Episode 21 October 2018

Getting Personal: Omics of the Heart

Speaker 1: Hi, everyone. Welcome to episode 21 of Getting Personal, Omics of the Heart from October 2018. I'm Jane Ferguson, an Assistant Professor at Vanderbilt University Medical Center and an Associate Editor at Circulation: Genomic and Precision Medicine. We have a great issue this month. So, let's dive straight in.

First up, an article on "Loss-of-Function ABCC8 Mutations in Pulmonary Arterial Hypertension" from Michael Bohnen, Wendy Chung and colleagues from Columbia University. In pulmonary arterial hypertension, or PAH, compromised pulmonary arterial function can raise pressure in the pulmonary artery which leads to increased pulmonary vascular resistance. This ultimately results in right heart failure.

While PAH is relatively rare, it has a high rate of mortality. Some genetic underpinnings have been identified, notably the KCNK3 gene identified by the same research group where they find that mutations result in potassium channelopathy. However, here the authors hypothesized that other genetic contributors also exist and that identification of these could highlight new therapeutic targets to improve treatment and outcomes in PAH.

In their study, the authors performed exome sequencing for discovery of novel disease variants in 233 PAH patients, 99 of whom had pediatric-onset and 134 with adult-onset. They sequenced a replication sample of 680 individuals with adult-onset PAH. They found a de novo missense variant in the ABCC8 gene in one patient and then found 10 more ABCC8 variants in other unrelated patients in the discovery and replication samples.

Half of these were novel mutations and all were located in conserved regions and predicted to be deleterious. They screened over 33,000 subjects from the Exome Aggregation Consortium and over 49,000 from the Regeneron-Geisinger DiscovEHR study and found significant overrepresentation on rare ABCC8 variants in the PAH cases compared with population controls. ABCC8 encodes sulfonylurea receptor ... part of the potassium ATP channel. The authors determined that it is expressed in lungs in both PAH and healthy individuals and is particularly localized to alveolar macrophages and proximal pulmonary arteries.

They expressed eight of the newly discovered ABCC8 mutations in COS cells, which are a monkey-derived, fiberglass-like cell line and they assessed the effects on function. They used patch-clamp experiments to assess potassium ATP channel activity and recorded efflux rates of Rubidium-86. Every mutation was associated with impairments in one or both functional assays, suggesting that mutations in ABCC8 are responsible for PAH by a modulating potassium channel function and flux.

An existing drug, Diazoxide, targets ABCC8 and has anti-hypertensive and insulin-lowering effects. The authors find that all mutants were pharmacologically activated by Diazoxide in the functional assays. Now, whether this drug would be safe or effective in PAH remains unknown, but these findings open up targeting of ABCC8 as a possible treatment in PaH and highlight the importance of potassium channels in PAH.

Our next paper also used whole-exome sequencing for novel discovery. Marzia de Bortoli, Alessandra Rampazza and colleagues from the University of Padua in Italy published "Whole-Exome Sequencing Identifies Pathogenic Variants in TJP1 Gene Associated With Arrhythmogenic Cardiomyopathy".

Arrhythmogenic Cardiomyopathy, or ACM, is one of the most common causes of sudden unexpected death in athletes and young people. It is known to be frequently caused by mutations in genes encoding mechanical junction proteins of the intercalated disks within the cardiac muscle. However, some individuals with ACM do not have any mutations in known genes.

This research group was interested in finding novel causal gene mutation and they use whole-exome sequencing to identify mutations from a single patient in Italy. They used InSilica tools to screen for potentially damaging mutations which brought their list of candidate mutations down to 52 and this was topped by a novel mutation in the TJP1 gene which was predicted to be highly deleterious using various algorithms.

Using Sanger sequencing, they found that this mutation was also present in several family members. A second mutation in TJP1, also predicted to be damaging, was identified in a second Italian family. They then screened a sample of 43 Dutch and German subjects diagnosed with ACM and found that, once again, mutations in TJP1 topped the list as predicted to be damaging.

The TJP1, or tight junction protein 1, encodes the intercalated disk proteins ZO1. The identified mutations may affect folding and local interactions within the protein, affecting protein-protein interactions and gap junction organization. Well, within this paper, they were not able to fully disentangle the mechanisms linking these mutations to disease, given that the prevalence of TJP1 mutations in their ACM samples was almost 5%. Screening for TJP1 mutations in ACM cohorts may identify many additional affected subjects.

Further research into TJP1 is needed to identify how these variants may cause ACM. If you want to read more about this paper, you can check out the accompanying editorial from Jason Roberts ... Western University, Ontario ... in this same issue.

Next up is a paper from Natsuko Tamura, Yasuhiro Maejima, Mitsuaki Isobe and colleagues from Tokyo Medical and Dental University entitled "Single-nucleotide Polymorphism of the MLX Gene Is Associated With Takayasu Arteritis".

