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The role of infections in Behçet disease and neuro-Behçet syndrome
Monica Marta1, Ernestina Santos2,5, Ester Coutinho2,6, Ana Martins Silva2,4,5, João Correia4,
Carlos Vasconcelos4 e Gavin Giovannoni1
1. Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary
University London, 4 Newark Street, London E1 2AT, UK
2. Neurology Department, Centro Hospitalar do Porto, Largo Prof. Abel Salazar, 4099-001
Porto, Portugal
3. Unidade de Imunologia Clínica, Centro Hospitalar do Porto, Largo Prof. Abel Salazar, 4099001 Porto, Portugal
4. Unidade Multidisciplinar de Investigação Biomédica, Instituto de Ciências Biomédicas de
Abel Salazar, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
5. Present address: Nuffield Department of Clinical Neurosciences. Level 6, West Wing, John
Radcliffe Hospital, Oxford OX3 9DU, UK
Corresponding author: Monica Marta, MD, PhD
[email protected]
Neuroimmunology Unit, Neuroscience and Trauma Centre
Blizard Institute, Queen Mary University London
4 Newark Street, Whitechapel
London E1 2AT
Phone no: +44 20 78822677
Fax no: +44 20 78822180
Ernestina Santos: [email protected]
Ester Coutinho: [email protected]
Ana Martins Silva: [email protected]
João Correia: [email protected]
Carlos Vasconcelos: [email protected]
Gavin Giovannoni: [email protected]
Abstract
Infections are considered an environmental trigger for exacerbations of immune-mediated
diseases.
We aimed to establish if common viral infections could be identified as a precipitant of Behçet
disease (BD) with or without neurological involvement and to assess the variability of the
immune response to common viruses. We also investigated whether cytokines and chemokines
could be markers of neurological involvement. Finally, we explored if anti-basal ganglia
antibodies (ABGA) would be associated with neurological involvement in BD. Our study
included 14 individuals with BD with neurological involvement (neuroBehçet syndrome - NBS),
16 individuals with BD without neurological involvement and 18 healthy controls (HC).
Overall we found a tendency for increased levels of anti-viral IgG antibody levels in BD, more
evident in NBS patients versus HC. Epstein-Barr viral-capsid antigen IgG titres were increased
in NBS patients versus other BD patients (p=0.032). Anti-measles antibody titres induced by
vaccination were similarly elevated. ABGA were not detected in the serum of our cohort. Raised
levels of serum IL-8 in some BD patients did not reflect clinical activity or severity.
In conclusion, there was evidence for a polyclonal immune activation rather than a specific virus
effect in the sera of individuals with BD or NBS.
Keywords: Behçet, infections, EBV, IL-8
Abbreviations: BD: Behçet disease; NBS: neuroBehçet syndrome; CSF: cerebro-spinal fluid;
MS: multiple sclerosis; ABGA: anti-basal ganglia antibodies; CNS: central nervous system; BBB:
blood-brain barrier; EBV: Epstein-Barr; VZV: varicella zoster; CMV: cytomegalovirus; HSV-1:
herpes simplex type 1; ASO: antistreptolysin O; VCA: viral capsid antigen; EBNA-1: EBV
Nuclear Antigen-1
1. Introduction
NeuroBehçet syndrome (NBS) is defined as the presence of neurological symptoms associated
with signs and/or with imaging and/or abnormal cerebro-spinal fluid (CSF) in an individual who
fulfils the Diagnostic Criteria for Behçet Disease (BD) 1, after exclusion of other possible causes
2
.
Neurological involvement ranges from 3.2 to 49% in BD series and may present as headache
(the most frequent initial neurological symptom), meningoencephalitis, multiple sclerosis (MS)–
like illness, stroke, pseudotumor cerebri, organic confusional syndrome or a combination of the
above 3. Neurologic involvement is one of the most serious complications, leading to severe
disability and ensuing high fatality rate.
BD is a multisystem vasculitis that affects small vessels causing focal or multifocal intraparenchymal disease in the central nervous system (CNS) 4,5; it also affects the peripheral nerves
in a distal and symmetrical way or as mononeuritis multiplex. Frequently, BD involves large
vessels presenting with thrombosis of the major cerebral venous sinuses 6.
