ADAMTS6 in the Heart

Question 1

What is sudden cardiac death

Answer 1

Sudden Cardiac Death - >50% of all CHD deaths, up to 20% of all deaths

Abnormal heart rhythms develop with increasing age, congenital heart defects

Rare arrhythmias e.g. Long QT syndrome, cardiomyopathy e.g. hypertrophic CM

Question 2

How can we investigate cardiac function

Answer 2

Genetic risk - candidate gene studies, GWAS

ECG (Electrocardiogram) - intermediate marker, measured in ms

PR interval : atrial depolarisation
QRS interval ventricular depolarisation
QT interval : ventricular de and repolarisation
RR interval : 1 cycle (heart rhythm)

Question 3

What is QRS and ventricular depolarisation, and how are they risk factors

Answer 3

Ventricular depolarisation - QRS Interval

QRS interval depends on age, sex (males have longer QRS)

Increased QRS duration is associated with increased risk of SCD

Question 4

What is GWAS

Answer 4

SNP’s are dispersed across the genome, mainly non-coding regions, and can be linked to regions of disease

Increasing sample sizes allows us to more confidently measure variability
That leads to an increased ability to discern smaller differences (SNP effect sizes β)

If association found, then further study can follow to
Determine the causal variants
Understand mechanism of action and disease aetiology in individuals
Characterize relevance and/or impact in more general population

Question 5

What are causal variants

Answer 5

Often the causal variant (responsible for the association signal at a locus) is not included on the SNP chips
This is usually because it is likely much rarer

Further work is required to narrow down the region of association and identify the causal variant
There are sets where you can investigated only lower frequency SNPs found in exons to find rare variants

As you are looking at very rare variants you need a large cohort from across the world
Investigation confirmed known QRS loci as well as novel loci

Question 6

What are collapsed SNP/gene analyses

Answer 6

Single variant tests - hard to find a statistically significant association when variants are rare
Low population to investigate

Instead use a region based analysis - test the joint effect of rare variants in a gene/region
Look at all the rare variants in a given gene and its joint effects

Question 7

Describe the ENU mouse model for ADAMTS6

Answer 7

Randomly mutated mice, some found with homozygous mutations in ADAMSTS6

These mice had cardiac defects, and die early in development (also seen in 85% of homozygous mouse mutants)

Homozygous ADAMSTS6 Ser149Arg mutant has double outlet right ventricle and atrioventricular septal defect

Question 8

Discuss gap junctions in the heart

Answer 8

Ventricular conduction relies on cardiomyocyte coupling through gap junctions, with connexin 43 (Cx43) being the predominant myocardial gap junction protein in the human and mouse heart

GJA1 (encoding Cx43) knockout mice exhibit slow conduction, QRS prolongation and increased susceptibility to ventricular arrhythmias

Homozygous mutants have severe structural heart defects and Cx43 deficiency

Heterozygous mutants have a reduction in Cx43 in ventricles without defects in cardiac morphogenesis
Suggests haploinsufficiency (one allele cannot rescue the phenotype)
Also suggests slower ventricular conduction in humans with rare 'pathogenic' ADAMTS6 variant could arise form impaired myocardial connectivity due to Cx43 reduction

Question 9

Describe in vitro investigations of ADAMSTS6

Answer 9

Myc-tagged ADAMTS6 human variants (p.Ser90Leu and p.Arg603Trp) were expressed in HEK293F cells
‘Benign’ human missense variants p.Ser210Leu and p.Met752Val used as a control along with mouse variant p.Ser149Arg

‘Damaging’ variants show reduced secretion, suggesting heterozygous individuals have reduced proteolytic activity which could affect cell-cell and cell-matrix interactions essential for efficient Cx43 gap junction assembly

Question 10

Discuss the results from the collapsed SNP/gene analysis - ADAMTS6

Answer 10

Found 12 SNP’s associated with ADAMTS6 with one significant signal

ADAMTS6 is a metalloprotease that mediates extracellular proteolytic processing of extracellular matrix components and other secreted

Individual results did not produce significant thresholds, but altogether it did
Also as exome chip has less SNP the threshold is slightly lower, at 10-6 rather than 10-7

The variant with the strongest p-value has MAF 0.2% (a rare variant)
The SNP β (effect size) showed that for every copy of this allele QRS duration changes by 2.6ms

If this was inherited alongside other variants increasing the risk it could add up

Missense variants involved in protein folding and secretion