Pulmonary arterial hypertension Flashcards
What does pulmonary hypertension provide?
A unique window into regulation of the lung vasculature
What are the clinical features of pulmonary hypertension?
27% reported fatigue
15% reported fainting or light headedness
22% reported chest pain
86% reported shortness of breath
13% reported palpitations
21% reported edema (swelling)
What accounts for 3% of all pulmonary hypertension?
Pulmonary arterial hypertension
Estimated prevalence: 10 – 50 cases per million
What is pulmonary hypertension?
Pulmonary hypertension = high blood pressure in the lungs
What is Pulmonary arterial hypertension ?
Chronic disease caused when the arteries in the lungs become narrowed, thickened or stiff, impeding blood flow from the heart
What are the stages of Pulmonary arterial hypertension?
Progressive and chronic occlusion of pulmonary arterioles
Forces the right ventricle to work harder to pump blood into the lungs
Right ventricular hypertrophy progresses to RV dilation and eventually right heart failure
What is Pulmonary arterial hypertension diagnosed by?
6-minute walk test and right heart catheterisation:
- Mean pulmonary artery pressure (mPAP) >20 mmHg
- Elevated pulmonary vascular resistance (PVR) ≥3 Wood units
- In presence of normal left atrial pressure
Occlusion of pulmonary arterioles caused by vascular remodelling in PAH.
What does this involve?
Increased Apoptosis:
Pulmonary artery endothelial cells undergo apoptosis
Some cells become apoptotic-resistant and hyper-proliferative
Increased Proliferation:
Endothelial cells proliferate by monoclonal expansion (cancer-like)
Smooth muscle cells are recruited leading to thickening of the vessel wall and vascular obstruction
Increased Permeability:
Monolayer integrity reduced leading to increased leukocyte transmigration
What remains the ‘gold-standard’ treatment?
Heart-lung transplant
Explain the genetics of PAH.
- Typically autosomal dominant inheritance or sporadic
- Features of complex disease:
- Reduced penetrance
- Gender bias – favours females
- Variable age of disease onset – Les s than 1 to patients in 70s, typical onset in 30s
What are at least twice as common in women compared to men?
Idiopathic and heritable forms of PAH
PAH is most diagnosed in who?
Women aged 30-60
What is the first major causal gene for PAH?
BMPR2
- Catalytic serine-threonine kinase domain: aa.205-508
- C-terminal cytoplasmic tail domain
- Signal peptide: aa.1-26
- Extracellular (ligand-binding) domain: aa.60-131
- Transmembrane domain: aa.152-174
- Over 800 independent mutations reported in the literature
Explain the Canonical BMP signalling pathway?
BMP signalling activated by binding of one of the ligands, leads to formation of a heterotetrametric complex with one of the type 1 receptors. Formation of this complex activates phosphorylation cascade whereby the SMAD intermediaries are phosphorylated and translocate to nucleus where they can then regulate transcription of downstream genes.
Mutations in BMPR2 gene, encoding the bone morphogenetic protein type II receptor, were identified by what?
Positional cloning (Lane et al. 2000)
BMPR2 is a receptor of the TGF-β signalling pathway
True or false
True
What is the major genetic risk factor for PAH ?
BMPR2 haploinsufficiency (Machado et al. 2001) – in autosomal dominant disease only one copy of the disease gene is present, thus only half amount of normal BMPR2 which is insufficient for the protein to function normally in the cell
BMP signalling has roles in what?
Embryogenesis, control of differentiation, apoptosis and proliferation of many adult cell types
BMPR2 mutation accounts for what?
~70% of hereditary PAH and ~25% of idiopathic PAH cases
Explain the distribution of BMPR2 mutation?
- BMPR2 mutations have been identified in 53-86% of familial PAH and 14-35% of idiopathic PAH cases
- The majority of amino acid substitutions cluster in exons encoding the ligand-binding domain and key catalytic regions of the kinase domain (exons 2-3, 6-9 and 11)
- Exons 1, 4, 5 and 13 (regions of uncertain importance) have a low frequency of missense mutation
- Mutations are distributed across all mutation categories
- High frequency of recurrent mutations in exon 12
What is the impact of Ligand-binding domain missense mutations in BMPR2?
