behcet`s disease study status report 2012final(2)

Center for Biomarker Research
Study of Behçet’s Disease
Overview
Behçet’s Disease (BD) is a chronic, multisystem inflammatory disease which occurs in
genetically susceptible individuals as a result of poorly understood environmental challenges.
These environmental challenges may include infectious agents, environmental toxins, or other
unknown entities which appear to cause a harmful and recurrent inflammatory reaction by the
patient’s immune system. The inflammatory reaction often results in recurrent genital ulcers,
skin lesions, uveitis1, as well as inflammation in large vessels, the central nervous system, and
the gastrointestinal tract. Unfortunately, Behçet’s Disease takes years to diagnose and has
unpredictable remissions and relapses throughout a patient’s life. The disease is rare in the
United States, ~5 cases /100,000, but is more prevalent in the Eastern Mediterranean countries.
Onset usually occurs between the ages of 15 and 45 with equal distribution between men and
women, and progresses as a series of remissions and relapses. Currently there are no
biomarker tests for BD and diagnosis is based on clinical manifestations which are quite varied
and not specific for BD. Without biomarkers, remissions and relapses, which occur over the
lifetime of the patient, cannot be effectively monitored. This study is designed to develop
specific diagnostic tests to help patients and the medical community better manage the disease.
Research in better diagnostic biomarkers for rare diseases, which are often difficult to diagnose,
is limited by the availability of patient samples. Keck Graduate Institute of Applied Life Sciences
(KGI) and the American Behçet’s Disease Association (ABDA) are using social networking to
obtain samples directly from patients in order to identify differences in serum proteins between
BD patients and controls. ABDA obtains signed informed consent forms, under the Center for
Biomarker Research (CBR) approved IRB, from patients interested in participating in this study.
ABDA then acts as the firewall between CBR and patient participants. Patients simply ask for an
extra tube of serum and whole blood during their normal blood draw and the samples are
shipped overnight to CBR. Patient and normal serum are labeled with different tags, mixed and
analyzed by gel electrophoresis. Bands that show differential migration will be further analyzed
to identify the exact protein present in each band. Based on these findings, immunoassays,
which are more precise, rapid and quantitative than gel electrophoresis, will be developed and
used in clinical trials to validate their effectiveness as biomarkers to better manage patients with
BD.
Behçet’s Disease
Behçet’s Disease (BD) was first described in 1931 by the Greek ophthalmologist Benedict
Adamantiades (1). In 1937 Hulusi Behçet, a Turkish dermatologist, gave a more complete
clinical description of the disease (2). This chronic vascular inflammation is characterized by
oral and genital ulcers, anterior uveitis, arthritis, skin and ocular lesions, gastrointestional
bleeding and neurological disorders (3). BD is more prevalent in Turkey, Iran, Saudi Arabia,
Japan, Israel and China than western European countries and is believed to have its origins
along the Silk Road.
Currently, doctors primarily rely on clinical symptoms to diagnose Behçet’s patients.
Unfortunately, many of these clinical symptoms are similar to other diseases, such as lupus and
multiple sclerosis. This challenge often leads to inaccurate diagnoses, extended time course for
diagnosis, and frustration for patients and their families. In 1990 the International Study Group
for Behçet’s Disease recommended a set of clinical criteria to diagnose BD: the presence of oral
1
Uveitis refers to inflammation of the uvea, a portion of the eye which includes the iris, ciliary body, and choroid.
ulcers and at least two of the following; skin lesions, vascular lesions, positive pathergy test,
genital ulcers or ocular lesions (4). These were modified in 2001 to remove the requirement for
oral ulcers and to place more weight on genital lesions and ocular lesions (5). These changes
increased sensitivity (percentage of true positives correctly diagnosed) from 86.2% to 96.7%
and specificity (percentage of true negatives correctly diagnosed) from 91% to 94.5% for the
diagnosis of BD. Unfortunately, in spite of the high sensitivity and specificity, diagnosis of BD
based on clinical symptoms may take years, delaying treatment and causing tissue damage due
to the recurring vascular inflammation. Consequently, the diagnostic and medical community
has been looking for improved Behçet’s specific diagnostic biomarkers for several decades.