Takayasu Arteritis, or TAK, is an autoimmune disease causing aortic vasculitis that is poorly understood and disproportionately affects young Asian women. In previous genome-wide associations, study of TAK in Japanese individuals conducted by this group, indicated SNPs in the MLX gene. In this paper, the authors aim to identify mechanisms linking MLX mutations with TAK. The top GWAS SNP rs665268 is a missense mutation causing L-Glutamine Arginine substitution in the DNA binding site of MLX.

They found that this SNP was associated with severity in disease in TAK. With additional copies of the risk alleles associated with more severe aortic regurgitation and greater number arterial lesions. In mice, the highest expression of MLX was found in the aortic valves. Using crystallography, they found that the missense mutation likely stabilizes a complex formed between MLX and MondoA. Immunoprecipitation experiments confirmed that the missense mutation was associated with enhanced MLX MondoA heterodimer formation and MLX transcriptional activity. This resulted in upregulation of TXNIP and higher TXNIP expression is associated with increased intracellular oxidative stress and the authors found for increased oxidative stress in cells carrying the MLX mutation.

Further, additional cell experiments showed evidence of this MLX mutation reduces autophagy and stimulates inflammasome activation. Overall, through a series of really elegant experiments, the authors demonstrate that a missense mutation in MLX leads to inflammasome activation and accumulation of cells within the aorta, potentially underlying the pathophysiology seen in TAK patients and highlighting novel causal pathways that may be probed therapeutically.regular

Our next paper from Danxin Wang, Wolfgang Sadee and colleagues from the University of Florida and The Ohio State University, also delves into the functional impact of disease-associated SNPs. In their paper, "Interactions Between Regulatory Variants in CYP7A1 Promoter and Enhancer Regions Regulate CYP7A1 Expression", they used a series of experiments to demonstrate how SNPs in CYP7A1 ... which have been associated with cholesterol and cardiovascular disease ... are related to gene function.

CYP7A1 is a gene which coordinates a key pathway for cholesterol removal from the body because it encodes an enzyme which is rate-limiting for bioassay synthesis from cholesterol. Although several SNPs in the gene have been associated with cardiovascular phenotypes, the reported effects on gene function have been inconsistent and/or unclear. Because of the linkage disequilibrium between SNPs, it has been hard to understand which SNP or SNPs are actually functional. What this team set out to do was to systematically screen functionality of individual CYP7A1 SNPs to understand the independent effects of each functional variant.

First, they used chromatin conformation capture, or 4C assay, to identify regions that associated with a CYP7A1 promoter. They found three distinct regions with evidence of enhancer function and [phonetic 00:09:05] active A>G regulation. They, next, used CRISPR Cas9 to delete each of the three regions in HepG2 cells and assess effects on CYP7A1 expression. One region had no effect, while one led to increased expression and one led to decreased expression ... thus, identifying the presence of both enhancer and repressor regions. Using reporter gene assays, they confirmed the effects seen in CRISPR experiments.

Based on reported SNP associations, they narrowed down candidate functional SNPs within the regions and constructed reporter assays containing haplotypes of potential functional SNPs. They were able to identify two SNPs acting together to determine differences in CYP7A1 gene expression. Because these SNPs are in LD, but the minor alleles have effects in opposite directions, considering genotype at both SNPs is required to understand the effects on gene expression. This explains why previous studies found inconsistent results. Both during the functional experiments, they went to human samples and they assessed the combined effect of the two SNPs on clinical phenotypes. Designating people as high or low activity based on the two SNPs, they found significant differences in cholesterol and in the likelihood to reach cholesterol targets on statin, as well as in the risk of MI.

This paper is a lovely example of how careful functional interrogation can tease out a complex problem and I think it highlights how much more of this type of work needs to be done for the many other genomic regions with confusing or discord in associations.

The last full-length article concerns the "Effect of Ascertainment Bias on Estimates of Patient Mortality in Inherited Cardiac Diseases" and comes from Eline Nannenberg, Imke Christiaans and colleagues at the Academic Medical Center, Amsterdam.

They were interested in how much ascertainment bias and the tendency to publish findings from more severe disease cases affects the mortality estimates that are used to guide clinicians and genetic counselors when helping patients understand their disease prognosis. They revisited three inherited cardiac diseases including idiopathic ventricular fibrillation associated with a mutation in DPP6, SCN5A overlap syndrome associated with SCN5A mutations, and Arrhythmogenic Cardiomyopathy caused by a founder PLN mutation.

They analyzed mortality over 2-10 years of clinical screening and cascade screening and found that the median age of survival quickly increased in all three conditions. In many cases, the reason that a mutation was identified was because of severe disease in that patient or family, but as the authors highlight here, this can bias publications towards associating the variant with more severe phenotypes and higher mortality. Following up the initial findings with additional screening and tracking of affected individuals is important to subsequently give a more accurate estimation of the effect of the mutation which can be used to inform treatment plans.

Moving on to this month's research letters, Catherine Hajek, Jerome Rotter and colleagues from LA BioMed and the University of South Dakota, published the results of their study, "A Coronary Heart Disease Genetic Risk Score Predicts Cardiovascular Disease Risk in Men, Not Women: The Multi-Ethnic Study of Atherosclerosis".

