Keywords: Hazelnut allergy; Cor a 1; Cor 2; Cor a 8; Cor a 9; Cor a 14; Component-resolved diagnosis

Transcription

1 Columbia International Publishing Journal of Contemporary Immunology (2014) Vol. 1 No. 1 pp Review Hazelnut Allergy as a Paradigm of the Advancements in Component Resolved Diagnosis: from Characterization of New Allergenic Components to Their Clinical Implications Fassio Filippo 1,*, Massaro Ilaria 2, Giudizi Maria Grazia 3, Almerigogna Fabio 3, Parronchi Paola 1,2, Rossi Oliviero 3, Maggi Enrico 1,2 Received 27 December 2013; Published online 22 February 2014 The author(s) Published with open access at Abstract Hazelnuts are one of the most common triggers of IgE-mediated food allergies worldwide, and they account for a large part of fatalities due to food allergies. Therefore, in the hazelnut-allergic patient, it is crucial to evaluate the risk and predict the severity of reaction on future exposure to the allergen. Component-resolved diagnosis (CRD) is an approach used to map the allergen sensitization of a patient at a molecular level, using purified natural or recombinant allergenic molecules (allergen components) instead of allergen extracts. Many advancements have been accomplished in recent years for CRD in hazelnut allergy, with regard to epidemiology, identification of species-specific vs cross-reactive patients and evaluation of risk for severe reactions. These advancements pave the way to new therapeutic approaches, which are currently being developed. This article summarizes the most relevant progresses of CRD in the field of hazelnut allergy. In parallel, a peculiar case of hazelnut allergy will be presented. Keywords: Hazelnut allergy; Cor a 1; Cor 2; Cor a 8; Cor a 9; Cor a 14; Component-resolved diagnosis 1. Introduction Hazel (Corylus avellana) is a shrub, native to Europe and Asia. It is a member of the family Betulaceae that also comprehends a number of widespread trees, which include birch, alder and hornbeam (Rigby et al., 2008). *Corresponding fassio.filippo@gmail.com 1* Immunology and Cell Therapies Unit, Dept. of Biomedicine, AOU Careggi, Florence, Italy 9 2 DENOTHE Center, Dept. Of Experimental and Clinical Medicine, University of Florence, Florence, Italy 3 Immunoallergology Unit, Dept. of Experimental and Clinical Medicine University of Florence, Florence, Italy

2 Its nuts, hazelnuts, belong to the peanut and tree nut group, including walnut, almond, cashew, and Brazil nut, which although botanically unrelated are often the cause of clinically associated reactions (Pastorello et al., 2002). Hazelnuts may be eaten fresh, after drying to improve storage, or roasted. Their characteristic flavour means that they are widely used to produce baked products and confectionery, so they can be found as hidden ingredient in many common or adultered foods (Arlorio et al., 2010; Rigby et al., 2008). Even though an exact evaluation of hazelnut allergy prevalence is hindered at least in part by the lack of availability of standardized allergens for clinical use, in Western countries it is perceived to range from 0.4 to 1.4% in adults, and up to 2.3% in children (Zuidmeer et al., 2008). A significant variation of prevalence in hazelnut allergy as well as severity of symptoms has been reported in relation to geographic and age distribution (De Knop et al., 2011). Hazelnuts are one of the most common triggers of IgE-mediated food allergies worldwide (Hansen et al. 2009). In the majority of cases, hazelnut allergy is a life-long condition with symptoms that can vary from mild to severe, including anaphylaxis (Pastorello et al., 2002). It has to be noted that hazelnut, together with peanut and other tree nuts, accounts for 90% of fatalities due to food allergies (Bock et al., 2001). As food allergy represents such an important health issue, it has to be underlined that primary food allergy mainly affects young children whereas adults frequently develop food allergy as a consequence of an inhalant sensitization. Pollen-related food allergy has become the most frequent form of food allergy in adolescent and adult individuals in Europe (Bohle, 2007); a typical example is the birch-fruit-vegetable-syndrome (Sastre, 2010). Hazelnut allergy is seen both with and without relation to pollen allergy. Molecular-based allergy diagnostics often also referred to as component-resolved diagnosis (CRD) is an approach used to map the allergen sensitization of a patient at a molecular level, using purified natural or recombinant allergenic molecules (allergen components) instead of allergen extracts (Canonica et al., 2013). It is very useful in food allergy as it helps to evaluate the risk and predict the severity of reaction on exposure to different allergen sources. Significant efforts have been carried out in recent years to identify and characterize at the molecular level hazelnut allergens. Overall, hazelnut allergy can be considered a shining example of the advancements of CRD about epidemiology, identification of species-specific vs cross-reactive patients and risk stratification for severe reaction. These advancements pave the way to new therapeutic approaches, which are currently being developed. This article summarizes the most relevant progresses of CRD in the field of hazelnut allergy. In parallel, a peculiar case of hazelnut allergy will be presented. 2. Patterns of molecular sensitization in hazelnut allergy The literature suggests different routes of sensitization between pollen-related plant food allergy and plant food allergy in patients without pollinosis (Valenta and Kraft, 1996). In patients with hazelnut allergy but no pollen allergy, the sensitization to the plant food is assumed to take place by 10

