Gene Activity Analysis of Whole Blood Finds 3 Major PAH Subgroups

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by Steve Bryson, PhD |

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Gene activity analysis of whole blood collected from people with pulmonary arterial hypertension (PAH) identified three patient subgroups associated with distinct clinical features and outcomes, a study concluded.

These findings may provide molecular insights into the development of PAH, improve disease risk assessments, and potentially guide individual treatment strategies, the scientists noted.

The study, “Biological heterogeneity in idiopathic pulmonary arterial hypertension identified through unsupervised transcriptomic profiling of whole blood,” was published in the journal Nature Communications.

In PAH, the pulmonary arteries that transport blood through the lungs narrow, restricting blood flow and increasing blood pressure (hypertension).

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The cause of idiopathic PAH (IPAH) is currently unknown, and diagnosis is usually confirmed after other forms of PAH have been ruled out, such as infections, the use of certain medications, or other diseases. As a result, IPAH patients show a wide range of differences in disease progression, treatment responses, and survival.

Several studies have found mutations in genes associated with both IPAH and inherited forms of PAH (HPAH), as well as disease-related signatures in protein production and gene activity that have identified biomarkers to aid in diagnosis and prognosis (outcomes).

However, the molecular heterogeneity (variability) within IPAH and HPAH cases has not been reported so far.

Now, researchers based at the University of Sheffield in the U.K., along with colleagues in the U.K. and the U.S., collected whole blood samples from IPAH and HPAH patients and analyzed global gene activity using machine learning to investigate the biological heterogeneity of PAH.

Participants were recruited from the National Cohort Study of Idiopathic and Heritable Pulmonary Arterial Hypertension in the U.K. (NCT01907295), designed to explore genetic and environmental contributions to the disease. Information was collected every six months as a part of routine clinical care.

Gene expression (activity) and identification were determined by RNA sequencing from whole blood samples isolated from 359 IPAH and HPAH patients, plus 13 patient relatives and 21 unrelated healthy controls. The study included both new (incident) and prevalent cases diagnosed at least six months before the study.

The analysis identified five distinct subgroups of patients based on different gene expression levels.

The largest of these groups contained 129 participants (subgroup I), who had the poorest survival — 53% were alive after a median of five years. The second-largest group (subgroup II) of 112 patients demonstrated the best survival (78% survival at a median five years).

Subgroup V, with 89, demonstrated a mixed gene expression pattern and average survival outcome compared with subgroups I and II. Subgroups III, with 19 patients, and IV, with 10 patients, also had distinct gene expression patterns, but statistical analysis was not possible due to the small size of these groups; thus, the team focused on subgroups I, II, and V.

A detailed examination of gene expression profiles identified 57 genes with specific associations to each subgroup. The ALAS2 gene, related to the disease porphyria, appeared in expression signatures for subgroups I and II, but was about twofold higher in group I (poor prognosis) than group II (good prognosis).

Several genes that encode for antibody proteins (immunoglobins) were “key markers” for each subgroup, which either had low expression (downregulated) in subgroup I or high expression (upregulated) in subgroup II.

In subgroup II, upregulation was high for Noggin, a protein that blocks the action of BMP4, which is associated with the development of PAH, “underlining its association with good prognosis,” the researchers wrote. In contrast, antibody genes and Noggin were downregulated more than twofold in subgroup I, “fitting with contemporary understanding of perturbed BMP and inflammatory signalling in PAH pathogenesis [disease development],” they added.

In addition to higher antibody gene expression, immune cell analysis found a higher abundance of lymphocytes (B-cells and T-cells) and memory B-cells in subgroup II. In poor prognosis (subgroup I), there was a lower proportion of lymphocytes and a higher number of neutrophils, which was statistically significant. A higher neutrophil-lymphocyte ratio is a known indicator of poor overall survival.

Moreover, a significantly higher number of patients carried a variant of HLA-DP, a protein in the surface of immune cells that is essential for immune responses and has been shown to be associated with survival in a large IPAH study.

An assessment of clinical characteristics across gene expression subgroups found patients in subgroup I were significantly older (45 to 70) than the other subgroups; had the highest levels of NT-proBNP, a marker for heart damage; and the lowest distance walked in six minutes (6MWD).

Subgroup I also had the highest proportion of patients (50.4%) within the functional class (FC) III, who experience shortness of breath and fatigue with normal activities. In contrast, subgroup II had a higher number of patients with FC I (16.5%) or II (41.3%) with either no symptoms or symptoms upon exertion.

Important clinical features that described different gene expression signatures in subgroup I included C-reactive protein (CRP), a sign of inflammation; creatinine, a marker for kidney function; age at diagnosis; 6MWD; and body mass index (BMI), a measure of body fat.

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In subgroup II, clinical features were CRP, creatinine, age at diagnosis, BMI, 6MWD, oxygen level in the blood (before-6MWD), and right atrial area, associated with PAH outcomes.

A higher CRP was a marker for subgroup I, and lower levels indicated subgroups II and V. Conversely, a shorter 6MWD was associated with subgroup I and a longer distance with subgroups II and V. An older age at diagnosis, and a higher BMI, right atrial area, and creatinine were associated with subgroup I.

Finally, the results were validated using these clinical features associated with gene expression levels to classify 197 patients without whole-blood RNA profiling. Again, three groups were classified similar to subgroups I, II, and V and showed differences in their 10-year survival outcomes.

“These findings independently validated provide evidence for the existence of 3 major subgroups (endophenotypes) within the IPAH classification, could improve risk stratification and provide molecular insights into the pathogenesis of IPAH,” the scientists wrote.

“Furthermore, these data hold promise that these molecular endophenotypes may be tractable to existing therapies, may offer an alternative approach to tailor, and assess individual treatment response, in PAH,” they added.