Genomes, CNVs, and EQ-Induced Broken Hearts
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1) The document summarizes research into potential genetic factors that may contribute to stress cardiomyopathy (Takotsubo syndrome), a condition where emotional or physical stress triggers temporary weakening of the heart muscle. 2) Analysis of exome sequencing data from 24 Takotsubo patients did not find any strongly causal genes, suggesting a polygenic basis. However, array CGH analysis found a surprisingly high rate (44%) of large, rare copy number variants (CNVs), affecting genes involved in ion channels, cell membrane function, and transcription. 3) Further research into whole genome sequencing and cellular models is suggested to better define the role of CNVs in Takotsubo predisposition and heterogeneity.
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Genomes, CNVs, and EQ-Induced Broken Hearts
- 1. Genomes, earthquakes, and broken hearts Prof. Martin Kennedy Department of Pathology University of Otago, Christchurch
- 2. Christchurch earthquakes • 4th Sept 2010, 4:36am (mag 7.1) • 22nd Feb 2011, 12:51pm (mag 6.4)
- 3. EQ-induced stress cardiomyopathy • Nature of coronary admissions altered markedly • Multiple cases of stress cardiomyopathy • Annually expect only 6 patients • 4th-11th Sept: 9 patients • 22nd-26th Feb : 21 patients
- 4. EQ-induced stress cardiomyopathy • All cases were female • 28/30 postmenopausal • One recurrent case in 2nd quake • Many received critical care in the CCU • All survived • All consented to take part
- 5. Takotsubo syndrome • Various names: – Stress cardiomyopathy – Transient left ventricular apical ballooning – “Broken heart syndrome” • “Heart attack” without traditional risk factors • 80% postmenopausal women • Emotional or physical trigger • “Sporadic” or associated with major disasters
- 6. Mechanism? • Brain appears to be trying to kill you! • Elevated catecholamine levels – Noradrenaline, adrenaline, dopamine • Myocardial stunning? • Can induce in rodents with adrenergic agents • Capture myopathy in animals • Poorly understood
- 7. “Do you think genetics might be relevant?” • Not whether, but to what degree! • Could it be a monogenic predisposition with strong environmental triggers? • That we can test…!
- 8. Any prior evidence for genetics? Two sisters Mother and daughter
- 9. Hypothesis • Mendelian (single gene) predisposition • ~300,000 people exposed --> 30 cases • Postmenopausal + earthquake induced stress • Can test hypothesis by exome sequencing
- 10. Individual genome variation • Each genome differs from reference at: – 10,000 nonsynonymous SNPs – 10,000 synonymous SNPs • Variants of high impact: – in frame indels (190-210) – premature stop codons (80-100) – splice site disrupting variants (40-50) – deletions that shift reading frame (220-250) • Heterozygosity for 50-100 disease mutations
- 11. Exome sequencing • 24 TAK exomes in two runs • Illumina TruSeq Exome Enrichment kit • HiSeq2000, 2x100bp PE reads • Good quality metrics, good DNA – >20 mill. sequence reads per patient – Mean seq. qual score (Phred) = 37 • ~50Gb of compressed sequence files
- 12. Data processing steps (Black/Cadzow/Merriman, Otago; Lehnert, Auckland) • Alignment to reference genome (BWA) - GRCh37.64 • Mark duplicates (Picard) • GATK pipeline (Broad) – Local realignment around indels – Recalibration of sequence quality scores – SNP calling, Indel calling – Variant recalibration • VCF file
- 13. SNP functional annotation tools CADD scores
- 14. Population allele frequencies SeattleSNP Exome Variant Server ExAc Browser
- 16. Analysis of VCF file • Used multiple variant filtering strategies: • Low population allele frequencies • Predicted functional effects • Over-representation in Takotsubo exomes
- 17. SEPT8 variant, rs30510 • Septin GTPase involved in neuron polarity, vesicle trafficking • Expressed in brain, heart, ovary • Triallelic – C/G/T • “T” allele extremely rare (.045%) • But T is the reference allele in GRCh37.p5! • Rarest allele was the reference…
- 18. “Rollercoaster of hope and disappointment”!