3 the oral route. In patients with pollen-related hazelnut allergy, the sensitization is more likely to occur through inhalation of pollen allergens and subsequent development of food-induced allergic symptoms caused by cross-reactivity between IgE raised against pollen allergens with homologous proteins in plant foods (Hansen et al., 2009). Differences therefore exist in sensitization patterns between hazelnut allergic patients from Northern and Southern Europe. In Northern Europe where prevalence of birch pollinosis is elevated allergy to hazelnuts is more often found in patients with birch pollen allergy. In contrast, in the Mediterranean area, patients are more frequently sensitized to nonspecific lipid-transfer proteins (LTPs), a member of class I food allergens, probably by primary sensitization after ingestion of other LTPs containing foods, primarily peach (Asero et al., 2002). Sensitization to hazelnut within this group of patients appears independent from allergy to birch or other pollens. Primary sensitization and allergy to tree nuts is much less common, at least in Italy (Asero et al., 2009a; Asero et al., 2009b), although of great clinical relevance due to the extreme severity of clinical consequences in allergic subjects (Asero et al., 2013). A number of different IgE-binding components from hazelnut have been proposed as candidates for sensitization and elicitation of allergic reactions, either in patients with and without pollinosis. Some of the most intensively studied molecules in hazelnut over the last decade are the Bet v 1 homologue Cor a 1.04, the hazelnut profilin Cor a 2 and the LTP Cor a 8 (Pastorello et al., 2002; Sastre, 2010). 3. Pollen-related hazelnut allergens: Cor a 1 and Cor a 2 Cor a 1.04 is the major allergen in hazelnuts. As the major birch pollen allergen Bet v 1, it belongs to the pathogenesis-related-10 (PR-10) allergen family (Hansen et al., 2009). In a previous study, of 50 subjects with sensitization to Bet v 1, 93% were also demonstrated to be sensitized to Cor a 1 (Bohle, 2007). Interestingly, it appears that the epitopes of the hazelnut allergen Cor a 1.04 are less related to the hazel pollen allergen Cor a 1.01 than to Bet v 1 from birch pollen (Lüttkopf et al., 2002). Cor a 2, the profilin from hazelnuts, is homologous to the birch pollen allergen Bet v 2, and has been cloned and described as a minor food allergen among hazelnut allergic patients from Central Europe with concomitant birch pollen allergy (Hansen et al., 2003). The large extent of crossreactivity among plant profilins is due to their high amino acid sequence identity (77 91%) and similar tertiary structures (Radauer et al., 2006). Sensitization to Cor a 1, and to a lesser extent Cor a 2, is therefore related to pollen sensitization, especially to birch pollen components Bet v 1 and Bet v 2, respectively. The prevalence of the pollen-related type of hazelnut allergy seems related to the prevalence of pollen allergy and may therefore be rather extensive, at least in Northern Europe (Flinterman et al., 2008). Cor a 2 is not resistant to thermal and protease treatment, while Cor a 1 shows a limited resistance to heat and proteases (Lauer et al., 2008; Masthoff et al., 2013). Therefore, cooked and industrially 11

