Identification of genomic biomarkers associated with hypertrophic cardiomyopathy (HCM) in cats
Lead Research Organisation:
Royal Veterinary College
Department Name: Clinical Sciences and Services
Abstract
Rationale: Hypertrophic cardiomyopathy (HCM), the most common cardiac disease in humans and cats, occurs spontaneously in 15% of domestic cats and may cause heart failure, cardioembolic complication or sudden death. HCM is heritable in many cat breeds, and two causative mutations in the MYBPC3 gene have been identified in Maine coon and Ragdoll cats using a candidate gene approach based on human studies. Further genetic studies could not only provide the means to control the disease, via genetic testing and selective breeding programmes, but they could also help identify new therapeutic targets. However, over the past decade the candidate gene approach has failed to identify further mutations. Genome-wide genotyping technologies might offer a useful alternative.
This project is aimed at investigating the genetic architecture and the underlying molecular mechanism of HCM susceptibility in cats. Our hypothesis is that HCM is a complex disease of polygenic inheritance controlled by several variants in both protein-coding genes and regulatory elements.
The objectives of this project are:
(1) Map and evaluate genomic regions affecting HCM susceptibility in cats.
(2) Identify gene expression signatures for HCM in cats.
(3) Integrate results and identify genetic markers and biomarkers for prediction of HCM susceptibility.
Experimental Design and Methods: We will conduct a genome-wide association (GWA) analysis to detect loci affecting HCM susceptibility using 200 meticulously phenotyped cats from 2 pedigree breeds, Birman and British shorthair, chosen on the basis of their diversity regarding disease onset, clinical expression and prognosis. We will analyse the disease as a binary trait (case vs. control) and using continuous variables based on echocardiographic measurements and histopathological findings.The study will include GWA, regional heritability mapping and selective sweep analyses across and within the two cat breeds (obj.1). In addition, the gene expression profile associated with disease will be assessed using RNA-sequencing in 20 myocardial samples collected from cases and controls (elderly, to avoid false-negatives, and sex matched) from the two cat breeds, followed by differential expression and pathway enrichment analysis (obj.2). Systems biology, artificial intelligence, and machine learning approaches will be used to integrate the results of the genomic/ transcriptomic analyses and shed light on the underlying HCM mechanism (obj.3). Main results will be validated using qRTPCR and Western Blot. Thus, the student will be trained in cutting-edge molecular, genetic and bioinformatic technologies as well as advanced echocardiography and histopathology.
This project is aimed at investigating the genetic architecture and the underlying molecular mechanism of HCM susceptibility in cats. Our hypothesis is that HCM is a complex disease of polygenic inheritance controlled by several variants in both protein-coding genes and regulatory elements.
The objectives of this project are:
(1) Map and evaluate genomic regions affecting HCM susceptibility in cats.
(2) Identify gene expression signatures for HCM in cats.
(3) Integrate results and identify genetic markers and biomarkers for prediction of HCM susceptibility.
Experimental Design and Methods: We will conduct a genome-wide association (GWA) analysis to detect loci affecting HCM susceptibility using 200 meticulously phenotyped cats from 2 pedigree breeds, Birman and British shorthair, chosen on the basis of their diversity regarding disease onset, clinical expression and prognosis. We will analyse the disease as a binary trait (case vs. control) and using continuous variables based on echocardiographic measurements and histopathological findings.The study will include GWA, regional heritability mapping and selective sweep analyses across and within the two cat breeds (obj.1). In addition, the gene expression profile associated with disease will be assessed using RNA-sequencing in 20 myocardial samples collected from cases and controls (elderly, to avoid false-negatives, and sex matched) from the two cat breeds, followed by differential expression and pathway enrichment analysis (obj.2). Systems biology, artificial intelligence, and machine learning approaches will be used to integrate the results of the genomic/ transcriptomic analyses and shed light on the underlying HCM mechanism (obj.3). Main results will be validated using qRTPCR and Western Blot. Thus, the student will be trained in cutting-edge molecular, genetic and bioinformatic technologies as well as advanced echocardiography and histopathology.
