Dr.

Jolene Fisher
is an Interstitial
Lung Disease
fellow and Clinical
Epidemiology
Master of Science
student at the University of
Toronto. Her research focus is
evaluating outcomes in patients
with interstitial lung disease
using administrative databases.
She completed her Respirology
training in 2013 at the University
of Toronto.
Past newsletters:
Idiopathic Pulmonary Fibrosis:
optimizing the diagnosis and
multi-disciplinary decision-
making by Dr. Gerard Cox, MD,
MB, FRCPCI, FRCPC
IPF: understanding the natural
history and epidemiology of this
fatal disease by Dr. Charlene D.
Fell, MD, MSc, FRCPC, FCCP
How recent randomized
controlled trials inform clinical
treatment of IPF: the therapeutic
trials roller coaster ride by Dr.
Charles K.N. Chan, MD, FRCPC,
FCCP, FACP
Upcoming newsletters:
The next several newsletters will
be based on presentations from
the US "PFF Summit 2013: From
Bench to Bedside" and the ATS
2014 International Conference.
If you wish to receive the
newsletters by email, wish to
subscribe or unsubscribe, or have
any questions, please contact:
Anna Liachenko, MSc
514-435-7860
anna@z-zinc.com
Genetics in Idiopathic Pulmonary Fibrosis: Impact
on Outcome and Patient Management
by Jolene Fisher, MD, FRCPC
Idiopathic pulmonary fbrosis (IPF) is a chronic, fbrosing interstitial lung disease (ILD) of unknown cause.
1
The
median survival is poor, ranging from 2.5 to 3.5 years after the diagnosis.
2
Furthermore, recent data suggests
that the incidence and prevalence of IPF is increasing independently of demographic factors.
3
Smoking, male sex
and environmental exposures have been identifed as risk factors for the disease, however the pathophysiology
remains incompletely understood.
1
For other pulmonary conditions, such as lung cancer and cystic fbrosis,
the study of genetics has provided valuable insights into disease behavior and remarkable advancements in
therapies. Recently, evolving research has identifed genetic factors as an important player in the development
of both sporadic and familial IPF (affecting 2 or more members of the same family). The discovery of genetic
associations in IPF may lead to a better understanding of IPF disease pathogenesis, ability to identify targets
for effective therapies and improved accuracy of individual patient prognostication. This article is based in
part on the information presented by Christine Kim Garcia, MD, PhD, at the Pulmonary Fibrosis Foundation
Summit 2013 From Bench to Bedside.
Tis is a newsletter series in the continuing educational program
designed to bring you articles by leading respirologists who are experts
in fbrosing diseases.
Surfactant protein C mutations
A surfactant protein (SP)-C gene mutation causing pulmonary fbrosis was frst described in 2001 in
2 members of the same family who developed the ILD. Te afected mother was diagnosed with the desquamative
interstitial pneumonia while the daughter was diagnosed with the non-specifc interstitial pneumonia. Both
possessed a heterozygous mutation in the SP-C gene.
4
SP-C was subsequently tested as a candidate gene in a
large kindred with pulmonary fbrosis, involving 14 afected members across 6 generations. A mutation in the
SP-C gene was found in all afected family members but was not present in the 4 unafected members or in
88 controls.
5
Additionally, abnormal staining in the lung tissue of the afected family members suggested alveolar
type II cell injury as a potential mechanism of the disease (Figure 1).
Surfactant protein A mutations
Wang et al. used genome-wide linkage to identify two rare mutations in the gene encoding for pulmonary
SP-A2 that were associated with familial pulmonary fbrosis (FPF) and lung cancer in two kindreds.
6
Te
pathogenesis of these mutations is thought to involve an increased endoplasmic reticulum stress of the type II
alveolar epithelial cells.
7
Telomerase gene mutations
Telomerase is a reverse transcriptase responsible for maintaining telomere length.
8
Mutations in telomerase-
encoding genes have been described in several diseases, including pulmonary fbrosis, and are characterized by
short telomere length.