BD patients have a preferential genetic background with a significant association with
HLA-B51 (B101) and the MICA6 allele in most populations. But apart from this genetic
susceptibility, the aetiology remains unknown 7.
An important role is recognized for infections acting as environmental triggers in the
induction or promotion of immune-mediated diseases in genetically predisposed individuals 8.
Further, infections and stress may lead to disruption of the blood-brain barrier (BBB) and lead to
neurological involvement 9,10. Common virus such as Epstein-Barr (EBV), varicella zoster
(VZV), cytomegalovirus (CMV), herpes simplex type 1 (HSV-1) or measles cause infections that
range from clinically asymptomatic to life threatening, mostly when they occur in healthy infants
or in immunocompromised individuals respectively 11. The indirect effects of the viral infection,
either due to the long-term presence of latent virus or to the large proportion of the immune
system they engage, are associated with autoimmunity through mechanisms like molecular
mimicry, polyclonal activation or superantigens. These are the mechanisms that in some cases
link infections to autoimmunity, and that counter aspects of the hygiene hypothesis 12.
Several studies have associated BD with infections, in particular streptococcal infections
but also altered microbiota of the gut 13. Streptococcal infection is usually confirmed by culture
or by demonstrating a raised antistreptolysin O (ASO) or anti-DNAse B antibody titre. Further,
acquired hypersensitivity to streptococcal antigens plays an important role in the aetiopathology
of BD. Persistently high ASO titers in BD patients could indicate that streptococcal infections
such as pharyngitis are related to BD symptoms like erythema nodosum-like lesions 14.
Group A beta-hemolytic streptococcal infections cause immune-mediated delayed
rheumatic fever in a proportion of individuals and Sydenham's chorea is part of that phenotype.
A type of anti-neuronal antibodies, anti-basal ganglia antibodies (ABGA), are associated with a
wide spectrum of post-streptococcal neuropsychiatric disorders 15. The possible aberrant immune
response may be triggered by streptococcal infection and the auto-antibodies cross-react with
human brain tissue and some individuals have dopamine-2 receptor antibodies 16,17.
The overall aim of the study was to identify an infection that would be associated with
BD with or without neurological involvement. In particular, we looked at (a) common viral
infections as possible precipitants or (b) a variation in the immune response to a common viral
agent, (c) cytokines and chemokines that may play a role or be a marker of permeability of the
BBB and finally at (d) the possibility that streptococcal infection could elicit the presence of
ABGA, which have been associated with neurological syndromes.
2. Methods
2.1 Sample collection and ethical approval
The study was approved by the Ethics Committee of Hospital Santo Antonio, Centro Hospitalar
do Porto, Portugal. Every participant signed an Informed Consent Form. Clinical and
epidemiological data of individuals with BD only and NBD followed in the Clinical
Immunology Unit and Neuroinflammation Clinic was retrospectively compiled. There was an
independent healthy control group.
2.2 Measurement of serum immunoglobulin G (IgG) antibodies to EBV (EBNA1 and VCA),
VZV, CMV, measles and HSV-1
Cross-sectional measurement of serum immunoglobulin G (IgG) antibodies to EBV (EBNA1:
Epstein–Barr virus nuclear antigen and VCA: viral capsid antigen), VZV, CMV, measles and
HSV-1 was done with commercial enzyme linked immunoassays. Control and standard samples
(supplied) and samples diluted 1:100 (1:400 for EBNA1 and 1:1000 for VCA) were added to the
microtest strips pre-coated with the antigen (supplied) and incubated for one hour at 37C in
moist chamber and washed thoroughly; the anti-human-IgG-alkaline phosphatase-conjugate
solution was then added, incubated for 30 minutes in the same conditions and washed
thoroughly; the p-nitrophenyl phosphate substrate solution was then added and incubated for up
to 30 minutes and finally HCl was used to stop the reaction. The optical density of the plate was
read at 405nm.