People |
ORCID iD |
| Androniki Psifidi (Primary Supervisor) | |
| Tom Smedley (Student) |
| Description | The computational work undertaken to determine the effect of a known disease-causing mutation has informed on a potential mechanism by which this mutation may cause the disease in question, hypertrophic cardiomyopathy (HCM). |
| Exploitation Route | The results of this work are informative with regard to how mutations may cause HCM. Additionally, the pipeline developed will be applicable to mutations identified in the future. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Genetic control of hypertrophic cardiomyopathy (HCM) in cats: genomic and transcriptomic characterisation |
| Amount | £147,389 (GBP) |
| Organisation | Petplan Charitable Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 10/2021 |
| End | 09/2024 |
| Description | LIDo Conference Fund |
| Amount | £1,000 (GBP) |
| Organisation | London Interdisciplinary Doctoral Biosciences Consortium |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 01/2023 |
| End | 02/2023 |
| Title | Echocardiography for phenotyping cats |
| Description | Echocardiography is performed to measure left-ventricular free-wall thickness in order to diagnose HCM. This determines individuals as HCM cases or controls. All LVFW measurements were taken from echocardiographic studies performed by cardiology residents in training at the Queen Mother Hospital for Animals (QMHA) using standardised equipment (GE Vivid E9 echo machine; GE systems, Hatfield, Hertfordshire, UK). Measurement results were stored in EchoPac (Version 110.0.2; GE Medical Systems, Hatfield, Hertfordshire, UK) and from this 'Echo Reports' are generated which contain patient details; breed, age, sex, heart measurements and final clinician comments/diagnosis. |
| Type Of Material | Physiological assessment or outcome measure |
| Provided To Others? | Yes |
| Impact | Echocardiography is the best current tool available for diagnosis of HCM and measurement of LVFW thickness. The cardiology residents at QMHA, Professors Virginia Luis Fuentes and David J Connolly are leaders in the field and have been involved in the development and improvement of this method for heart examination and diagnosis of cardiomyopathies, an example being the cut-off measurements used for determining HCM-positive or negative. |
| Title | Dataset of RNA-Seq. data from RNA collected from meticulously phenotyped cats with HCM and healthy controls |
| Description | RNA-Seq data obtained from RNA from individuals described in the Dataset; "Dataset of myocardial samples collected from meticulously phenotyped cats with HCM and healthy controls for RNASeq analyses". Consists of 28 samples sent for total RNA-seq with Novogene. Dataset will be used to determine differential expression of genes relating to HCM. Quality control has been performed using FastQC before and after adapter removal and general trimming using Trimmomatic. Alignment has been performed using STAR and differential expression will be determined using DESeq2. This is ongoing analysis. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2023 |
| Provided To Others? | No |
| Impact | Analysis ongoing. |
| Title | Dataset of blood samples collected from meticulously phenotyped cats with HCM and healthy controls for GWAS analyses |
| Description | Dataset created identifying appropriate cases and controls for GWAS analyses. This is a set of ~130 individuals, cases and controls, from the British Shorthair and Birman cat breeds, which have been phenotyped by echocardiography and that we have EDTA Blood samples for. Echocardiography is performed to measure left-ventricular free-wall thickness in order to diagnose HCM. This determines individuals as HCM cases or controls. All LVFW measurements were taken from echocardiographic studies performed by cardiology residents in training at the Queen Mother Hospital for Animals (QMHA) using standardised equipment (GE Vivid E9 echo machine; GE systems, Hatfield, Hertfordshire, UK). Measurement results were stored in EchoPac (Version 110.0.2; GE Medical Systems, Hatfield, Hertfordshire, UK) and from this 'Echo Reports' are generated which contain patient details; breed, age, sex, heart measurements and final clinician comments/diagnosis. DNA has been extracted from these samples using the Qiagen DNeasy kit with an optimised protocol. So far 130 DNA samples have been extracted, this is a work in progress with a total target number of 200 individuals. These samples will then be sent for sequencing. |
