Pulmonary arterial hypertension

Question 1

What does pulmonary hypertension provide?

Answer 1

A unique window into regulation of the lung vasculature

Question 2

What are the clinical features of pulmonary hypertension?

Answer 2

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)

Question 3

What accounts for 3% of all pulmonary hypertension?

Answer 3

Pulmonary arterial hypertension

Estimated prevalence: 10 – 50 cases per million

Question 4

What is pulmonary hypertension?

Answer 4

Pulmonary hypertension = high blood pressure in the lungs

Question 5

What is Pulmonary arterial hypertension ?

Answer 5

Chronic disease caused when the arteries in the lungs become narrowed, thickened or stiff, impeding blood flow from the heart

Question 6

What are the stages of Pulmonary arterial hypertension?

Answer 6

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

Question 7

What is Pulmonary arterial hypertension diagnosed by?

Answer 7

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

Question 8

Occlusion of pulmonary arterioles caused by vascular remodelling in PAH.

What does this involve?

Answer 8

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

Question 9

What remains the ‘gold-standard’ treatment?

Answer 9

Heart-lung transplant

Question 10

Explain the genetics of PAH.

Answer 10

1. Typically autosomal dominant inheritance or sporadic
2. 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

Question 11

What are at least twice as common in women compared to men?

Answer 11

Idiopathic and heritable forms of PAH

Question 12

PAH is most diagnosed in who?

Answer 12

Women aged 30-60

Question 13

What is the first major causal gene for PAH?

Answer 13

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

Question 14

Explain the Canonical BMP signalling pathway?

Answer 14

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.

Question 15

Mutations in BMPR2 gene, encoding the bone morphogenetic protein type II receptor, were identified by what?

Answer 15

Positional cloning (Lane et al. 2000)

Question 16

BMPR2 is a receptor of the TGF-β signalling pathway

True or false

Answer 16

True

Question 17

What is the major genetic risk factor for PAH ?

Answer 17

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

Question 18

BMP signalling has roles in what?

Answer 18

Embryogenesis, control of differentiation, apoptosis and proliferation of many adult cell types

Question 19

BMPR2 mutation accounts for what?

Answer 19

~70% of hereditary PAH and ~25% of idiopathic PAH cases

Question 20

Explain the distribution of BMPR2 mutation?

Answer 20

• 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

Question 21

What is the impact of Ligand-binding domain missense mutations in BMPR2?

Answer 21

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.

Question 22

What is the functional impact of BMPR2 mutation?

Answer 22

1. Truncating mutation:
   - Majority degraded by nonsense mediated decay. This leads to haploinsufficiency, haploinsufficiency is the primary molecular mechanism of disease.
2. 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

Question 23

What increase cell-membrane expression of both wild-type BMPR-II and the ligand-binding mutation p.C118W?

Answer 23

Trafficking agents, thapsigargin and glycerol

Question 24

Candidate gene analysis has implicated what?

Answer 24

7 additional BMP pathway genes in PAH pathogenesis

*Mutations in ALK1 and ENG also cause hereditary haemorrhagic telangiectasia (HHT):

Question 25

BMPR2 accounts for 85% of reported variation vs…

Answer 25

other BMP / TGF-β pathway variants = 15%

Question 26

Although BMPR2 accounts for the majority of HPAH…

Answer 26

~75% of IPAH cases have no apparent mutation in BMP pathway genes – thus for many patients no genetic cause identified

Question 27

Linkage analysis can be combined with what?

Answer 27

Whole-exome sequencing, to narrow down the region of the genome that needs to be examined for the disease gene.

Question 28

What steps are involved in variant detection by exome sequencing?

Answer 28

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

Question 29

What do CAV1 knockout mice demonstrate?

Answer 29

Features of PAH

Question 30

What did exome sequencing in 3 members of a HPAH family find?

Answer 30

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

Question 31

TBX4 mutations found at higher rates in whom?

Answer 31

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

Question 32

Exome sequencing has identified what genes?

Answer 32

CAV1, KCNK3 (and EIF2AK4 in PVOD/PCH)

Question 33

WGS has identified what genes?

Answer 33

Whole-genome sequencing:

ABCC8, ATP13A3, AQP1, GDF2, SOX17, TBX4

Question 34

A WGS concerning Idiopathic PAH: Rare variant analysis used gene burden testing and concluded what?

Answer 34

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)

Question 35

What is the significance of GDF2?

Answer 35

BMP ligands are secreted as mature disulfide-linked dimers

• GDF2 mutations prevent secretion of the BMP9 ligand

Question 36

Why may BMP9 be a potential treatment of PAH?

Answer 36

BMP9 prevents apoptosis and vascular permeability in endothelial cells

Pulmonary hypertension in Bmpr2+/R899X knock-in mice is reversed by BMP9

Question 37

What is SOX17?

Answer 37

SOX17 is an endothelial transcription factor

SOX17 mutations predicted to lead to loss-of-function

SOX17 mutations have also been detected in CHD-PAH

Question 38

What is SOX17 associated with?

Answer 38

PAH and congenital heart disease

Question 39

What has Idiopathic PAH: Common variant analysis shown?

Answer 39

The HLA-DPB1 lead SNP is associated with improved survival in patients with IPAH

Question 40

What has in silico analysis suggested?

Answer 40

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