Discussion
CFTR mutations are one of the factors that determine the severity of
CF.20 However, there are many studies performed with
patients bearing the same mutation but with different clinical
severity.13,14,15,18,19 Research regarding clinical
heterogeneity in twins and siblings with same CFTR mutation suggests
that modifier genes could play a role in disease severity. Studies which
use transcriptomic approaches determined that clinical severity is
associated with many molecular mechanisms such as CFTR protein folding,
function, and trafficking, inflammation, immune response, epithelial
cell differentiation, ion transport and oxidative stress.
In a study performed with nasal epithelial cells of F508del homozygotes
patients and healthy controls, the expression of genes involved in cell
proliferation (MMP1, ADM, AREG, GJA1, RUNX2) was increased and the
expression of genes involved in cilia formation (DNAH9, DNAH12, DNAI1,
DNAI2 and DNAAF1) was decreased in patients.13 In
another transcriptomic study, nasal and bronchial samples obtained from
same patients have been used, and as a result the primary affected
molecular pathways in nasal and bronchial epithelial cells were found to
be amino acid metabolism and inflammatory response,
respectively.19 In another recent study conducted by
Kormann et al. it was found that genes involved in type-1 interferon
response and ribosomal stalk proteins that interact with rRNA have an
effect on disease severity.18 Similar to this study,
Wright et al. have shown that genes involved in lipid metabolism,
protein ubiquitination mechanism, protein metabolism and immune response
are related to disease severity.15
So far, there is no transcriptomic study that investigates phenotypic
variability between mild and severe siblings with the same mutation. In
our study we aimed to explain clinical heterogeneity by using targeted
transcriptome analysis of nasal epithelial cells to investigate
differentially expressed genes and associated pathways of between mild
and severe siblings. For this purpose, we determined three families that
include siblings with different clinical severity determined by
recurrent lung infection, pulmonary involvement and comorbidity. The
mutations of Family 1 (F508del/G85E) and Family 2 (F508del/F508del)
belonged to Class II group whereas Family 3 (I1234V/I1234V) had
mutations of Class IV.
As a result of comparison between mild and severe siblings with Class II
mutation, seven genes (TNFRSF11A, KCNE1, STX1A, SLC9A3R2, CXCL1, CFTR,
CXCL2) were found to be differentially expressed. TNFRSF11A, KCNE1,
STX1A and SLC9A3R2 genes were up regulated while CXCL1, CFTR and CXCL2
genes were down regulated in severe patients. TNFRSF11A also known as NF
kappa B activator, is a member of the TNF receptor family and triggers
the transcriptional activation of NF kappa B pathway mediated
proinflammatory cytokines and chemokines such as CXCL1-2-8 and
IL1B.21 Similarly, in our study the reason for up
regulation of TNFRSF11A (4,03 fold), may be to increase the inflammatory
response to infection in severe patients with Class II mutation. The
mRNA expression of the SLC9A3R2 (2,02 fold) and SCNN1G (2,77 fold) genes
in the aldosterone regulated sodium reabsorption pathway were also
increased in severe patients. Studies have shown that the ion
transporters, negative CFTR regulators SCL9A3R2 (SLC9A3 regulator) and
SCNN1G (ENaC subunit) are associated with decreased CFTR
expression.22,23 STX1A was determined in SNARE
interactions in vesicular transport pathway. Up regulated STX1A, have
been associated with CFTR expression and its
conductance.24,25 The decreased expression of CXCL1
(-5,03 fold), CXCL2 (-2,65 fold), IL1B (-3,22 fold), CXCL8 (-10,59
fold), HSP90AA1 (-2,09) and PTGS2 (-3,61 fold) in inflammation related
pathways (IL-17 signaling, NF-kappa B signaling, cytokine-cytokine
receptor interaction, TNF signaling, NOD-like receptor signaling and,
chemokine signaling pathways) suggested that the inflammatory response
could be suppressed in severe patients with Class II mutation. Some
studies have shown increased expression of chemokines and cytokines
detected in CF patients compared to controls.13,19,26But some reported conflicting results, similar to our findings. Ideozu
et al. showed that the expression of CXCL1 and IL1B were decreased in CF
patients compared to healthy controls.27 Similar to
this study, Kormann et al. and Wright et al. showed that CXCL8
expression was decreased in severe CF patients.15,18In our study, the decreased chemokine expression detected in severe
patients with Class II mutation could also be related to endoplasmic
reticulum stress and chemokine secretion.28 Pathway
enrichment analysis showed that NOD-like receptor signaling pathway was
among the most prominent ones. Significantly down regulated CXCL1 and
CXCL2 genes were found to be functional in NOD-like receptor signaling
pathway in severe patients. In a study conducted on primary bronchial
epithelial cell culture and monocytes of CF patients, it was detected
that as a result of decreased CFTR protein expression, ENaC expression
was increased. This caused stimulation of NLRP3 inflammasome and the
release of IL18 and IL1B.29 This finding supported the
formation of the inflamasomme mechanism in nasal epithelial
cells.29,30 The decrease in the expression of genes in
this pathway in severe patients could be elucidated by the decreased
CFTR expression and ER stress.
