The functional consequences of the 57 kb deletion for the TRPV1-receptor in cystinosis patients

Cystinosis is a rare autosomal recessive disorder, characterized by the
abnormal accumulation of cystine in the lysosomes. This accumulation occurs in
almost all cells and tissues, including the conjunctivae, corneas, liver,
spleen, lymph nodes, intestines, brain and kidneys.1 The most common and
severe form is infantile nephropathic cystinosis. Patients are usually
asymptomatic at birth, but at the age of 6 months they present with failure to
thrive, vomiting, constipation, polyuria, excessive thirst, dehydratation and
sometimes rickets. Cystinosis is mostly caused by mutations in the cystinosin
gene (CTNS). The major mutation, which is present in almost 50% of the
cystinosis patients, is a 57-kb deletion. This deletion removes the first 9
exons and a part of exon 10 of the CTNS gene. Exon 10 of the CTNS gene is a
upstream 5* region that encodes for the CARKL gene and also for the first two
noncoding exons of the transient receptor potential channel, vanilloid
subfamily member 1 (TRPV1) gene.3
TRPV1 belongs to the transient receptor potential (TRP) superfamily of cation
channels. TRPV1 is primarily expressed in sensory nerves and is activated by
heating (>43°C) and a wide range of chemical stimuli. One of these chemical
stimuli is capsaicin, the pungent ingredient in hot chilli peppers.4 This
receptor is expressed on a subpopulation of primary sensory neurons consisting
of A*- and C-fibre nociceptors. Though several putative endogenous ligands of
the TRPV1 receptor have been identified, their physiological and
pathophysiological effects in and outside the sensory nervous system remain
unclear.5 In contrast, the effect of binding of the exogenous ligand capsaicin
with the TRPV1 receptor is well known to provoke the release of a number of
bioactive substances including calcitonin gene-related peptide (CGRP).6,7 These
substances, in turn, act on target cells in the surrounding tissue such as mast
cells, immune cells and vascular smooth muscle cells. The resulting response is
characterized by redness and warmth (secondary to vasodilatation), swelling
(secondary to plasma extravasation) and allodynia (i.e. hypersensitivity to
heat and touch secondary to alterations in the excitability of primary sensory
neurons). Collectively, these changes are referred to as *neurogenic
inflammation*, that is, inflammatory symptoms resulting from the release of
substances from the afferent fibres of primary sensory neurons.

The present study wants to test the following hypotheses:

(i) The DBF response to topical applied capsaicin is decreased in cystinosis
patients, compared to matched control subjects.

(ii) The skin sensitivity response after topical applied capsaicin is decreased
in cystinosis patients, compared to matched control subjects.

(iii) The temperature sensitivity is decreased in cystinosis patients, compared
to matched control subjects.

(i) To compare the DBF response to capsaicin between cystinosis patients,
homozygous and heterozygous for the 57-kb deletion, and matched controls.
(ii) To compare the skin sensitivity response after capsaicin application
between cystinosis patients,homozygous and heterozygous for the 57-kb deletio,n
and matched controls.
(iii) To compare the temperature sensitivity between cystinosis
patients,homozygous and heterozygous for the 57-kb deletion, and matched
controls.

This is a single-blinde study, consisting of 1 visit of approximately 2 hours.
During this study visit, the following tests will be performed:
1. The capsaicin test
During this test, 3 rubber O-shape rings will be placed on the volar surface of
the forearm. In the 2 most proximal rings, 1000µg/20µL capsaicin (adults) or
300µg/20µl (<18 years old) will be applied. In the most distal ring, placebo
will be applied. Thereafter, the change in dermal blood flow will be measured
with the use of Laser Doppler Perfusion Imaging on baseling (i.e. 30 minutes
after acclimatization) and on 10,20,30 and 40 minutes after capsaicine/placebo
application in each ring.
2. The Von Frey test
During this test, the skin sensitivity will be tested, 40 minutes after
capsaicin application, with the use of Von Frey filaments. These filaments are
standarised fine-gauge metal wires, to test skin sensititivity to pinch and
mechanical stimuli.
3. Temperature test
Temperature sensitivity will be tested by applying gradual increasing and
decreasing temperatures to determine the temperature sensitivity threshold.

1. Topical application of capsaicin can cause temporarily redness, irritation,
warmth and hypersensitivity of the skin.
2. Subjects can experience hypersensitivity of the skin during the Von Frey
test and temperature test.