7
Te relationship between telomerase dysfunction and pulmonary fbrosis was initially
identifed in a kindred with dyskeratosis congenita, a telomerase reverse transcriptase (TERT) mutation and
short telomere length.
9
Four of the 7 afected family members were also diagnosed with pulmonary fbrosis.
Te theory that pulmonary fbrosis is caused by a short telomere length was tested on a separate cohort of FPF
patients who were sequenced for mutations in telomerase genes.
10
Six of 73 kindreds (8%) were found to have
mutations resulting in short telomeres (Table 1). Five of these were TERT mutations and one was a mutation in
the gene encoding for the RNA component of telomerase (TERC). Additional sequencing of TERT and TERC
in over 100 unrelated kindreds with FPF found that 18% had TERT mutations and just under 1% had TERC
mutations.
11
Interestingly, TERT mutations have also been described in the absence of family history of lung
disease in individuals with sporadic ILD.
12
Short telomere length appears to be associated with pulmonary fbrosis even outside of TERT and TERC
mutations. When individuals with these two mutations are excluded from consideration, over 20% of FPF
Learning objectives:
1. Review the current data regarding genetic associations in IPF
2. Discuss the potential role of genetics in IPF diagnosis,
prognostication and management
3. Discuss the role of genetics in the study of IPF disease pathogenesis
Sponsored by an unrestricted educational grant from InterMune.
The Honeycomb Network
THE IPF EXPERT EXCHANGE
See Page 3 for Protein-based biomarkers in IPF: current
evidence and future directions by Deborah Assayag, MDCM.
kindreds and individuals with sporadic pulmonary fbrosis still have
telomere lengths below the 10th percentile.
12

A common MUC5B promoter polymorphism
While mutations in SP-C, SP-A2, TERT and TERC are strongly
associated with the development of pulmonary fbrosis, they are relatively
rare, representing a small proportion of patients with the disease. Using
linkage and fne mapping, Seibold et al. were able to identify a common
polymorphism in the promoter region of mucin 5B (MUC5B) that
was associated with IPF.
13
Te minor allele of the single-nucleotide
polymorphism (SNP) rs35705950, located 3 kb upstream of MUC5B,
was present in 38% of the 492 subjects with IPF and 9% of the 322
controls. Te minor allele of this SNP conferred an odds ratio for disease
of 9.0 for heterozygotes and 21.8 for homozygotes. Te mutation was
also associated with an increased lung expression of MUC5B in both
afected and unafected individuals. However, those with IPF had 14
times higher MUC5B expression in the lung compared to those without
the disease (Figure 2).
Genome wide association study (GWAS) for
pulmonary fbrosis
A case-controlled genome wide association study of 1616 patients
with fbrotic idiopathic interstitial pneumonias and 4683 controls
without the disease was conducted to identify susceptibility loci for
pulmonary fbrosis.
14
TERT, MUC5B and TERC were again identifed,
as well as seven new loci. Tese included the family with sequence
similarity 13, member A (FAM13A) gene at 4q22, the desmoplakin
(DSP) gene at 6p24, the oligonucleotide-binding fold containing 1
(OBFC1) gene at 10q24, the ATPase, class VI, type 11A (ATP11A)
gene at 13q34, the dipeptidylpeptidase-9 (DPP9) gene at 19p13 and
chromosomal regions 7q22 and 15q14-15 (Figure 3). Te authors
suggested that based on this data, genes involving the functions of host
defense, cell-cell adhesion and DNA repair may contribute to the risk of
developing pulmonary fbrosis.
Conclusions
While the genetics of pulmonary fbrosis are complex, evolving data
suggests that they play an import role in IPF disease pathogenesis. As the
understanding of IPF genetics improves, there is increasing optimism
that it will lead to enhanced ability to diagnose IPF (via genetic and
molecular markers), improved prognostication of individual patients
and more efective therapies targeting specifc disease mechanisms.