2.3 Measurement of serum cytokines
Cross-sectional measurement of 11 serum cytokines: IL (interleukin)-2, IFN-gamma, IL-12p70
and TNF-beta (type-1 cytokines); IL -4, IL-5, IL-6, IL-10 and IL-13 (type-2 cytokines), IL-1b,
IL-8 and TNF with a flow cytometry multiplex platform (Human Th1/Th2 11plex FlowCytomix
Multiplex, Bender MedSystems, eBioscience, Austria) 18 according to suppliers instructions. In
brief, the bead mixture and the biotin-conjugate mixture were added to the samples, standards
and blank tubes, mixed and incubated at RT for one hour protected from the light. Buffer was
added to all tubes and spun then the supernatant was discarded leaving 100μL. Streptavidin-PE
solution was added to all tubes, mixed and incubated protected from the light for one hour.
Buffer was added, spun and the supernatant was discarded twice. It was then analysed in the flow
cytometer. The bead mixture included beads of two different sizes (4 and 5 μm) and each had 9
or 11 populations internally dyed with different intensities of a fluorescent dye for individual
detection and coated with antibodies specifically reacting with the analyte.
2.4 Detection of ABGA in the sera
Detection of ABGA in the sera by Western blotting was described elsewhere 19. In brief, human
basal ganglia with no pathology were physically disrupted and homogenized with protein
extractor and protease inhibitors (Sigma-Aldrich Co., UK). The protein fraction was then
collected and stored at −80 °C until required. The homogenate and a molecular weight ladder
were loaded onto a Nu-Page gel (Invitrogen, Life Technologies Ltd, UK) and electrophoresis
was run at 200V, 120mA and 25W for about 1 hour. The proteins were blotted onto
nitrocellulose membranes (Bio-Rad Laboratories, Inc., UK) and blocked with 2% milk proteins
for 2 hours. Samples were diluted at 1:300, applied to the blot with the help of a sample manifold
saving one well for the mix of control antibodies. The membranes were incubated overnight at 4
°C and after ten wash cycles and incubated for 2 hours with rabbit anti–human IgG, FAB
fragments conjugated with horseradish peroxide diluted at 1:1,000 (Dako, Cambridge, UK).
After washing, the substrate 4-chloro-1-naphthol (Sigma-Aldrich Co., UK) was added and the
blot was allowed to develop for 20 minutes.
2.5 Statistical analyses
Mann-Whitney without corrections for multi-testing, were done with GraphPad Prism 5.
3. Results
3.1 Characteristics of the patients and clinical manifestation of BD and NBS
The group studied originated from the same Clinical Immunology Unit and Neuroimmunology
Clinic and included 14 individuals with BD and neurological involvement (NBD) and 16
individuals with BD. The clinical and radiological characteristics of the NBD patients were
detailed in a previous publication 20. The neurological syndrome included cognitive impairment,
visual defects, hemiparesis (2 patients), aseptic meningitis, psychosis, meningoencephalitis
involving brain stem (2 patients) or hemispheres (2 patients), cerebellar syndrome (2 patients),
cerebral venous thrombosis (CVT) and vasculitic polyneuropathy. All individuals except the one
with cerebro-venous thrombosis had multiple small hyperintense lesions on T2/FLAIR
predominantly in the meso-diencephalic and ponto-bulbar regions.
Serum samples were collected in a cross-sectional way, independent of the activity of disease or
treatment and a healthy control group from relatives and friends of patients attending the
Neuroimmunology Clinic was matched for sex and area of origin.
The BD clinical syndrome and the treatments used at time of sample collection are
described in Table 1.
3.2 Immune response to common viral infections
The IgG antibodies to EBV, VZV, measles, HSV-1 and CMV were measured in a serum sample
from all thirty individuals with BD with and without neurological involvement and in eighteen
healthy controls. Comparisons were made between the titres in healthy controls (HC) and all the
individuals with BD and between the individuals with and without neurological involvement.
3.3 EBV serology
The prevalence of latent infection with EBV in the general population is 85-90% and increases
with age. Elevated titres of EBNA1 IgG are associated with an increased risk for MS.
Only one person in the healthy control group was negative for EBV infection (negative EBNA1
and VCA serology). There was a non-significant increased titre of EBNA1 in NBS patients
compared to BD patients, but this was not apparent when all individuals with BD were compared
with HC. The titres of VCA IgG were increased in NBS patients when compared to BD patients
(p=0.032) but the difference was not significant when all individuals with BD were compared to
HC (Fig1).