Currently, the field believes that one potential cause of Behçet’s Disease is the inappropriate
overreaction of a patient’s immune system to a pathogen or other immune threat, resulting in
excessive and harmful inflammation of tissues and veins. The pattern of remission and relapse
is variable between patients and no specific infectious agents have been reproducibly isolated
from BD patients (6). This has led to the concept that a pathogen may act as a “trigger” to
activate the immune system leading to systemic inflammation. The trigger may be different for
different patients. Candidate triggers include herpes simplex virus type 1 (7), Streptococcus
sanquis (8), hepatitis virus, Borrelia burgdorferi, Escherichia coli, and Saccharomyces fungus
(3). Heat shock proteins (HSP) have also been implicated as triggers for BD. Human HSP 60
has about 50% homology with HSP 65 of mycobacteria and T cell epitope mapping identified
four HSP peptides in BD patients (9). A summary of the pathogenesis of BD is illustrated below
(Figure 1).
Figure 1. Pathogenesis of BD (10-13)
The geographical distribution of patients along the Silk Road, the higher prevalence in Asia and
Mediterranean countries, and familial pediatric clustering in different ethnic groups support a
genetic basis for the pathogenesis of BD. The strongest genetic association with BD is within
the human leukocyte antigen (HLA) region on the short arm of the 6 th chromosome. This region,
about 3.6 MB in length, codes for the Major Histocompatibility Complex (MHC) family of
receptors and proteins of the immune system. MHC genes are divided into three classes: class I
contains genes for the α-chain of HLA-A,-B and -C; class II contains genes for the α- and βchains of HLA-DR, -DP and -DQ; and class III contains genes that code for complement
proteins and some cytokines. All of these molecules are important components of the immune
system and polymorphisms in these genes have been associated with numerous autoimmune
diseases. MHC HLA-B has been shown to be strongly associated with BD in numerous studies
(14). There are many polymorphisms in HLA-B, and the HLA-B510101 subtype has the highest
association with BD. However, the HLA-B510101 subtype is only present in about 60% of BD
patients and is present in about 50% of control subjects (15).
Research Approach
Research to develop diagnostic biomarkers for rare diseases is often difficult because of the
limited availability of patient samples. A high school student, KGI professor and the American
Behçet’s Disease Association (ABDA) used social networking to obtain patient samples to
identify unrecognized differences in serum proteins between patients and controls. ABDA
obtains signed informed consent forms, under the CBR approved IRB, from patients interested
in participating in this study. ABDA then acts as a firewall between CBR and patient, providing
anonymity for the patient. If patients contact CBR directly, Jim Osborne serves as the firewall
between patients and CBR laboratory researchers. Patients ask for an extra tube of serum and
whole blood during their next normal blood draw and the samples are shipped prepaid overnight
to CBR (Figure 2). Patient and normal serum are labeled with different fluorescent dyes, mixed
and analyzed by 2D electrophoresis. Bands that show differential migration will are analyzed by
Mass Spec to identify potential diagnostic biomarkers. Based on the findings with serum, we
may want to investigate nucleic acids and cells in whole blood. In that case, ABDA will interface
with patients and request additional samples of whole blood. Results of the study will be shared
with the scientific community and communicated to patient families through ABDA.
The collaboration with ABDA has resulted in 92 Behçet’s Disease patients and their unaffected
family members participating in the study. CBR continues to solicit samples from patients and
their unaffected family members. CBR has prepaid overnight FedEx shipping of samples to KGI.
ABDA is the firewall between CBR and patient identity and maintains signed informed consent
forms so we may obtain additional samples from selected patients. ABDA sends a redacted
consent form to CBR for tracking specific samples. CBR maintains a file with patient gender,
age, diagnosis and current treatment and symptoms. The following flow chart is sent to patients
to aid in sample processing.
Figure 2. ABDA-Patient-KGI sample flow chart
As we discussed earlier, Behçet’s disease is a likely the result of the complex interplay between
external immune activation by bacterial, fungal or viral pathogens or other environmental
stimulants and the inappropriate response by the patient’s immune system(16). At some point
in the disease, the patient’s immune system targets “self” through an inappropriate activation or
unsuccessful immune suppression resulting in vasculitis, thrombosis or other “self-targeted”
destruction takes. CBR is initially investigating two aspects of the valuable patient samples
provided through our partnership with ABDA. Firstly, an aggressive search for serum protein
differences between disease patients and normal populations is being pursued and secondly,
both mRNA differences and protein differences within the T-cell population of diseased and
normal samples will be examined. By targeting both serum and cellular components of the
patient and normal population general markers of disease (in serum) will be pursued.
Additionally, we will examine a central function (T cells) likely to be involved in the immune
communication causing Behcet’s disease.