- 19. Exome analysis conclusions • No convincing candidate genes detected • Unlikely SNVs or small indels in one or a few genes underlie EQ-SCM • Caveats: – Variable coverage (both capture and sequencing) – Absence of most regulatory regions – Indels poorly annotated
- 20. “…these data support genetic heterogeneity in TC susceptibility and a likely polygenic basis, conferring a cumulative effect on adrenergic pathway dysregulation in a subset of individual subjects.”
- 23. aCGH analysis of Takotsubo cases • Surprising rate of large, rare CNVs (44%) – “potential clinical” or “unclear” significance – Deletions, duplications – Affecting 1 exon through to many genes • Childhood developmental disorders: ~30% • Rate amongst Takotsubo cases >> than rate in unselected population • No overlaps or recurrent CNV
- 24. CNV affected genes in Takotsubo patients • ANX3 • CHODL • CHRNA7 • COL4A5 • DLEU1 • DLEU2 • DLEU7 • ENOX1 • FRAS1 • GP6 • GPBP1L1 • GPC5 • GRID2 • IPP • IRGM • IRS4 • ITGBL1 • ITPR1 • KCNRG • LOC100505702 • MAST1 • MIR15A • MIR16-1 • NID67 • NLRP2 • NLRP7 • NRG1 • PIK3R3 • RBFOX1 • RDH13 • RNASEH2B-AS1 • ST13P4 • SUMF1 • TCF7L2 • TMEM69 • TRIM13 • ZNF300
- 26. Functional categories of CNV genes Term P-Value Genes ionic channel 8.22E-04 GRID2, TRIM13, CHRNA7, KCNRG, ITPR1 cell membrane 0.001638 IRS4, FRAS1, GPC5, MAST1, GP6, IRGM, GRID2, CHRNA7, NRG1, ENOX1 glycoprotein 0.048268 FRAS1, GPC5, GP6, SUMF1, GRID2, CHODL, CHRNA7, NRG1, ITPR1, COL4A5, ITGBL1
- 28. Transcription factors • UCSC_TFBS GATA6 • 12 CNV genes • P=6.33E-04 • FRAS1, GPC5, DLEU1, DLEU2, GRID2, PIK3R3, NRG1, TCF7L2, ITPR1, COL4A5, ENOX1, ITGBL1 • These 12 genes are in 11 different CNV • GATA6 important cardiac TF
- 29. Many CNV genes have cardiac links • RBFOX1 – ~135kb deletion removes one exon
- 30. Other CNV conditions • Autism • Schizophrenia • Developmental delay • Heterotaxy (congenital heart disease) • Some cases of early onset Alzheimer’s disease • In general large, rare CNVs involved
- 31. Conclusions • Exome analysis inconclusive • aCGH suggests may be a CNV disease – Many different, large, rare deletions or duplications – No overlap between CNVs (as yet) – Considerable heterogeneity – GATA6 transcription factor may be a common link Is Takotsubo predisposition due to CNVs?
- 32. Where next? • Whole genome sequencing – Better coverage of exome – Better definition of CNVs, especially smaller ones • Continue recruiting sporadic cases • GWAS? • Patient fibroblast iPSCs > cardiomyocytes – Cellular models with relevant CNVs – Study transcriptomics +/- adrenergic agents – Study physiology
- 33. Acknowledgements • Kit Doudney • Paul Bridgeman • Murray Cadzow • Vicky Cameron • Cameron Lacey • Allison Miller • Alan Aitchison • Bridget Kimber • Julie Zarifeh • Peter George • Klaus Lehnert • Vivienne Bickley • Roger Mulder • Tony Merriman • Mik Black
- 35. “it seems like women respond differently to an event like this. The reason why is something we’ll never know.”