Previous broad therapies targeting infammatory and fbrotic pathways
have proven disappointing in altering the course of the disease in a
meaningful way. It stands to reason that by taking a personalized
FIGURE 1. Photomicrographs of immunohistochemistry. (A) Lung of normal
adult immunostained for proSP-C. Type II cell shows predominantly focal brown
staining of the cytoplasm adjacent to the lamellar bodies, which are evident as
clear vesicles unstained (arrows)(x175). (B) Lung immunostained for proSP-C,
taken from an explanted lung from FPF. Two cuboidal type II cells show diffuse
brown cytoplasmic staining. No obvious lamellar bodies are seen (x175). Am J
Respir Crit Care Med 2002;165:1322-8.
A B
FIGURE 2. Immunohistochemistry staining of MUC5B in lung tissue from subjects
with IPF and controls. MUC5B distribution in the cytoplasm of the secretory
columnar cells of the bronchi and larger proximal bronchioles in a specimen
of lung tissue from a control subject (A). In subjects with IPF, regions of dense
accumulation of MUC5B were observed in areas of microscopic honeycombing
and involved patchy staining of the metaplastic epithelia lining in the honeycomb
cysts (B). Accumulation was also observed in the mucus plugs within the cysts
(C). N Engl J Med 2011;364(16):1503-12.
B A
C
N Engl J Med 2007;356:1317-26.
TABLE 1. Mutations in Telomerase and Associated Clinical Features of the Six Probands.
Proband
No.
Presenting
Symptom
Smoking
History
Mutation Sex Age (yrs)
TLC FVC
liters (% of predicted value)
DLCO WBC
per mm
3
Hgb
g/dL
Platelet
per mm
3
At
Onset
At Time
of Study
Pulmonary Function
Findings on
Lung Biopsy
Complete Blood Count
AII.1
BIII.5
CII.7
DIII.2
EII.2
FIII.5
M
M
M
F
F
F
77
58
58
48
68
60
81
67
61
49
76
66
4.45 (68)
3.17 (44)
5.28 (69)
N/A
3.25 (68)
2.48 (51)
3.02 (68)
2.10 (44)
3.55 (66)
1.31 (43)
1.69 (47)
1.2 (45)
14.2 (76)
12.5 (49)
12.8 (47)
N/A
12.5 (54)
7.07 (32)
8,800
8,800
8,800
10,800
9,500
6,800
14.0
14.1
16.2
13.8
15.5
12.8
206,000
282,000
201,000
235,000
317,000
218,000
Dyspnea
Cough
Dyspnea
Dyspnea
Dyspnea
Dyspnea
None
None
30 pack-years
32 pack-years
None
None
Usual interstitial
pneumonia
Usual interstitial
pneumonia
Usual interstitial
pneumonia
Idiopatic interstitial
pneumonia
Usual interstitial
pneumonia
Usual interstitial
pneumonia
hTERT
CTGCAG
Leu55Gln
hTERT
IVS1+1 GA
hTERT
codon 112
del C
hTERT
IVS9-2 AC
hTERT
ACGATG
Thr1110Met
hTR
98 GA
approach and using IPF genetics to guide therapeutic development,
efective treatments may become available. Currently, genetic testing and
molecular markers in IPF are generally unavailable outside of research
setting. However, as the body of genetic research in IPF develops, the use
of genetic results in clinical practice will likely increase.
MUC5B
DSP
TERT
7q22
15q14-15
DPP9
17q21
20
15
10
5
0
1 2 3 4 5 6 7 8 9
1
0
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
1
9
2
0
2
1
2
2
x
Chromosome
-
l
o
g
1
0
(
p
)
FIGURE 3. Genome wide association study results at 439 828 single-nucleotide
polymorphisms (SNPs) with 1616 fbrotic idiopathic interstitial pneumonias
patients and 4683 controls under an additive model. SNPs above the red line
were genome wide signifcant at p < 5 x 10
-8
. Nat Genet 2013;45:613-20.
REFERENCES:
1. Raghu G, et al. An offcial ATS/ERS/JRS/ALAT statement: idiopathic pulmonary
fbrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care
Med 2011;183(6):788-824.
2. American Thoracic Society/European Respiratory Society International
Multidisciplinary Consensus Classifcation of the Idiopathic Interstitial Pneumonias. Am J
Respir Crit Care Med 2002;165:277-304.