Most cystine mutations domain identified change this amino acid to an alternative amino acid – important because cystines typically work in pairs to form disulfide bridges.Disulfide bridges form rigid stuctures which help to hold protein structure in place
Thus when cystine is replaced with an alternative amino acid, this bond is broken and this impacts folding of protein.
What is the functional impact of BMPR2 mutation?
- Truncating mutation:
- Majority degraded by nonsense mediated decay. This leads to haploinsufficiency, haploinsufficiency is the primary molecular mechanism of disease. - Kinase domain missense mutations:
a) Subcellular localisation
b) Luciferase assays measuring Smad activation
Cysteine mutations do not traffick to the cell membrane due to mis-folding
The majority of amino acid substitutions result in loss of Smad activity
All mutations cause a constitutive activation of p38MAPK and uncontrolled proliferation
What increase cell-membrane expression of both wild-type BMPR-II and the ligand-binding mutation p.C118W?
Trafficking agents, thapsigargin and glycerol
Candidate gene analysis has implicated what?
7 additional BMP pathway genes in PAH pathogenesis
*Mutations in ALK1 and ENG also cause hereditary haemorrhagic telangiectasia (HHT):
BMPR2 accounts for 85% of reported variation vs…
other BMP / TGF-β pathway variants = 15%
Although BMPR2 accounts for the majority of HPAH…
~75% of IPAH cases have no apparent mutation in BMP pathway genes – thus for many patients no genetic cause identified
Linkage analysis can be combined with what?
Whole-exome sequencing, to narrow down the region of the genome that needs to be examined for the disease gene.
What steps are involved in variant detection by exome sequencing?
Variant profile
Filtering strategy-
Filtering of whole-exome sequence data typically applies what is known about disease (e.g. rare, autosomal dominant):
Can can filter variants based on zygosity (autosomal dominant = heterozygous) and allele frequency (rare = less than 1% in control populations)
This will reduce the number of candidate variants to <100 variants, but using linkage data can further reduce list (e.g. to <10 variants)
This increases chances of finding the causal variant/gene (and is cheaper than sequencing many affected individuals!)
Causative variant identification
What do CAV1 knockout mice demonstrate?
Features of PAH
What did exome sequencing in 3 members of a HPAH family find?
KCNK3 gene: c.608G>A (p.G203D)
6 missense mutations found, impact function of potassium channel. Used pharmaceutical agents to reverse this phenotype, suggests potential therapeutic
TBX4 mutations found at higher rates in whom?
Paediatric cases compared to adults
TBX4 mutants previously identified in few childhood cases of PAH with small patellar syndrome
This study identified up to 8 percent of familial and idiopathic cases may be called by TBX4 defects
Exome sequencing has identified what genes?
CAV1, KCNK3 (and EIF2AK4 in PVOD/PCH)
WGS has identified what genes?
Whole-genome sequencing:
ABCC8, ATP13A3, AQP1, GDF2, SOX17, TBX4
A WGS concerning Idiopathic PAH: Rare variant analysis used gene burden testing and concluded what?
Higher frequency of ‘protein-truncating’ variants (PTVs) in ATP13A3 (6 cases) (Padj = 0.0346)
Significantly higher proportion of GDF2 variants (P = 0.001) in the combined PTV and missense analysis
SKAT-O association with rare variants in AQP1 (Padj = 4.28x10-6) and SOX17 (Padj = 6.7x10-5)
What is the significance of GDF2?
BMP ligands are secreted as mature disulfide-linked dimers
- GDF2 mutations prevent secretion of the BMP9 ligand
Why may BMP9 be a potential treatment of PAH?
BMP9 prevents apoptosis and vascular permeability in endothelial cells
Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9
What is SOX17?
SOX17 is an endothelial transcription factor
SOX17 mutations predicted to lead to loss-of-function
SOX17 mutations have also been detected in CHD-PAH
What is SOX17 associated with?
PAH and congenital heart disease
What has Idiopathic PAH: Common variant analysis shown?
The HLA-DPB1 lead SNP is associated with improved survival in patients with IPAH
What has in silico analysis suggested?
SOX17 risk variants lie within a putative enhancer region which contains only SOX17
PAH risk allele had reduced enhancer activity compared to non risk allele
Thus this is evidence that common variation in SOX17 larger gene region and rare variation identified by WGS data are both potentially risk factors for the same condition