In Class IV group, 24 genes (Up regulated 3 genes, down regulated 21
genes) were found to be differentially expressed in severe sibling.
Since Class IV group consisted of only one family (3 siblings), the
statistical significance of differentially expressed genes could not be
evaluated and the pathways were interpreted by means of biological
significance. As a result of pathway analysis, different from the Class
II group, specific to the Class IV group, AGE-RAGE signaling pathways in
diabetic complications and TLR signaling pathways were found.
Especially, the AGE-RAGE signaling pathway in diabetic complications was
the most important pathway in the severe patient. AGE-RAGE interaction
triggers the activation of inflammation-related pathways such as
NF-kappa B signaling.31 CCL2, CXCL8, TNF and IL1B were
included in this pathway and were found to be down regulated in the
severe patient (F3-P3). NF kappa B signaling pathway associated
inflammation has suggested that in this patient the inflammatory
response may be suppressed. Also, NOS3 gene was identified in the
AGE-RAGE signaling pathway and played a role in caspase-3 activation.
In order to identify common differentially expressed genes between
severe patients with Class II mutation and severe patients with Class IV
mutation Venn diagram was created. 13 genes (KCNE1, ACE, CFTR, SFTPB,
CXCR2, CXCL1, CXCL8, IL1B, PLA2G5, HSP90AA1, DUSP1, PTGS2, MBL2) which
were found to be common between Class II and Class IV severe patients
show that their expression change independent of mutation type (Figure
S1). Gene Ontology (GO) analysis showed that the common genes were
involved in neutrophil chemotaxis and regulation, inflammation response,
chemokine signaling pathway and immune response-related processes.
As a result of heat map interpretation, it was an interesting finding
that controls demonstrated similar gene expression profiles with mild
Class IV group with I1234V mutation. F3-P1, which is the mildest sibling
in the Class IV group, has a close relationship with C3. The common up
and down regulated genes between intermediate F3-P2 and severe F3-P3
siblings with Class IV mutation, were associated with hepatic
involvement. In addition, 17 genes (KIT, DEFB1, SNAP23, HSPA4, CLU,
AHSA1, LCN2, HSP90AA1, EZR, HSPH1, CCL2, TCF7L2, FAS, KCNE1, PPP2R4,
SERPINA1 and CXCL2) which showed distinct expression pattern between
F3-P2 and F3-P3 were responsible for cell chemotaxis, inflammation,
immune response, protein folding, stabilization, TNF, chemokine and
IL-17 signaling pathways. Also, it can be concluded that inflammation
and protein processing mechanisms may explain the clinical severity
between intermediate F3-P2 and severe F3-P3 siblings with Class IV
mutation. Another significant finding in the heat map was the close
correlation of mild F1-P1 and severe F1-P2 in Family 1. Genes that
showed a similar expression profile in Family 1, were the ones
responsible for LPS response, cytokine-chemokine activity and protein
folding processing mechanism. On the other hand, 32 genes which were
differentially expressed between mild and severe siblings in Family 1
were associated with hepatic involvement and may have a role in
determining clinical severity.
In order to investigate whether there was an association between
clinical parameters (recurrent lung infection, FEV1%, hepatic
involvement, CFRD) and differentially expressed genes in all CF
patients, correlation analysis was performed. The increase in expression
of 29 genes was found to have a significant correlation with an increase
in severe hepatic involvement and liver inflammation. Among the
significant pathways, AGE-RAGE signaling pathways in diabetic
complications (PRKCE, CCL2, TNF, NOS3, EDN1 and TGFB1) was determined as
the most outstanding one and may be regarded as a prognostic biomarker
for CFLD which is characterized by focal biliary cirrhosis and occurs in
5-10% of patients with CF.32 A recent study performed
by Paranjapye et al. has determined ABCA4, GSTP1, MBL2 and SERPINA1 as
modifier genes for CFLD.33 Also, Debray et al. has
confirmed SERPINA1 as a modifier gene for severe
CFLD.34 Also they concluded that ACE, TGFB1 and CXCL8
could be associated with CFLD severity. However, in our study, we did
not find an association between ABCA4, GSTP1, MBL2 and SERPINA1 genes
and hepatic involvement in CF patients. We also showed that ACE and
TGFB1 had a positive correlation with hepatic involvement.