The genetic risk scores are being increasingly applied to estimate disease risk in individuals. However, these scores are based on the GWAS discovery from specific populations which have often been disproportionately male and with individuals of European ancestry. In this letter, the authors wanted to understand whether coronary heart disease genetic risk scores performed the same in men and women of European ancestry. Using data from the MESA Study, they applied a 46 locus genetic risk score to over 2500 individuals. In men, this risk score was strongly associated with event rates. However, in women, there was no association. Given the known differences in disease pathophysiology and manifestation between men and women, this finding additionally highlights the need to conduct genetic studies in underrepresented groups so that we can design scores that accurately predict risk within specific groups.

Our next letter comes from Xiao Wang and Kiran Musunru at the University of Pennsylvania ... "Confirmation of Causal rs9349379- PHACTR1 Expression Quantitative Trait Locus in iPSC Endothelial Cells". They were interested in understanding the affect of a coronary disease SNP in the PHACTR1 gene on gene expression.

Previous efforts to investigate this had yielded conflicting results showing either a significant eQTL effect for PHACTR1 and vascular tissue or no effect on PHACTR1, but an effect on a distal gene EDN1 in endothelial cells. For this study, the authors used CRISPR Cas9 to introduce the SNP to iPS cells and then expanded isogenic lines at the major and minor allele homozygous and differentiated these into endothelial cells. They find that the major allele was associated with significantly higher factorial expression, but no difference in EDN1 expression. Thus, based on these experiments, it appears that PHACTR1 may indeed be the causal gene in that region underlying the GWAS signal and whether or not EDN1 is involved remains unclear.

Our next letter is a clinical letter from Nosheen Raza, Anjali Owens and co-authors at the University of Pennsylvania. They report on "ACTA1 Novel Likely Pathogenic Variant in a Family With Dilated Cardiomyopathy".

In this case report, they describe that the discovery of a mutation in ACTA1 in a family with dilated cardiomyopathy, but no skeletal muscle symptoms. As a gene that is predominantly expressed in skeletal muscle, ACTA1 mutations have previously been associated with skeletal muscle myopathies and would not have been expected to cause cardiac symptoms in the absence of skeletal muscle dysfunction. However, sequencing suggests that this variant is a causal mutation in this family, highlighting the need to consider potential mechanisms for cardiac muscle specifics of highly expressed skeletal muscle genes.

Our second clinical letter comes from Laura Zahavich, Seema Mital and co-authors from the Hospital for Sick Children in Ontario. They report a "Novel Association of a De Novo CALM2 Mutation With Long QT Syndrome and Hypertrophic Cardiomyopathy". They report finding mutation in the calcium transporter CALM2 gene in the child who presented with hypertrophic cardiomyopathy and ultimately died from sudden cardiac death.

While this patient also had some variants of un-insignificance, the CALM2 gene is highly conserved and mutations are likely to be pathogenic. The CALM2 is not on all of the clinical genetic testing panels and in this case, whole-exome sequencing was required to identify a mutation. CALM2 have been described in other individuals and together with the findings reported here, there's compelling evidence for inclusion of CALM2 on cardiomyopathy in clinical testing panels.

This issue also contains a perspective article from Michael Mackley, Elizabeth Ormondroyd and colleagues from the University of Oxford entitled "From Genotype to Phenotype: Clinical Assessment and Participant Perspective of a Secondary Genomic Finding Associated with Long QT Syndrome". They describe some of the challenges arising from more widespread genetic testing including how to deal with incidental findings.

A larger number of people including apparently healthy individuals are receiving sequencing results that highlight potential disease-related mutations, but with varying penetrance and uncertain effects. This perspective paper highlights the issues through case study and discusses future directions and challenges in this rapidly growing area.

Finally, we ride out this issue with an AHA scientific statement on "Cardiovascular Health in Turner Syndrome: A Scientific Statement From the American Heart Association" led by Michael Silberbach and Jolien Roos-Hesselink and a group of co-authors representing the American Heart Association Council on Cardiovascular Disease in the Young; Council on Genomic and Precision Medicine; and Council on Peripheral Vascular Disease.

In this statement, they discuss the cardiovascular complications that commonly occur in girls and women Turner syndrome. Cardiovascular disease contributes significantly to premature death in individuals with Turner syndrome. Because of the unique nature of the cardiac presentations in Turner syndrome, better clinical guidelines are needed to improve diagnosis and treatment of [phonetic 00:17:26] ischemia in these individuals. This statement takes a first step to outline suggestions to improve clinical practice and highlights the work that still remains to be done to inform disease management.

That rounds out the October issue of Circulation: Genomic and Precision Medicine. Thanks for listening!

You can go online to ahajournals.org/journal/circgen to access the latest issue and browse previous issues. As a last reminder, AHA Sessions is approaching fast and I hope to see many of you in Chicago, November 10-12.

This podcast was brought to you by Circulation: Genomic and Precision Medicine and the American Heart Association Council on Genomic and Precision Medicine. This program is Copyright American Heart Association, 2018.