- 37. A diversion - myotonic dystrophy • Sporadic Takotsubo case series • One Christchurch woman previously diagnosed with myotonic dystrophy type I • Very similar case from Vienna published 2014 – QT-prolongation – torsades de pointes
- 38. DMPK 3’ UTR repeat expansion
- 39. Could DMPK mutations contribute more widely to Takostubo cardiomyopathy risk? Repeat-primed PCR (Allison Miller)
- 40. DMPK 3’ repeat expansion • Pathologic expansion may be a risk factor for Takotsubo cardiomyopathy • May influence presentation • No other expansions in 34/35 sporadic cases • No expansions in 28/28 earthquake cases • Not commonly involved
Notes de l'éditeur
- Takotsubo First described medically in 1991 by Japanese doctors, the condition was originally called takotsubo cardiomyopathy."Takotsubo" is a type of pot used by Japanese fishermen to capture octopuses. When doctors take X-ray images of a person who's experiencing the syndrome,the left ventricle of his or her heart resembles the pot. The medical term is stress cardiomyopathy or "Broken heart syndrome". It is real and potentially deadly but recovery is quick.
- On 4 September, 2010, Christchurch, the second largest city in New Zealand with an urban population of 400,000, was struck by a 7.1 magnitude earthquake at 4:46 am. The city suffered significant damage to its infrastructure and buildings. A state of emergency was declared and financial loss was estimated to be as high as $5 billion. Fortunately, there were no direct fatalities. Six months and thousands of mild to moderate aftershocks later, Christchurch was again struck by a powerful earthquake on 22 February 2011 at 12:51 pm. Although smaller on the Richter scale than the first one, the epicentre of this magnitude 6.3 earthquake was 5 kilometres deep and 10 kilometres southeast of the city centre. It generated a vertical 2.2G force, the highest peak ground acceleration ever recorded in the world [1]. As many as 185 people died with many more injured and left homeless. More than one third of the buildings in the central business district were destroyed, including 2 multi-story buildings.
- SCM was first described in Japan in 1991 as an acute coronary event often presenting in association with emotional trauma 1, 2. It was not recognised outside Asia until 2003, and since then it has become a widely recognised clinical entity 3, 4. SCM patients typically present at the emergency clinic with chest pain and breathlessness, and they display ischaemic electrocardiogram changes and modest troponin elevation, symptoms resembling an acute coronary syndrome. Most but not all patients are post-menopausal females, and they generally lack pre-existing pathology such as obstructive coronary arterial disease About 70-80% of cases of Tako-tsubo Syndrome (TTS) occur in post-menopausal women under some form of extreme, exceptional and prolonged mental stress,... with no good way out, no relief and often feeling deep resentment (such as the loss of a dear one...) (Note: a word of caution... , in a minority of patients (<20%) the stress is physical (such as massive trauma, surgery or severe pain, or other type of stress. In very rare cases, no "cause" can be found). Tako-tsubo Cardiomyopathy or Syndrome is also known as: neurogenic myocardial stunning, stress cardiomyopathy stress-induced cardiomyopathy, transient left ventricular apical ballooning, "ampulla" cardiomyopathy "broken heart syndrome". "Tako-tsubo" is the japanese name foroctopus traps that fishermen still use to catch octopus. In this syndrome, the heart (left ventricle) takes the shape of an octopus trap (tako-tsubo). How about that!
