient had recessive

mutation in SLC26A3 (confirmed in 5/39 additional probands) Choi et al, PNAS 2009

• >20K variants identified per exome

• ~45% missense, 150 nonsense

• 150-250 unique variants / individual


HeartRepair Next Generation Sequencing

European FP6 consortium HeartRepair (www.heartrepair.eu)

• Identify 500 patients with „under developed heart“ CHD

Diagnosis

• Select 160 representative patients for large scale sequencing

• Screen 410 genes selected by hand, bioinformatic and

mouse models (1.6Mb sequence)

Patients selected from three different cardiology centers:

Newcastle, Amsterdam, Berlin


Phenotyping

• „Underdeveloped heart“

– Hypoplastic left heart 9

– Hypoplastic left heart syndrome 8

– Mitral atresia 9

– Isolated left ventricular noncompaction 14

– Double inlet left ventricle 22

– Hypoplastic right heart 18

– Tricuspid atresia 34

– Ebstein’s anomaly 27

– Pulmonary atresia 25

– Univentricular heart 16

LV

RV

LV/RV

178 main diagnoses (multiple main diagnoses possible)

157 patients


Initial analysis

Match sequence reads to chromosomes

• Perform QC

• Visualize the results

• Generate a list with variants

Tools:

• BWA, Samtools, Varscan

• Annotate-it


Coverage plot

~95% of targeted regions have an average coverage of >20-fold


Identification of pathogenic variations ~2000 variants called per patient

variants annotated in dbSNP

(mostly common variants)

~1-10 stop codons / non-synonymous

changes per patient

All in relevant genes!

2000

200

Intronic and synonymous changes

14

Common variants in 1000 genomes

10


• All samples included (not filtered for phenotypes)

• Unique variants only

• Looking for non-synonymous, non-sense and splice site variants

• Coverage >= 20

• Not in dbSNP, 1000G

• 140 patients sequenced / 100 analyzed

• 451 unique variants found in 229 genes

• Non-synonymous variants: 430

• Nonsense variants: 13

• Splice site variants: 8

Filtering variations


0

2

4

6

8

10

12

Unique variants per gene N

o. o

f u

niq

ue

vari

ants


List of unique non-synonymous, nonsense and

splice site variants detected in MYH7 gene

Sample Chromosome Position Gene_symbol Coverage Varfreq No. variant

reads Worst consequence

209 14 22972150 MYH7 54 35 19 Non-synonymous



T0866

(id T1405) 14 22970518 MYH7 38 55 21

Nonsense

(false positive)

T1918

(id T1009) 14 22956672 MYH7 140 57 80

Nonsense

(validated-maternal)

T3116 14 22962750 MYH7 194 51 99 Non-synonymous

3/6 MYH7 variants in Ebstein’s anomaly patients


Ebstein´s Anomaly

Adherence of the septal and

posterior leaflets to the underlying

myocardium

Downward displacement of the

functional annulus

Dilation of the atrialized portion of

the right ventricle

Dilation of the right atrioventricular

junction (true tricuspid annulus)

Morphology

•Most cases are sporadic; familial Ebstein´s anomaly is rare

•More common in patients with a family history of congenital

heart disease

•Mutations in Nkx2.5 have been described (Benson 1999)

Source: Attenhofer Jost et al., Circulation 2007


Ebstein´s Anomaly and Left Ventricular

Anomalies

• A significant number of patients with Ebstein´s anomaly have

morphofunctional abnormalities of the left ventricle (Monibi et al.,

1978)

• Left heart lesions in patients with Ebstein anomaly (Attenhofer

Jost et al., 2005)

– 18% had left ventricular noncompaction (LVNC)

• Large family with LVNC and 4 family members with Ebstein´s

anomaly (Budde et al., 2007)

• Mutations in sarcomere proteins in LVNC (Klaassen et al., 2008)

Hypothesis:

Do mutations in MYH7 lead to Ebstein´s anomaly?


Mutational analysis of cohort of Ebstein

patients

• 141 unrelated Caucasian individuals with Ebstein

(www.heartrepair.eu), mean age, 46 years

• ROCHE GS FLX Pyrosequencing

– Emulsion-based clonal amplification (emPCR)

– 43 amplicons, 200 megabases of sequence, mean

coverage 45 fold

• SNP Validation by MassARRAY MALDI-TOF (Sequenom)

• Confirmation by Sanger sequencing


Results

• Heterozygous mutations in MYH7 were identified in 8 of 141

probands (6 %) with Ebstein´s anomaly in our cohort

• Clinical phenotypes were assessed in all available family

members of the 8 probands with mutations and familial

structural congenital heart disease was found in 3 of them

• 7 distinct mutations were found of which 5 were novel and 2

were known to cause HCM. All mutations except for one 3-bp

deletion were missense mutations

• In 6/8 probands with MYH7 mutations LVNC was identified in

addition addition to Ebstein´s anomaly. In 133 probands

without MYH7 mutations there was only one individual with

additional HCM


Pedigrees


Mutated MYH7 residues


Conclusions

• Ebstein´s anomaly is within the diverse spectrum of cardiac

morphologies associated with mutations in the gene encoding

β-myosin heavy chain

• MYH7 mutations are common in patients with Ebstein´s

anomaly and LVNC

• Clinical and genetic evaluation is recommended to facilitate

the diagnosis of cardiomyopathy and congenital heart disease

in first-degree relatives

• The role of sarcomere proteins in congenital heart disease

should be subject to further investigation

Postma et al. Circ CVG 2011


Polygenic CHD?

a common situation:

multiple unique potential pathogenic variants

inherited from healthy parents

Gene Amino acid change

Conserved across

species

Present in Father

/ Mother

ZFPM2 G840S Yes Mother

TBX5 L243P Yes Mother

TBL2 E8Q No Father

PTPRJ V413I Yes Father

EVC P156A Yes Father

NRG1 R545W Yes Mother


Discussion

• Mutations may still be missed (genes, regions not covered, low

coverage at certain positions)

• Need for analysis of insertions / deletions / copy number

variation (within NGS data)

Conclusion

• Majority of (sporadic) CHD likely caused by combination of

multiple (inherited) mutations

• Need to develop novel bioinformatic tools to incorporate and

integrate all the data (array CGH, CNVs, common snps,

mutations, inheritance), e.g. pathway analysis involving

multiple hits


Acknowledgments • Heart Failure Research Center, AMC, Amsterdam

• AFM Moorman, K van Engelen, B. Mulder

• Department of Human Genetics, LUMC, Leiden

• P-B t Hoen, Y. Lai

• Institute of Human Genetics, Newcastle University

• J. Goodship, B. Keavney, T. Rahman, A. Topf

• Katholieke Universiteit Leuven

• A. Silfrim, Y. Moreau

• Max-Planck-Institute for Molecular Genetics, Berlin

• S. Sperling

• Max-Delbrueck-Center for Molecular Medicine, Berlin

• S. Klaassen

• National Reg. and Competence Network for Congenital Heart Disease

• U. Bauer

EU: FP6 HeartRepair, FP7 CHearteD

Documents

Next Generation Sequencing mapping mutations in congenital heart