3. Navaratnam V, et al. The rising incidence of idiopathic pulmonary fbrosis in the U.K.
Thorax 2011;66(6):462-7.
4. Nogee LM, et al. A mutation in the surfactant protein C gene associated with familial
interstitial lung disease. N Engl J Med 2001;344(8):573-9.
5. Thomas AQ, et al. Heterozygosity for a surfactant protein C gene mutation associated
with usual interstitial pneumonitis and cellular nonspecifc interstitial pneumonitis in one
kindred. Am J Respir Crit Care Med 2002;165(9):1322-8.
6. Wang Y, et al. Genetic defects in surfactant protein A2 are associated with pulmonary
fbrosis and lung cancer. Am J Hum Genet 2009;84(1):52-9.
7. Garcia CK. Idiopathic pulmonary fbrosis: update on genetic discoveries. Proc Am
Thorac Soc 2011;8(2):158-62.
8. Greider CW, Blackburn EH. Identifcation of a specifc telomere terminal transferase
activity in tetrahymena extracts. Cell 1985;43:405-13.
9. Armanios M, et al. Haploinsuffciency of telomerase reverse transcriptase leads
to anticipation in autosomal dominant dyskeratosis congenita. Proc Natl Acad Sci USA
2005;102(44):15960-4.
10. Armanios MY, et al. Telomerase mutations in families with idiopathic pulmonary
fbrosis. N Engl J Med 2007;356(13):1317-26.
11. Diaz de Leon A, et al. Telomere lengths, pulmonary fbrosis and telomerase (TERT)
mutations. PLoS One 2010;5(5):e10680.
12. Cronkhite JT, et al. Telomere shortening in familial and sporadic pulmonary fbrosis.
Am J Respir Crit Care Med 2008;178(7):729-37.
13. Seibold MA, et al. A common MUC5B promoter polymorphism and pulmonary
fbrosis. N Engl J Med 2011;364(16):1503-12.
14. Fingerlin TE, et al. Genome-wide association study identifes multiple susceptibility
loci for pulmonary fbrosis. Nat Genet 2013;45(6):613-20.
Protein-based biomarkers in IPF: current evidence
and future directions
by Deborah Assayag, MDCM
The pathogenesis of idiopathic pulmonary fbrosis (IPF) is still incompletely understood, however new insights
suggest that alveolar epithelial dysfunction and abnormal fbroblast proliferation leads to interstitial fbrosis
characteristic of IPF.
1
Understanding the biology of the disease can help identify biomarkers which can be used
clinically and for research in IPF. Biomarkers are measurable surrogates for meaningful outcomes in IPF. Clinical
utility includes help in establishing the diagnosis and predicting disease progression.. Biomarkers can also
potentially contribute to the early diagnosis of subclinical interstitial lung disease (ILD), and identify those at risk
of developing the disease. These biomarkers are present in both the peripheral blood and in bronchoalveolar
lavage fuid of IPF patients. They are different targeted molecules such as proteins, lymphocyte subsets and
circulating fbroblasts. This article is based in part on the presentation by Dr. Ivan O. Rosas at the Pulmonary
Fibrosis Foundation Summit 2013 From Bench to Bedside.
Dr. Deborah
Assayag is a
respirologist.
In 2014, she
completed
a fellowship
program in interstitial lung
disease at the department of
Pulmonary Medicine at the
University of California, San
Francisco. She previously
completed the Respiratory
Medicine Residency Training
Program and the Internal
Medicine Residency Training
Program at McGill University.
Dr. Assayag has published
several peer-reviewed articles
on Interstitial Lung Disease
and has presented at a number
of international conferences.
Learning objectives:
1. Review the best studied protein biomarkers in IPF
2. Discuss their role in identifying early stages of the disease,
establishing disease progression and predicting survival
Peripheral blood proteins may predict IPF prognosis and disease progression
Select peripheral blood proteins are increased in the serum of IPF patients compared to controls and have
been associated with the presence of disease and poor prognosis. Te amount of protein biomarkers is correlated
with IPF disease progression. Table 1 summarizes the evidence for these biomarkers.