- isoprenaline (isoproterenol; a non-selective beta-adrenergic agonist that is structurally similar to adrenaline
- A 44 year-old female presented with sudden onset of chest discomfort which started after she experienced severe emotional stress. The pain resolved with the administration of sublingual nitroglycerin in the emergency department. Initial electrocardiogram demonstrated sinus rhythm with diminished R wave amplitude in leads V1 through V3, and mild ST-segment elevation. The troponin T level was elevated at 0.09 (normal b0.01) ng/mL. Coronary angiography did not reveal significant coronary atherosclerosis or a culprit lesion suggestive of acute plaque rupture. The left ventriculogram demonstrated severe systolic dysfunction with akinesis of the mid-ventricle, hypokinesis of the apex and hyperdynamic basal function consistent with apical ballooning syndrome (ABS) [1] (Fig. 1). On further questioning, the patient stated that her mother experienced a similar illness presenting with acute chest pain following an intense emotional stressor when she was 49 years old. She was diagnosed with an acute myocardial infarction at that time possibly due to coronary spasm. Her coronary angiogram from 1991 was reviewed with her consent and did not reveal any obstructive coronary artery disease but showed wall motion abnormalities suggestive of ABS. (Fig. 2). In addition, she subsequently had an echocardiogram in 2007 that revealed normal left ventricular systolic function
- Base quality scores need to be recalibrated as the scores from the sequencers isn’t very accurate or helpful. Hi Martin, I have added in 2 steps that were also performed (marked with *) and added some additional notes to help you. I won't be at the NGS meeting but would be happy to catch up, what day/time would be good for you? I'm pretty flexible. I'm happy to provide you with any of the files you would like - the final vcf at the end of the pipeline is 3.5Gb. Easiest method is probably a usb stick when you're down if that works, otherwise we can work out a way to transfer via internet/uni network or even a DVD Data processing steps (Murray Cadzow, Otago) • Base calling and image analysis - hiseq2000 does this • Alignment to reference genome (BWA) - GRCh37.64 * mark duplicates (picard)- looks for all the pairs that aligned to the same place. selects best scored pair and marks rest as duplicates. • Local realignment around indels (GATK - does all the following steps too) • Recalibration of sequence quality scores • SNP calling • Indel calling * variant recalibration - uses known database of variants to generate probability it is true variant or machine artifact
- CADD is a tool for scoring the deleteriousness of single nucleotide variants as well as insertion/deletions variants in the human genome. While many variant annotation and scoring tools are around, most annotations tend to exploit a single information type (e.g. conservation) and/or are restricted in scope (e.g. to missense changes). Thus, a broadly applicable metric that objectively weights and integrates diverse information is needed. Combined Annotation Dependent Depletion (CADD) is a framework that integrates multiple annotations into one metric by contrasting variants that survived natural selection with simulated mutations. C-scores strongly correlate with allelic diversity, pathogenicity of both coding and non-coding variants, and experimentally measured regulatory effects, and also highly rank causal variants within individual genome sequences. Finally, C-scores of complex trait-associated variants from genome-wide association studies (GWAS) are significantly higher than matched controls and correlate with study sample size, likely reflecting the increased accuracy of larger GWAS. CADD can quantitatively prioritize functional, deleterious, and disease causal variants across a wide range of functional categories, effect sizes and genetic architectures and can be used prioritize causal variation in both research and clinical settings.
- Provides annotation of SNVs ) and indels, both known and novel. Includes dbSNP rs ID, gene names and accession numbers, variation functions (e.g. missense), protein positions and amino-acid changes, conservation scores, HapMap frequencies, PolyPhen predictions, and clinical association. Links to other annotation sites also provided.
- single protein of protein information ressource (SP_PIR)
- Regulates splicing of genes involved in muscle structure and function Specifically expressed in brain, skeletal muscle, and heart Alternative promoters, encoded by different 5’ exons including this one. Mouse M43 is exclusively used in heart and skeletal muscles, while B40 is predominantly used in brain http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1075922/
- Medical Definition of TORSADES DE POINTES : ventricular tachycardia that is characterized by fluctuation of the QRS complexes around the electrocardiographic baseline and is typically caused by a long QT interval DMPK
- The protein encoded by this gene is a serine-threonine kinase that is closely related to other kinases that interact with members of the Rho family of small GTPases. Substrates for this enzyme include myogenin, the beta-subunit of the L-type calcium channels, and phospholemman. The 3' untranslated region of this gene contains 5-37 copies of a CTG trinucleotide repeat. Expansion of this unstable motif to 50-5,000 copies causes myotonic dystrophy type I, which increases in severity with increasing repeat element copy number. Repeat expansion is associated with condensation of local chromatin structure that disrupts the expression of genes in this region. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.