Dear Editor,
Loss-of-function mutations in the serine protease inhibitor
lymphoepithelial Kazal-type related inhibitor (LEKTI) encoding gene,
SPINK5, determines a severe
autosomal recessive syndromic form of ichthyosis named Netherton
syndrome (NS, OMIM 266500)(1). NS is characterized by
the association of congenital
scaly erythroderma(2) with hair abnormalities(3) and severe atopic
manifestations, including atopic dermatitis-like lesions, incoercible
itch, environmental and food allergies, hypereosinophilia, and highly
increased levels of IgE(4). LEKTI deficiency results in unopposed serine
protease activity leading to premature degradation of corneodesmosomal
protein components highly accelerated stratum corneum desquamation and a
profound skin barrier defect with severe skin inflammation(4),(5).
We sought to extensively characterize the IgE sensitization profile in
10 Italian patients (6 female, mean age 13.8±12.2) whose diagnosis has
been confirmed by SPINK5 molecular analysis or anti-LEKTI
immunohistochemistry (Table 1). The study received ethical approval from
IDI-IRCCS Ethical Committee (106/05) and written informed consent was
obtained from all patients or caregivers before participation, following
the Declaration of Helsinki. Nine of 10 patients showed increased serum
total IgE levels (ranging from 155 to 16,300 kU/L), and blood
eosinophilia (ranging from 230 to 850/ μl; Table | 1).
It is widely known that when the total IgE is extremely high, the use of
the current singleplex systems can result in non-specific antigen
recognition (6). This problem does not occur with multiplexed microarray
analytical systems (7). We, therefore, evaluated the IgE sensitization
profile in our NS patients, with the ImmunoCAP ISAC® (Thermo Fisher
Scientific, Sweden) microarray system, without any background problems
resulting in impaired allergenic recognition due to extremely high IgE
levels found in these patients (Figure 1 | A). In 2 cases
(NS#2 and NS#4) the repetition of the microarray testing at one-year
intervals showed comparable values, demonstrating the reproducibility
and robustness of the data and also in complex cases with IgE levels
>10000 kU/L (data not shown).
Overall, a quite heterogeneous reactivity profile was observed in NS
patients: none of the subjects studied was negative, and the molecular
recognition ranged from 6.3% to 66.3% of the total allergen components
investigated (Figure 1 | B)
Analyzing the genuine molecules of the main indoor and outdoor
allergenic sources represented in the micro-chip, group 1 and 2
molecules from dust mites were by far the most frequently positive in
the NS population studied (9/10 patients), followed by the Ole e 1
allergen from the Olive tree (7/10). All other genuine molecules studied
were scattered among the patients, being heterogeneously recognized only
by some of them (i.e. Cup a 1 from the cypress tree in 3/10 cases, Alt a
1 from Alternaria alternata in 2/10, Art v 1, the major allergen of
mugwort pollen in 2/10, Can f 1 from Canis familiaris in 4/10, Fel d 1
from Felis domesticus in 3/11, group 1 and 5 molecules from the grasses
in 4/10, and Par j 2 from pellitory in 3/10).
Two patients, curiously the youngest (3-year-old) and the oldest of our
series (43-year-old) were strongly reactive to latex, being allergic to
Hev b 6 and Hev b 5 respectively, with an anaphylactic reaction after
exposure to the allergen, in the second case.
Three patients (NS#1, 2, and 7), had a serious allergy to cow’s milk,
with recognition of both thermolabile molecules (α-lactalbumin and
β-lactoglobulin) and thermostable casein (Bos d 8), in all cases
associated with immediate urticarial reactions after ingestion of the
food, both raw and cooked. The same 3 patients also had a clinical
history suggestive of adverse reactions (in 2 cases anaphylactic) after
ingestion of cooked hen’s egg (all patients were allergic to both the
thermostable protein Gal d 1, and the thermolabile Gal d 2 and Gal d 3).
None of the patients had reactivity for Gald5 α-livetine; indeed they
regularly ate poultry meat. Interestingly, the remaining 7 patients
studied never had any problems with egg or milk. NS patient #2 was also
reactive to wheat (Tri at 19; ω-5 gliadin). Four patients also scored
reactive for Act d 1 from kiwi. Two patients (NS#4 and 5) had severe
fish allergy confirmed by cod parvalbumin reactivity (Gad c 1), with a
positive clinical history of anaphylactic events. One of the patients
studied had a simultaneous reactivity for 11s globulin from hazelnut
(Cor a 9), peanut (Ara h 2), walnut (Jug r 1), and sesame (Ses i 1) 2s
Albumins, while another patient was mono reactive to the walnut 2s
Albumin-(Jug r 1).
The main plant panallergens available on the array (nsLTP, Profilin,
PR-10, and Polcalcin) were also studied. At least one out of the 8
molecules belonging to the nsLTP superfamily scored positive in 8/10
subjects studied, Pru p 3 from peach being the most frequently
recognized (5 patients). Eight/10 patients were immunoreactive to
profilin panallergen, whilst 3/10 scored positive to PR10 molecules.
Since none but one patient was monoreactive to LTPs, but also
immunoreactive to PR10 or Profilin, no cases of severe reaction to LTP
were recorded in our patients, probably due to the mitigating effect
exerted by the multiple vegetable panallergen recognition in LTP
allergic patients (8), similar to what already observed in Finnish
surveying of 10 patients with NS (9).
None of the NS patients was reactive to polcalcin or cross-reactive
carbohydrate determinants (CCDs).
Interestingly, when we considered the IgE reactivity profile overall and
applied a hierarchical analysis, we observed the formation of 2 main
reactivity clusters. The first, included 3 patients (# 4, # 8 and #
9), who were PR10 reactors, also sensitized to the olive tree, house
dust mite molecules and grasses, while the second cluster was
characterized mainly by dust mites, profilins and LTPs reactivity.
In conclusion, this study demonstrated the efficacy of a proteomic
microarray approach in the study of rare diseases presenting immune
defects and characterized by extremely high IgE levels, such as NS,
where the assessment of specific reactivity can be hampered due to the
limitations of an in vitro dosing with singleplex methods.
Moreover, constraints related to the availability of small amounts of
serum, especially in very young patients, do not represent a limit for
multiplexed microarray analytical systems, which allow for obtaining
reliable and reproducible results on hundreds of allergenic molecules
with a few microliters of the serum. By using this approach, we
succeeded in decoding comprehensive reactivity profiles, useful in the
clinical management of this rare but severe disease.
Table 1. Demographical and molecular features of Netherton
syndrome patients.