VZV serology
Varicella zoster is another common herpes family infection, which has a much lower prevalence
in the Portuguese population. In countries like the USA where the vaccination recommendation
is for universal coverage, the results would differ. The titres of VZV IgG were increased in NBS
when compared to BD patients (p= 0.0323) and in individuals with BD when compared with HC
(not significant) (Fig 2).
3.4 Measles serology
The majority individuals tested were under universal vaccination coverage for measles (in
Portugal since 1974). There were six individuals with BD who had titres below the positive
threshold for measles, but, according to their age all of them had been vaccinated. The sera IgG
titres showed a tendency to be higher in NBD patients when compared with BD patients and in
all individuals with BD when compared with HC (not significant) (Fig 3).
3.5 HSV-1 serology
HSV-1 persists in neural ganglia in a latent state after the initial primary infection. Different
stimuli cause the virus to reactivate and cause new lesions in the respective dermatome. The
percentage of people who are positive for HSV-1 infection as measured by IgG generally
increases with age and is higher in lower socioeconomic strata. The titres of HSV-1 IgG had a
tendency to be increased in NBS patients when compared to BD patients and in BD patients
when compared with HC (not significant) (Fig 4).
3.6 Cytokines in sera of patients with BD with and without neurological involvement and HC
Our results showed that IL-8, IL-2 and IL-10 were the only detectable serum cytokines in any
patient when we tested for IL-2, IFN-gamma, IL-12p70, TNF-beta, IL-4, IL-5, IL-6, IL-10, IL13, IL-1b, IL-8 and TNF-alpha with the multiplex platform used.
IL-8 was detected in the 11 of 17 BD patients and 7 of 13 NBS patients. The levels of IL8 had a tendency to be higher in all individuals with BD than in NBS patients. The two NBS
patients with the highest IL-8 levels also had detectable IL-2. There was no correlation between
activity of disease and IL-8 levels (Fig 5).
IL-10 was detected in only two BD patients without neurological manifestations. Both
patients were on colchicine and had moderately severe disease; there was no correlation of IL-10
with the levels of IL-8, which were undetectable in one patient and high in another.
3.7 Detection of anti-basal ganglia antibodies in sera of patients with NBS
BD has been associated with Streptococcal colonization and infections and post-streptococcal
ABGA are associated with many neuropsychiatric manifestations. We investigated whether BD
neurological involvement was associated with the presence of anti-neuronal antibodies, in
particular ABGA. We tested for presence of anti-aldolase-C (neuron-specific), anti-gammaenolase, anti-pyruvate kinase antibodies in sera of all individuals with BD with the routinely
used western blot and did not find a specific band.
3. Discussion
There is a continuing debate whether specific autoimmune diseases are triggered by infections.
In particular for diseases that do not fulfill the strict criteria for autoimmunity (characterized
antibody mediated diseases), the presence of infectious particles can trigger self-immune
recognition as a bystander phenomenon. Polyclonal activation, molecular mimicry, superantigens
and bystander activation are all mechanisms that have an effect and are independent of a
particular agent.
We chose to test EBV, CMV, VZV and HSV-1 IgG titres as they are neurotropic, often
found in a latent state after acute infection and have previously been associated with other
autoimmune diseases 11,21,22. On the other hand, measles can rarely cause subacute sclerosing
panencephalitis, a progressive infection caused by persistent mutated virus, but is part of the
routine infant vaccination program in most countries, and so most people have antibodies to the
vaccine.
The increased levels of VCA IgG indicate that the immune responses to the main protein that
characterize the lytic phase of EBV infection may be associated with NBS. This will need to be
confirmed, but it is an interesting observation as EBNA1 IgG levels, rather than VCA, are
associated with an increased risk for MS 23–25. Overall, for all tested viral infections, there was a
tendency towards higher IgG levels in people with BD than HC. Similarly, the titres of antimeasles IgG detected were also increased. They probably represent the post immune response
childhood vaccination and not exposure to or response to infection, which makes us believe in a
plasmocyte polyclonal activation rather than a specific increased exposure or response to viral
infections. Nevertheless, it was interesting that the patient with most severe residual neurological
disability had increased IgG levels for all viruses except for measles.
