scienze della vita roma, 22-23 ottobre 2012 - SIF

SERIAL GENE EXPRESSION ANALYSIS IN PERIPHERAL BLOOD
MONONUCLEAR CELLS OF PATIENTS WITH IDIOPATHIC AND
HERITABLE PULMONARY ARTERIAL HYPERTENSION
S. Cantoni 1,2 , M. Galletti 1,2 , R. Tassinari 1,2 , A. Guffanti 3 , E. Brini 3 , F. Zambelli 1 , C. Ventura 1,2 , N.
Galiè 2
1 National Institute Biostructures and Biomolecules, Bologna Italy; 2 Cardiovascular Department
University of Bologna, Bologna Italy; 3 Genomnia s.r.l.- Milano, Italy.
Introduction
Pulmonary arterial hypertension (PAH) is a rare disorder characterized by progressive increase in
pulmonary vascular resistance, right ventricular failure and premature death. PAH can be idiopathic
(IPAH), heritable (HPAH), or associated with other conditions, such as scleroderma or congenital
heart disease.
Studying pulmonary vascular diseases, such as PAH, access to affected tissue is especially
problematic for investigators. Due to high risk for complication, lung biopsies are not routinely
performed, prompting for alternative cell sources and molecular approaches. In this contest the
prominent role of inflammation in the development of PAH lead to consider peripheral blood
mononuclear cells (PBMCs) as a valuable tool for investigating pathologic processes, representing a
convenient source for the assessment of alteration in gene associated with immune-related diseases.
So far, only a few transcriptional studies for PAH have been performed with PBMCs using
microarray analysis. Compared to this technique, Serial Analysis of Gene Expression (SAGE) has
remarkable advantages. Since it does not require prior knowledge of the sequences to be analyzed,
SAGE can be used to obtain complete transcriptional profiles of expressed genes, albeit unknown
genes.
Methods
SAGE allows quantitative analysis of thousands transcripts, relying on the principle that a short
oligonucleotide sequence (tag) can uniquely identify mRNA transcripts. Tag frequency in SAGE
library reflects transcript abundance. For the study we enrolled 15 PAH patients (5 IPAH nonresponder,
5 IPAH responder, 5 HPAH), 4 healthy BMPR2 mutation carriers and 6 healthy subjects
(HS). Patients were diagnosed according to the 2009 ESC/ERS guidelines and blood samples were
collected before PAH therapy initiation.
Results
Starting from 6 million of tags, corresponding to 15,000 transcripts, comparative analysis of
different groups revealed that significant differential expression was only restricted to a hundred of
down- or up-regulated genes. Interestingly, computational analysis revealed that genes can be
clustered into functional networks, sharing a number of crucial features in cellular homeostasis and
signaling (migration, proliferation, inflammation). Overlapping analysis revealed genes shared
between IPAH responder/ non-responder patients, and between HPAH patients/ healthy mutated
carriers.
Conclusions
SAGE can provide affordable analysis of genes amenable for molecular dissection of PAH using
PBMCs as a
sentinel, surrogate tissue.
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