The systematic search for biomarkers in serum is a challenging activity. Both serum and
plasma contain millions of different proteins and protein variants. Furthermore, the highly
complex serum or plasma proteome is known to have a quantitative dynamic range of over 10 10
making it difficult to identify proteins like IL-6 at 40 pg/ml in the presence of albumin at 40
mg/ml(17), see Figure 3 (18).
Figure 3. Abundance of plasma proteins (18)
Findings
Serum or plasma proteins may show specific signs of disease with changing concentration of
cytokines, chemokines or inflammatory markers or they may simply reflect the effects of the
therapeutic treatments attempting to suppress the disease. None-the-less, it is essential to
examine the changes which take place in serum and relate these changes to the disease state.
To approach this challenge we will rely on two proteomic approaches: 1, Two-dimensional
differential gel analysis using fluorescent cyanine dyes with mass spectrographic analysis (2DDIGE)(19); and 2, filtration isolated low-molecular weight serum proteins followed by trypsin
digestion and two-dimensional chromatographic separation (using both ionic and hydrophobic
interaction) followed by mass spectrographic analysis (LMW-2D)(20,21). It is hoped that these
approaches will allow CBR to aggressively identify changes in both high- and low-molecular
weight proteins.
2D-DIGE: Two-dimensional differential gel analysis (2D-DIGE) using fluorescent cyanine dyes
combined with immune-depletion of high abundance proteins is now considered a standard
approach for differential analysis of proteins found at moderate concentrations. These studies
use 2D-DIGE with and without immunodepletion of high abundance proteins such as albumin
and IgG. Gel scans will be performed using the Typhoon Trio (GE/Amersham) instrument and
difference analysis will be performed using DeCyder software (GE/Amersham). Excised gel
pieces containing proteins of interest will be dehydrated, reduced and alkylated prior to in gel
digestion, elution and desalting with a C-18 ZipTip (Millipore). NSI-MS spectra and MS/MS will
be deconvoluted to identify proteins of particular interest (Thermo Finnigan LCQ Deca). By
using immunodepletion, the serum proteome will be examined to a moderate depth for proteins
greater than 15,000 daltons.
Figure 4. Serum Protein Analysis Overview.
One of the challenges in analyzing BD serum is that the disease is cyclic in nature, i.e. recurring
relapses and remissions change serum protein profiles. Another issue is selection of the proper
normal serum for comparison. In the 2D image below (figure 5), protein profiles of a randomly
selected normal serum were compared with the serum from a patient with Magic Syndrome, a
very rare combination of BD and relapsing polychrondritis, a chronic inflammation of cartilage.
Some proteins were the same (yellow) in these samples, however major differences in normal
(green) and patient (red) protein profiles were also observed. These results are interesting but
have little use in selecting specific biomarkers for further study.
Figure 5. Behçet’s patient CBR #2 (stained red, Cy5) against normal female control (stained
green, Cy3). IEF (3-10 NL, Immboline dry strip, Amersham GE). MW markers are labeled on
the right.
The 2D image shown in Figure 6 (below) represents the serum of a female Behçet’s patient
which was differentially labeled (Cy5) and co-migrated with the serum of her unaffected sisters
(Cy2, and Cy3). The overlapping protein profiles (yellow) dominate the image but some
differences can be seen in the diseased patient profile (red). One low molecular weight protein
shows a significant difference in serum concentration (the red spot 1 in 3d). This spot is a much
better candidate for additional studies.
We are now analyzing spots on this gel and others by mass spec to identify protein differences
that are shared between BD patients. Precise identification of candidate biomarkers will require
removal of high abundance proteins prior to 2D-DIGE as indicated in proteomic approach 2
summarized above. This will enrich the samples in low molecular weight proteins, like the one in
the above gel, and give higher protein concentrations in spots selected for mass spec analysis.
Higher protein concentrations allow more accurate identification of peptide fragments in
selected spots.
6a
6b
6c
6d
Figure 6. Behçet’s patient CBR #24 (3a stained red, Cy5) against unaffected sisters, (CBR#23,
3b stained blue, Cy2; CBR#27, 3c stained green, Cy3) and the combined image, 3d. The inset
is an image of spot 1 from a software program we use to quantify differences in amounts of
protein in the samples.
Jamie Liu with the Mass Spec
Craig Adams
Jim Osborne
Jim Osborne, Maggie King analyze data
Emily Putnam John Cvitkovic Alice Lai
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