It is possible that IgG responses are dampened by the use of steroids but the differences
between BD and NBS patients could not be accounted for. None of our patients were in flare and
there is no accurate measure of clinical activity or severity between BD and NBS patients, hence
no correlation can be made. Viral infections are good candidates for environmental promoters of
autoimmunity 26,27. The number of individuals in the groups would need to be increased to reach
statistical significance.
Streptococcal infection is a well-known cause of autoimmune disease: a delayed postinfectious inflammatory disease that affects the heart muscle and valves, large joints, subcutaneous tissue and brain. Acute rheumatic fever and Sydenham’s chorea are well established
but PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal
infections), which presents with tics and/or obsessive compulsive behavior soon after infections,
has yet to be widely accepted as an specific disease entity. Streptococci are more frequently
isolated from the oral cavity of people with BD, who also have more frequent streptococcal
infections. Initially, S sanguis was the identified strain but others have been isolated 28,29. We
hypothesized that if BD is triggered by a streptococcal infection, anti-neuronal antibodies could
mediate some neurological involvement and/or constitute a biomarker to help clinical therapeutic
decisions. ABGA WB recognises four protein bands: the 40kDa antigen is aldolase-C (neuronspecific), the 45 kDa is gamma-enolase, the 60 kDa is pyruvate kinase and the 98 kDA that
possibly is a dimmer of gamma- and alpha-enolase. The negative findings can either be because
of the presence of ABGA in higher detectable quantity in the CSF rather than sera unlike other
diseases, or because the individuals were not in acute stage and the ABGA had lower levels.
ABGA are associated with typical phenotype that includes hyperkinetic movement disorders
with a relatively characteristic neuropsychiatric syndrome 19. The NBS patients in this study did
not present with this phenotype.
In systemic inflammatory diseases potentially driven by antibodies, disruption of the
BBB by cytokines or chemokines is needed for access of inflammatory cells and antibodies to
the brain. The mechanism that increases the permeability of the BBB may also be related to
cellular entry; peripheral blood phenotyping was not performed in this study. Autoantibodies,
binding molecules exposed on the surface of neurons, lead to a neurotoxic effect 1,30. Increased
levels of TNF, IL-6 31, IL-18 32 and IL-8 have been reported in cultures of monocytes from
people with BD 33,34. IL-8 in particular attracts and activates neutrophils and other leukocytes
and is thought to mediate endothelial damage in BD. The association between IL-8 and activity
of BD has not been tested in a NBD cohort 35. Surprisingly, serum TNF-alpha or IL-6 were not
detected in serum. We would expect increased levels of TNF-alpha as infliximab and other TNFalpha inhibitors have proven to be effective in severe BD 36–38. Probably TNF-alpha does not act
at a systemic level, but at cellular level, and the titres detected in serum do not correlate with
clinical disease.
In conclusion, our results do not identify increased IL-8 levels as markers for
neurological involvement. Further, our analysis of the clinical picture at the time of sample
collection did not demonstrate a correlation between IL-8 levels and clinical severity. It is likely
though the activity of BD was not overt or the treatment has an effect in the symptoms but not in
this inflammatory mediator. Alternative facilitators of BBB permeability are anti-endothelial cell
antibodies. These may activate the endothelium inducing the synthesis of pro-inflammatory
cytokines and chemokines and expression of adhesion molecules, leading to increased
permeability of the BBB 39.
Ideally, our study would have collected serum and CSF samples at the presentation of
neurological manifestations and in a prospective way. The numbers in our cohort are limited and
make it difficult to achieve sufficient power for robust statistical analyses. The wide number of
virus and possibly bacteria that have not been tested is large and the causal infectious etiology
may yet to be identified 40.
Overall we found a tendency for increased levels of anti-viral IgG antibody levels in BD
and more so in NBS patients than in HC. The observation that also anti-measles antibodies
elicited by vaccination where elevated in a similar fashion argues for a polyclonal activation
rather than a specific viral effect. ABGA were not detected in the serum of our cohort. The high
levels of serum IL-8 detected in some of the individuals did not reflect clinical activity or
severity and were not increased in NBS patients.
Conflict of Interest: “The authors declare(s) that there are no conflicts of interest regarding the
data and publication of this paper.”
Acknowledgements: We thank Dr Andrew Church for technical help with the ABGA assay.