| Type Of Material | Data handling & control |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | These datasets underpin all the genetic analysis we will do for this project. |
| Title | Dataset of genotyped DNA samples collected from meticulously phenotyped cats with HCM and healthy controls for GWAS analyses |
| Description | Dataset created from genotyping of Feline DNA from HCM case and control individuals using the Illumina Infinium iSelect 63K Cat DNA Array. Dataset consists of 167 individuals from the British Shorthair and Birman breeds described in the: "Dataset of blood samples collected from meticulously phenotyped cats with HCM and healthy controls for GWAS analyses". |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | Ongoing analysis. GWAS analyses have identified SNPs of interest. Further analysis will consist of Fst and RHM. |
| Title | Dataset of myocardial samples collected from meticulously phenotyped cats with HCM and healthy controls for RNASeq analyses |
| Description | Dataset created identifying appropriate cases and controls for RNASeq analyses. This is a set of ~30 individuals, cases and controls from the British Shorthair and Domestic Shorthair cat breeds, which have been phenotyped by either by echocardiography or post-mortem histopathology and that we have myocardial tissue samples for. Collected samples are stored either in RNALater or frozen at -80 degrees. RNA will be extracted from these samples using the Qiagen RNeasy kit with an optimised protocol. This will be sent for sequencing. This collection is still in progress in order to get a better balance of controls to cases. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | Samples to be sent for sequencing and analyses will be performed subsequently. |
| Title | Dataset of protein domain models of the C6 domain of cMYBPC3 for discrete molecular dynamics analysis to determine mutant effect |
| Description | Dataset of homology models produced using SwissModel for the C6 Fn3 domain of cardiac myosin-binding protein from amino acid sequence data from cardiomyocyte cell lines edited using the CRISPR/Cas9 system to contain the HCM-causing variants R820W and KDel814. These are found in cats and humans and humans only respectively. Models were produced for the wild-type domain and variant-containing domains. Models were secondarily produced using the more recently available AlphaFold software in order to validate our models. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | These models were run through discrete molecular dynamics simulations in order to identify the impact of a known, HCM-causing mutation. This work is being prepared for publishing. |
| Title | Discrete Molecular Dynamics Simulation Pipeline for analysis of the cMYBPC3 C6 domain and its association with HCM |
| Description | In order to investigate the impact of two known HCM-associated mutations; R820W in cats and humans and KDel814 in humans, we have developed a pipeline to produce homology models from amino acid sequence data gained from iPSC-derived cardiomyocyte cell lines edited using the CRISPR/Cas9 system to contain these single nucleotide mutations. The pipeline has multiple processes, starting with the generated model and; 1) Running denaturation simulations 2) Running Replica-Exchange simulations on a) Folded and b) Unfolded versions of the models 3) Displaying the protein folding landscape of models 4) Extracting key structures from simulation files in order for structural and surface comparisons to be made. This enables conclusions to be drawn on the impact of these HCM-causing mutations on the structural stability of the domain and changes to protein-folding specificity. This will inform on potential pathomechanisms such as haploinsufficiency or a poison polypeptide effect. |
| Type Of Material | Data analysis technique |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | The analysis of the models produced has identified potential pathomechanisms of a known HCM-causing mutation. In combination with laboratory work performed by other team members on wild-type and mutant-containing cardiomyocyte cell lines this data suggests the pathomechanism in action is a poison polypeptide mechanism where mutant proteins are still incorporated into the protein complex but have a deleterious effect. A draft has been prepared and will be submitted this year. |
| Title | Genome Wide Association Study Data from Genotyped Feline DNA |