CC Chemokine ligand 18 (CCL18)
CCL18 is a small protein derived from alveolar macrophages. It is a marker of macrophage activation. Studies
found that CCL18 is increased in the bronchoalveolar lavage fuid of IPF patients compared to healthy controls.
Also, CCL18 is negatively correlated with the severity of pulmonary physiologic impairment, specifcally the
total lung capacity (TLC) and the difusion of the lung for carbon monoxide (DLCO).
2
In a longitudinal
study, baseline levels of serum CCL18 were correlated with worsened TLC and forced vital capacity (FVC) at 6
months.
3
Baseline levels of CC18 were associated with survival. Patients
with low levels of serum CCL18 (<150 ng/mL) had signifcantly better
survival than those with CCL18 levels >150 ng/mL. Te risk of mortality
remained signifcantly elevated in the low CCL18 group after adjusting
for age, sex and pulmonary function.
3

Matrix Metalloproteases 1 and 7 (MMP1, MMP7)
Matrix metalloproteases are a family of proteins involved in the
breakdown of extracellular matrix. MMP1 and MMP7 genes were found
to be overexpressed in the lung tissue of IPF patients. Concentrations of
MMP1 and MMP7 were also signifcantly higher in bronchoalveolar
lavage fuid and in the serum of IPF patients compared to controls.
4,5

Tese controls included patients with other lung diseases such as
chronic obstructive pulmonary disease, sarcoidosis and hypersensitivity
pneumonitis. In this study, high concentrations of serum MMP7
(>1.99 ng/mL) correctly classifed 69 out of 74 patients (93%) as
having IPF. In addition, levels of MMP7, but not of MMP1, negatively
correlated with the disease severity as measured by FVC and DLCO (%
predicted). In their model, for each increase of 1 ng/mL in serum MMP7,
FVC and DLCO (% predicted) both decreased by about 4 percentage
points. In another study, high levels of MMP7 were associated with a
decreased transplant-free survival and a two-fold increase in the risk of
death, which persisted after adjusting for age, sex, and baseline FVC.
6

Surfactant Proteins A and D (SP-A, SP-D)
Pulmonary surfactants are lipoproteins synthesized by alveolar
epithelial cells. Tey reduce surface tension in the alveoli leading to an
easier lung expansion. Te diagnostic and prognostic value of surfactant
proteins A and D have been examined in IPF. Both SP-A and SP-D are
increased in the BAL fuid of IPF patients.
7,8
Notably, these surfactants
are also increased in patients with the acute respiratory distress syndrome.
As well, SP-A is increased in the BAL of patients with ILDs (other than
IPF).
9
In the same study, the concentration of both biomarkers was also
associated with increased hazard ratios of death (HR 1.73 and 2.04 for
SP-A and SP-D, respectively). Another study showed that serum SP-A,
but not SP-D, levels were associated with an increased risk of death (3
fold increase for each 49 ng/mL increase in SP-A).
10
Krebs von den lungen-6 (KL-6)
KL-6 is a glycoprotein expressed at the surface of alveolar and
bronchial epithelial cells. It is thought to promote migration and
proliferation of fbroblasts. Serum KL-6 levels are signifcantly elevated
in IPF compared to healthy controls.
8
However, elevated KL-6 are also
associated with other ILDs. KL-6 levels greater than 465 U/mL provide
the best sensitivity, specifcity and diagnostic accuracy for IPF compared
to SP-A and SP-D.
8
Composite protein biomarkers
Combinations of blood proteins can potentially be more useful in
predicting prognosis and disease progression, compared to individual
biomarkers. A combination of 5 plasma proteins (MMP1, MMP7,
MMP8, IGFBP1 and TNFRSF1A) was highly specifc and sensitive
for a diagnosis of IPF, compared to MMP7 alone.
4
In another study,
high concentrations of MMP7, ICAM1, IL8, VCAM1 and S100A12
predicted an overall poor IPF patient survival.