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Figure 1. EBV IgG antibodies against EBNA1 and VCA. There was no significant difference in
the number of positive results or levels of IgG levels between BD patients, NBS patients or HC.
The top graphs show the levels of IgG in individuals with Behçet with (NB; n=14) and without
(B; n=16) neurological features. The bottom graphs show the levels of IgG in all Behçet patients
(BD) compared with healthy controls (HC; n=18). The titres of VCA IgG were increased in NBS
when compared to BD patients (p=0.032), but this was not significant after multi-testing.
Figure 2. VZV IgG antibodies. There was no difference between BD only, NBS patients or HC.
The graph on the left shows the levels of IgG in Behçet patients with (NB; n=14) and without (B;
n=16) neurological features. The graph on the right shows the levels of IgG in all Behçet patients
(BD) compared with healthy controls (HC; n=18).
Figure 3. Measles IgG antibodies. There was no difference between BD only, NBS patients or
HC. The graph on the left shows the levels of IgG in Behçet patients with (NB; n=14) and
without (B; n=16) neurological features. The graph on the right shows the levels of IgG in all
Behçet patients (BD) compared with healthy controls (HC; n=18).
Figure 4. HSV-1 IgG antibodies. There was no difference between BD only, NBS patients or
HC. The graph on the left shows the levels of IgG in Behçet patients with (NB; n=14) and
without (B; n=16) neurological features. The graph on the right shows the levels of IgG in all
Behçet patients (BD) compared with healthy controls (HC; n=18).
Figure 5. IL-8 levels in Behçet’s patients with (NB; n=14) and without (B; n=16) neurological
involvement.
U/mL
Figure 2. VZV IgG antibodies.
BD
NB
HC
U/mL
U/mL
10
3000
00
60
00
70
00
0
20
4000
0
60 0
8000
00
U/mL
80
12 0
0
16 0
00
VZV
40
0
B
50
0
HC
0
25
0
U/mL
10
0
80
20
0
NB
20
0
U/mL
10
0
50
0
Behcet
Behcet
B
0
0
10
0
20
0
30
0
40
0
50
0
80
0
90
10 0
00
11
0
12 0
0
13 0
0
14 0
00
15
00
16
00
17
00
18
00
Behcet
EBNA1
VCA
B
NB
EBNA1
VCA
BD
BD
HC
Figure 1. EBV IgG antibodies against EBNA1 and VCA.
VZV
Measles
Measles
BD
NBS
HC
U/mL
10
00
0
15
00
0
0
50
00
U/mL
75
0
10 0
00
0
15
00
0
50
00
25
00
0
Behcet
BD
Figure 3. Measles IgG antibodies.
HSV1
HSV1
BD
NBS
HC
Figure 4. HSV-1 IgG antibodies.
U/mL
40
5000
10 00
00
0
30
00
20
00
0
10
00
U/mL
10
0
15 00
00
0
60
00
40
00
20
00
0
Behcet
BD
IL-8
Behcet
B
NB
0
0
20
0
40
00
6
U/mL
0
80
00 00
10 20
Figure 5. IL-8 levels in Behçet’s patients with (NB; n=14) and without (B; n=16) neurological
involvement.
Table 1. Clinical characteristics of people with BD with and without neurological involvement
and medication at time of blood collection
Sex
NBS
BD
10 females 4 males
10 females 6 males
Age BD diagnosis 34 for females, 31 for males
Neurological
involvement
4.5 years (from -4 to 18 years)
orals ulcers (OU) only (4)
BD clinical
syndrome
32 for females , 29 for males
oral ulcers only (4)
OU and erythema nodosum (1) OU and OU + arthralgia (1)
vaginal ulcers (1)
arthritis only (1)
cyclosporin only (1), steroids only (1),
colchicin only (6)
Drugs at time of
Colchicin (C) + clopidogrel (1)
steroids only (3) (2.5 – 30mg
blood collection
C + steroids (2), C + sulfasalazine (1)
prednisolone)
talidomide (1), aspirin (1)
Colchicin and prednisolone (4)
Highlights

polyclonal immune activation in individuals with BD or NBS

raised sera IL-8 levels in individuals with BD or NBS even under treatment

no specific virus effect

no differences in sera IgG or cytokines values for individuals with BD or NBS