| Description | Genome Wide Association Study (GWAS) Data from "Dataset of genotyped DNA samples collected from meticulously phenotyped cats with HCM and healthy controls for GWAS analyses". GWAS analyses were performed on; the entire cohort, British Shorthair only and Birman only data as well as in combination with existing genotyped samples from other pedigree breeds and Domestic Shorthairs. GWAS were performed using Plink and Gemma incorporating meta-data including age and sex as covariates. Additionally, Principal Component Analysis (PCA) has been performed to determine the genomic relationships within and between groups. The initial GWAS results have identified several genomic markers associated with HCM across-breeds as well as within breeds. Some of these are located close to potential candidate genes linked, for example, to actin and myosin-binding. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | Ongoing work, this will continue with Fst and RHM analyses and will be integrated with transcriptomic analyses in 2023. |
| Title | Pipeline for production of protein model and subsequent molecular dynamics simulations and analysis |
| Description | Protein models were produced for a wild-type and mutant protein, molecular dynamics simulations were performed in order to compare. This enabled computational functional analysis of a previously identified genetic variant. This work is currently being prepared for publishing. |
| Type Of Material | Computer model/algorithm |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | Pipeline will be used in the future of this project and also will be available for others to use for functional analysis of any identified mutations. |
| Description | iCASE Studentship partnership - Zoetis |
| Organisation | Zoetis |
| Country | United States |
| Sector | Private |
| PI Contribution | This PhD studentship is an iCASE studentship partnered with Zoetis. We have created two datasets of phenotypes cats for which we have 1) blood samples for GWAS analysis and 2) tissue samples for RNASeq analysis. Samples are still being collected, when target sample numbers are reached we will be sending them for sequencing and performing genetic and genomic analyses. |
| Collaborator Contribution | This PhD studentship is an iCASE studentship partnered with Zoetis. Zoetis have contributed to funds for the sequencing analyses for this project and will be providing advice and feedback on the analysis performed. Additionally they will be facilitating a 3 month placement. |
| Impact | Creation of two datasets of phenotyped cats for the GWAS and RNASeq analysis. These datasets are still growing. |
| Start Year | 2019 |
| Description | BSAS 2021 Abstract and Poster Presentation |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | Online presentation to researchers and students attending BSAS 2021. On a topic unique to this conference (protein modelling), sparked follow-up questions. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Invited Plenary Talk "Feline Hypertopic Cardiomyopathy: Unravelling the genetics of a puzzling disease" at Conference organised by the Hellenic Veterinary Association in March 2022 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | Invited plenary talk entitled: " Feline Hypertopic Cardiomyopathy: Unravelling the genetics of a puzzling disease" presented in a one day conference on feline cardiomyopathies organised by the Hellenic Veterinary Association in March 2022. Speaker; A. Psifidi |
| Year(s) Of Engagement Activity | 2022 |
| Description | LIDo Annual retreat Poster Presentation |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Poster presentation to LIDo peers, facilitated discussion and questions, gained useful advice. |
| Year(s) Of Engagement Activity | 2021 |
| Description | LIDo iCASE Gala 2021 Poster Presentation |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Poster presentation to other LIDo post-graduate students and supervisors. Discussion of research with peers, led to useful ideas on future direction and options. |
| Year(s) Of Engagement Activity | 2021 |
| Description | LIDo iCASE Gala 2022 Presentation |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | Formal presentation to other LIDo post-graduate students and supervisors of 3 years of PhD research. Discussion of research with peers and investigators led to useful ideas on future direction and options. |
| Year(s) Of Engagement Activity | 2022 |
| Description | PAG 2023 Abstract and Poster Presentation |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Poster presentation at the Plant and Animal Genome Conference in San Diego January 2023. |
| Year(s) Of Engagement Activity | 2023 |
| URL | https://plan.core-apps.com/pag_2023/abstract/963d7d6c9252da3647857a6b5d016f8a |