4 processed foods are usually well-tolerated by Cor a 1- and Cor a 2-sensitized patients (Sastre, 2010). In most cases, sensitization to Cor a 2 is associated with mild if any symptoms, such as oral allergy syndrome (OAS), after consuming raw hazelnuts. Even sensitization to Cor a 1 is usually associated with mild symptoms; in a series of 17 patients with double-blind placebo-controlled food challenge-confirmed hazelnut allergy, 5/17 did not tolerate heat-processed hazelnuts, but no severe reactions were reported (Hansen et al., 2003). Anyway, severe or anaphylactic reactions in Cor a 1-sensitized patients although rare cannot be excluded (Le et al., 2013). 4. Pollen-unrelated hazelnut allergens: Cor a 8, Cor a 9, Cor a 12 and Cor a 13 Since the first LTP allergen Pru p 3 was fully characterized in peach (Prunus persica), LTPs have been identified as major allergens in a wide variety of plant food allergens(pastorello et al. 1999). LTPs are small proteins comprising 91 to 95 aminoacids, with 8 conserved cysteines forming disulfide bridges, basic isoelectric points, and a common α-helical structure; because of their stable structure, they have been supposed to be responsible for more severe allergic reactions to many foods, including hazelnuts (Schocker et al., 2004). The hazelnut LTP has been identified as major allergen in the Mediterranean population and denominated as Cor a 8 (Schocker et al., 2004). Recently, it was demonstrated that peach is the primary sensitizer to LTP from a large spectrum of botanically unrelated plant-derived foods including Cor a 8 from hazelnut (Ebo et al., 2012). It was remarkable that (1) almost all study populations in which LTPs were described as relevant allergens were from Mediterranean countries and that (2) LTP sensitization was found to be associated with severe food allergies in the majority of cases (Schocker et al., 2004). Combining these two results, it seems that the Mediterranean area has more severe clinical cases of hazelnut allergy than the northern parts of Europe; this perfectly fits with the different pattern of sensitization to pollen-related and pollen-unrelated hazelnut allergen components in geographical regions with different prevalence of birch pollinosis (Hansen et al., 2009). In a comprehensive study by Hansen et al. about CRD of hazelnut allergy, which evaluated patients from different regions in Europe, severe symptoms were reported in 5 (31%) of 16 LTP-sensitized and in 2 (5%) of 41 non LTP-sensitized subjects. Regarded the opposite way, LTP sensitization was present more frequently (71%) in patients with severe symptoms than in those with milder reactions (21%). Despite the fact that that some patients were LTP-sensitized but only showed mild reactions and a few reacted severely in the absence of LTP sensitization, these results suggested an association between severe reactions to hazelnut and LTP sensitization, even if not quite as strong as that reported for certain other foods(hansen et al., 2009). In addition, Cor a 9(Beyer et al., 2002), an 11 S globulin-like seed storage protein, Cor a 11 (Lauer et al., 2004), a 7 S vicilin-like protein, and oleosins (two isoforms with 17 KDa and KDa molecular weight, respectively named Cor a 12 and Cor a 13) (Akkerdaas et al. 2006) were recently described as allergens from hazelnuts, but their clinical relevance across Europe is still scarcely known (Lauer et al., 2008). 12

