Next Generation Sequencing. mapping mutations in congenital heart disease

Transcription

1 Next Generation Sequencing mapping mutations in congenital heart disease AV Postma PhD Academic Medical Center Amsterdam, the Netherlands

2 Overview talk Congenital heart disease and genetics Next generation sequencing HeartRepair NGS Ebstein s anomaly and MYH7 mutations Discussion

3 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

4 Genetics and Congenital Heart Disease 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

5 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

6 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 unique variants / individual

7 HeartRepair Next Generation Sequencing European FP6 consortium HeartRepair ( 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

8 Underdeveloped heart Phenotyping 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

9 Initial analysis Match sequence reads to chromosomes Perform QC Visualize the results Generate a list with variants Tools: BWA, Samtools, Varscan Annotate-it

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

11 Identification of pathogenic variations ~2000 variants called per patient 2000 variants annotated in dbsnp (mostly common variants) Intronic and synonymous changes 200 Common variants in 1000 genomes ~1-10 stop codons / non-synonymous changes per patient All in relevant genes!

12 Filtering variations 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

13 No. of unique variants Unique variants per gene

14 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 MYH Non-synonymous MYH Non-synonymous MYH Non-synonymous T0866 (id T1405) T1918 (id T1009) MYH MYH Nonsense (false positive) Nonsense (validated-maternal) T MYH Non-synonymous 3/6 MYH7 variants in Ebstein s anomaly patients

15 Morphology 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) Source: Attenhofer Jost et al., Circulation 2007 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)

16 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?

17 Mutational analysis of cohort of Ebstein patients 141 unrelated Caucasian individuals with Ebstein ( 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

18 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

19 Pedigrees

20 Mutated MYH7 residues

21 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

22 Gene Polygenic CHD? Amino acid change Conserved across species ZFPM2 G840S Yes Mother TBX5 L243P Yes Mother TBL2 E8Q No Father PTPRJ V413I Yes Father EVC P156A Yes Father NRG1 R545W Yes Mother a common situation: multiple unique potential pathogenic variants inherited from healthy parents Present in Father / Mother

23 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

24 Acknowledgments, 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