6
MMP7 and SP-A together
were signifcant predictors of survival in IPF in another study. Patients in
whom both biomarkers were elevated had a signifcantly shorter survival
and greater decline in lung function, compared to those with only one
elevated biomarker, or low levels of both.
11
Conclusion
Several peripheral blood proteins originating from the alveolar
and bronchial epithelium are elevated in IPF and may have prognostic
signifcance. A combination of these proteins is likely more useful than
individual proteins in establishing the IPF diagnosis, predicting patient
survival and IPF disease progression. At this time however, protein
biomarkers are not routinely measured by clinical laboratories and it
is still unclear how to use them in addition to the standard diagnostic
procedures (such as pulmonary function tests, CT scans and surgical lung
biopsies). Further research will reveal whether protein biomarkers will
be useful in diagnosing pre-clinical or asymptomatic IPF in susceptible
patients, such as those with familial predisposition or smokers.
REFERENCES:
1. Selman M, et al. Role of Epithelial Cells in Idiopathic Pulmonary Fibrosis. Proc Am Thor
Soc 2006;3:364-72.
2. Prasse A, et al. A vicious circle of alveolar macrophages and fbroblasts perpetuates
pulmonary fbrosis via CCL18. Am J Respir Crit Care Med 2006;173: 781-92.
3. Prasse A, et al. Serum CC-chemokine ligand 18 concentration predicts outcome in
idiopathic pulmonary fbrosis. Am J Respir Crit Care Med 2009;179(8):717-23.
4. Rosas IO, et al. MMP1 and MMP7 as potential peripheral blood biomarkers in idiopathic
pulmonary fbrosis. PLoS Med 2008;5:e93.
5. Fujishima S, et al. Production and activation of matrix metalloproteinase 7 (matrilysin 1)
in the lungs of patients with idiopathic pulmonary fbrosis. Arch Pathol Lab Med
2010;134:1136-42.
6. Richards TJ, et al. Peripheral blood proteins predict mortality in idiopathic pulmonary
fbrosis. Am J Respir Crit Care Med 2012;185:67-76.
7. Phelps DS, et al. Increased surfactant protein-a levels in patients with newly diagnosed
idiopathic pulmonary fbrosis. Chest 2004;125(2):617-25.
8. Ohnishi H, et al. Comparative Study of KL-6, Surfactant Protein-A, Surfactant Protein-D,
and Monocyte Chemoattractant Protein-1 as Serum Markers for Interstitial Lung Diseases.
Am J Resp Crit Care Med 2002;165:378-81.
9. Greene KE, et al. Serum concentrations of surfactant proteins a and d predict mortality
in patients with idiopathic pulmonary fbrosis. Chest 2001;120(1_suppl):S72.
10. Kinder BW, et al. Serum surfactant protein-a is a strong predictor of early mortality in
idiopathic pulmonary fbrosis. Chest 2009;135:1557-63.
11. Song JW, et al. Blood biomarkers MMP-7 and SP-A: predictors of outcome in
idiopathic pulmonary fbrosis. Chest 2013;143:1422-9.
TABLE 1. Biomarkers and their role in IPF
Biomarker Role as biomarker Clinical correlates Compartment References
CCL18
MMP1
MMP7
SP-A
SP-D
KL-6
Diagnostic
Prognostic
Diagnostic
Diagnostic
Prognostic
Diagnostic
Prognostic
Diagnostic
Prognostic
Diagnostic
Increased in serum and BAL of IPF patients
Correlates with increased mortality and disease progression (TLC, FVC and DLCO)
Increased in serum and BAL of IPF patients
Increased in plasma and BAL of IPF patients
Negatively correlated with pulmonary physiology (FVC, DLCO)
Increased in BAL and serum of IPF patients
Associated with increased risk of death
Increased in BAL and serum of IPF patients
Associated with increased hazard of death
High sensitivity and specifcity for IPF diagnosis
[2, 3]
[4]
[4, 5, 11]
[7, 9, 10]
[7, 9]
[8]
BAL
Serum
BAL
Serum
BAL
Serum
BAL
Serum
BAL
Serum