5 5. A peculiar case of hazelnut allergy: diagnostic workup 3 years ago In 2010 a 19-year-old girl with adverse reactions to hazelnut was referred to our Unit. Over her life, since she was a child, she experienced several episodes of generalized urticaria, angioedema and gastrointestinal symptoms after eating raw or roasted hazelnuts, even in tiny amounts. Frequently she had to recur to the Emergency Department after eating hazelnuts-containing foods; anyway no respiratory or cardiocirculatory symptoms ever developed and epinephrine was never administered. She could tolerate all other kinds of food, including other nuts, and her medical history was otherwise unremarkable. Extracts-based diagnostic work-up for food allergy had already confirmed isolated sensitization to hazelnut, either trough prick testing and ImmunoCAP (3,07 KUA/l). Total IgE were 37 KU/l (both in-vitro tests from Phadia, Uppsala, Sweden). No other sensitization was found on a wide panel of food or respiratory allergens, including Corylacee pollens, either with prick testing or ImmunoCAP. CRD diagnostic workup was performed with an allergen microarray (ISAC test, Phadia, Uppsala, Sweden), a multiplexed test which in the version available at that time comprehended several hazelnut components (Cor a 1, Cor a 8 and Cor a 9) as well as a wide panel of cross-reactive allergenic molecules, including the profiline Bet v 2 (homologue to Cor a 2) and the storage protein Ara h 1 (7S vicilin homologue to Cor a 11). In our patient, the ISAC test resulted completely negative. These results were also confirmed by ImmunoCAP testing with single components (except for Cor a 9 which was not commercially available as a single ImmunoCAP component at that time). In this patient, we diagnosed food allergy to hazelnuts (raw and processed), with moderate-severe reactions, but could not demonstrate allergic sensitization for commercially-available hazelnut allergenic components, either specific or cross-reactive. This made stratification of risk and prediction of the severity of reaction upon future exposure to the trigger allergen very challenging in our patient. The patient s serum was also tested with an experimental hazelnut storage protein preparation (courtesy of Phadia, Uppsala, Sweden), which demonstrated the presence of serum specific IgE at a concentration of 2,2 KUA/l. Hazelnut storage proteins were therefore identified as the target of our patient s IgE, even if a single specific component could not be identified at that time. Patient was prescribed epinephrine autoinjector, to be used in case of anaphylactic reaction. 6. Characterization of a new hazelnut-specific component: Cor a 14 More recently, a new allergen component was identified and characterized in hazelnut. It is a storage protein which belongs to the 2S albumin family (Garino et al., 2010). Even though the presence of a 2S albumin as a putative allergen in hazelnut had been previously reported (Pastorello et al., 2002), its identification, cloning and characterization was completed only 13

6 recently. The 2S albumin was shown to bind IgE of 11 out of 36 hazelnut-allergic patients with moderate to severe allergic reactions, and immunoblot inhibition experiments indicated that the recombinant version had the potential to be used as a mimic of the purified natural allergen (Garino et al., 2010). These results were submitted to the WHO/IUIS Allergen Nomenclature Sub-committee and the allergen was named Cor a 14. A subsequent study (Masthoff et al., 2013), demonstrated that sensitization to hazelnut Cor a 9 or Cor a 14 is highly specific for hazelnut allergy with objective symptoms among Dutch hazelnutallergic children and adults, and it as a marker for a more severe hazelnut allergic phenotype. Cor a 9 and Cor a 14 are both abundant seed storage proteins in hazelnut. Seed storage proteins have been identified as major allergens in several other tree nuts and peanut, and sensitization to the peanut 2S albumin Ara h 2 (homologue to Cor a 14) has been shown to be a good predictor of peanut allergy as well (Nicolaou et al., 2010). 7. Hazelnut-allergic patient: 2013 diagnostic update Shortly after these advancements were achieved, our patient s serum was tested for IgE against Cor a 14, which were present at a concentration of 2,0 kua/l (courtesy of ThermoFisher Scientific, Waltham, MA, USA). Finally, the component responsible for reactions in our hazelnut-allergic patient could be identified. No data is available about IgE cross-reactivity to 2S albumins of different allergenic sources, and our patient except for hazelnut tolerated all kinds of peanut and tree nuts, and no crossreactivity was demonstrated with in vivo and in vitro tests. She was therefore advised to exclude hazelnuts from the diet, but she was allowed to continue to eat all other kinds of nuts. She was also advised to promptly report any new symptom which would develop after eating nuts, and epinephrine autoinjector prescription was confirmed. No need for epinephrine administration was reported up to date. 8. Conclusion Here we reported a peculiar case of primary hazelnut allergy, in a patient with isolated sensitization to Cor a 14. This is an uncommon condition, as sensitization to cross-reactive and pollen-related Cor a 1 and Cor a 2, or to the class-i LTP allergen Cor a 8, account for the vast majority of reactions in hazelnut-allergic patients. Another interesting feature of this case, is that the patient reacted to even tiny amounts of raw and roasted hazelnuts, but could tolerate any other kind of nuts and foods. Moreover, both in vivo and in vitro IgE tests were negative for all the other tested extracts and components. More data is needed to extrapolate food-avoidance indications for hazelnut-allergic patients sensitized to Cor a 14, but this case suggests that cross-reaction among 2S albumins from different foods could be limited. Even if severe anaphylactic reactions did not occur in our patient up to date, sensitization to Cor a 14 should be considered as a marker for a more severe hazelnut allergic phenotype (Masthoff et al., 2013). 14

