Study of individual N-acetyltransferase 1 activity in persons with contact allergy for para-phenylene diamine (PPD).

In a recent study (on NAT polymorphisms and PPD) there was found a significant
difference in acetylation activity (through NAT-1, a phase II enzyme which
provides detoxification) between PPD allergic subjects and healthy volunteers.
In the allergic group, half of the acetylation products were found, compared to
the healthy subjects. However, no difference was found between the NAT-1
genotype between both groups, i.e. both PPD allergic patients and healthy
subjects were indentified as being normal acetylators and not being slow
acetylators. For the time being, the difference in acetylation activity between
the two groups can't be explained by the known genetic facor. There appears to
be unknown genetic, epigenetic and/or environmental factors playing a role in
the deviating N-acetylation. That's why we request for expanding the study
"Study of n-Acetyltransferase-1 en -2 (NAT-1, NAT-2) and other polymorphisms in
persons with contactallergy for p-Phenylenediamine (PPD)", NL19111.042.07.

The aim of this study is to detect the hitherto unknown factors which possibly
can explain the difference in acetylation. Therefore we need to collect blood
from PPD allergic patients and healthy volunteers, to determine not only the
genotype, but also N-acetylation activity of whole blood and peripheral blood
mononuclear cells (PBMCs). Besides determining N-acetylation activity of PBMCs
as a whole group, different subpopulations of T-cells will be analyzed too, to
determine their specific role. Other, possible explanations for the differences
in acetylation activitynare differences in NAT-1 protein level in leucocytes
and T-cells and the methylation status of NAT-1's promotorregion. The blood
will be analyzed for these variables too.

In this study an amount of 300ml blood is needed from patients allergic to PPD.
Each patient that has a positive reaction on PPD in the routinely taken
patch-test at the department of Dermatology in the University Medical Centre
Groningen (UMCG) are asked if they are prepared to give a blood sample for this
study. Also patients patchtested positive for PPD in the past year, will be
approached in writing. Both groups don't have to answer a questionnaire, their
information is known already through their regular visits to the Dermatology
department. De control group will consist of healthy volunteers who dyed their
hair in the past, but didn't develop an allergic reaction anamnestically. They
will partly be recruted from known PPD negative patchtested patients, with a
positive history of hair, which visited the Dermatological department in the
past year. They don't have to be patchtested again. When there is insufficient
response, there will be an advertisement on bulletin boards in the UMCG in
order to obtain healthy volunteers.(First of all via bulletin boards of the
dermatology department, if needed via general bulletin boards in the UMCG, or
via an advertisement in local newspapers). These volunteers will be patchtested
with PPD where appropriate, to exclude a PPD allergy. For these volunteers,
there has to be a period of at least 3 weeks in between the patch test and the
blood drawing, to obtain reliable results. The control group has to answer a
short questionnaire. Verbally as well as in writing the patients and controls
will be explicitly told that he can quit participating in the study, without
this having any negative influence on the treatment of the patient in the UMCG.
They have one week to think about participation. When a patient decides to
participate in the study, written informed consent will be asked by signing the
informed consent form both by the patient and the investigator. When permission
is given, 300ml of blood will be drawn by an experienced doctor. This will be
processed within 24 hours and then will be stored frozen (-80 C or in nitrogen)
until further analysis will be executed. The stored blood samples are stored
anonymous. To achieve a large enough case-series, considered the amount of
patients that have a positive reactions to PPD, the collection of blood samples
will take a minimum of 3 years. According to the power-calculation 30 blood
samples from PPD-allergic patients and from 30 healthy subjects will be needed.
Processing and analysis of the anonymous blood samples will take place in
cooperation with the department of Toxicology/Ecotoxicology from the University
of Trier (Germany).

All patients that are considered for participating in this study because of the
results of the allergy-test will initially be informed verbally (when
patchtested routinely) or in writing (when they had a positive patchtest on PPD
in the past year). Subsequently the patient will receive written information
signed by the investigator and an independent physician, in which the objective
and design is further explained. Verbally as well as in writing the patient
will be explicitely told that he can quit participating in the study, without
this having any negative influence on the treatment of the patient in the UMCG.
When a patient decides to participate in the study, written informed consent
will be asked by signing the informed consent form both by the patient and the
investigator. The effort of patients will be kept to a minimum and will
comprise in all cases of a single blood donation of 300ml, obtained through a
venous puntcion by an experienced doctor. In case of healthy subjects with no
allergic reaction towards hairdyeing anamnestically, a PPD patchtest will be
applied to exclude a PPD allergy. A PPD patchtest doesn't comprise a big risk;
in fact it is regarded as safe and not causing active sensitization (Contact
Dermatitis 2007; 57(2):133-4). Where appropriate, a short questionnaire has to
be completed. The investment in time in total will comprise a maximum of 15
minutes per patient and will at least include donating blood and in some cases
completing a short questionnaire and/or a PPD patchtest. The PPD patchtest
comprises the application of the patch in the hopital (2.5 minutes), whereupon
subjects can return to their homes. After 48 hours the patch can be removed by
the patient himself, after 72 hours the patchtest reading will be performed in
the UMCG by a doctor (2.5 minutes). At least 3 weeks after the PPD patch test,
the blood drawing may take place. In total, these subjects are coming to the
UMCG three times.