7 One of the most important clinical features of molecular-based allergy diagnosis is its ability to reveal the allergens to which patients are sensitized, including primary (species-specific) allergens and markers of cross-reactivity (panallergens). Identifying whether the sensitization is genuine in nature (primary, species-specific) or if it is due to cross-reactivity to proteins with similar protein structures helps to evaluate the risk of reaction on exposure to different allergen sources (Sastre 2010). However, CRD adds a considerable amount of complexity to the allergologic diagnostic work-up. Hence the need that a specific training for allergists, in molecular-based allergy diagnosis, should not be overlooked. Moreover, as every food source is composed of several components, many efforts are needed to characterize their allergenic content and their importance related to epidemiology, heat- and protease-stability, prediction of risk for severe allergic reactions. Hazelnut allergy can be considered a striking example of the advancements in the field of CRD. Over the last decade, a comprehensive panel of hazelnut allergenic components has been identified, characterized, and studied in the clinical setting (Table 1). It is therefore much easier for the allergist, today respect to ten years ago, to discriminate hazelnut-allergic patients from crossreactive patients and to predict the risk of severe reactions upon exposure to the allergen. Table 1 Most important allergen components in hazelnut. Allerge Allergen Molecular n name family weigh Clinical significance Cor a 1 PR kda Major allergen in hazelnuts, has limited resistance to heat and proteases. Sensitization to Cor a 1 is mostly related to pollen sensitization, especially to birch. Usually associated with mild symptoms; anyway, severe or anaphylactic reactions in Cor a 1- sensitized patients although rare cannot be excluded. Cor a 2 Profilin 14 kda Cor a 2 is the profilin from hazelnuts, it is homologous to the birch pollen allergen Bet v 2, and has been described as a minor food allergen among hazelnut allergic patients, usually associated with pollen sensitization. Not resistant to thermal and protease treatment. In most cases, sensitization to Cor a 2 is associated with mild if any symptoms, such as OAS, after consuming raw hazelnuts. Cor a 8 Lipid transfer protein 10 kda Cor a 9 Legumin (11- S globulin) 40 kda Scarcely known Cor a 11 Vicilin (7-S globulin) 48 kda Scarcely known Cor a 12 Oleosin 17 kda Scarcely known Cor a 13 Oleosin kda Scarcely known Cor a 14 2-S albumin 17 kda Identifed as a major allergen in hazelnuts in the Mediterranean population. Resistant to heat and proteases, Cor a 8-sensitization was found to be associated with severe food allergies in many cases. Highly specific for hazelnut allergy with objective symptoms in children and adults, it as a marker for a more severe hazelnut allergic phenotype. 15

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