Pollen-food allergy syndrome and component sensitization in adolescents: A Japanese population-based study

Allergic rhino-conjunctivitis with pollen allergy has been prevalent worldwide and Pollen-food allergy syndrome (PFAS) refers to individuals with pollen allergy who develop oral allergy syndrome (OAS) on consuming fruits and vegetables. The prevalence of PFAS varies by region and that in Japanese adolescents remains to be elucidated. In this cross-sectional study, we examined the epidemiological characteristics of PFAS in a general population of Japanese adolescents according to pollen allergy, OAS, and IgE component sensitization. Participants comprised adolescents, at age 13 years, from a prospective birth cohort study in Japan. We administered questionnaires to collect information from parents regarding pollen allergy, PFAS and OAS at each child’s age 13 years. ImmunoCAP ISAC was used to assess IgE component sensitization. Among 506 participants with a complete questionnaire and ISAC measurement results, 56.5% had a history of hay fever, 16.0% had a history of OAS, 51.0% had pollen allergy, and 11.7% had a history of PFAS; additionally, 72.7% were sensitized to one or more tree, grass, and/or weed allergens. The most common sensitization (95.7%) among adolescents with pollen allergy was to Japanese cedar (Cry j 1). The most common causal foods were kiwi and pineapple (both 39.0%). Knowledge levels about PFAS were poor among affected adolescents. We found a high prevalence of PFAS among adolescents in Japan. Although it affects approximately 1/10 adolescents in the general population, public awareness regarding PFAS is poor. Interventional strategies are needed to increase knowledge and to prevent PFAS in the general population.


Introduction
Oral allergy syndrome (OAS) was defined by Amlot et al. [1] in 1987 as immediate allergic symptom of the oral mucosa owing to food antigens. Thereafter, the term pollen-food allergy syndrome (PFAS) has been used in patients with pollen allergy who develop OAS after eating fruits and vegetables [2]. PFAS is caused by cross-reactivity between pollen allergens and fruit The questionnaires were developed by several certified allergists and epidemiologists. Rhinitis was assessed using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire [17][18][19]. IgE component sensitization was analyzed using ImmunoCAP ISAC (see Table 1).

Questionnaire survey
A paper-based questionnaire in Japanese, which included the ISAAC and clinical history of OAS and PFAS, was completed by participants' parents. The responses were used to evaluate the health and daily life of adolescents at age 13 years.

Blood sampling and IgE component measurement
Venous blood samples were obtained from 13-year-old adolescents. Allergen component-specific IgE antibody titres were measured using a multiplex array ImmunoCAP ISAC [20][21][22] by a private contract laboratory (Thermo Fisher Scientific, Tokyo, Japan). The ImmunoCAP ISAC enables measurement of IgE titres using a fixed panel of the 112 most relevant allergen components from 51 sources in a single test. Specimen management was conducted by a private contract laboratory (SRL, Inc., Tokyo, Japan).

Bias and study size
Participants were adolescents age 13 years who joined medical check-up of the T-Child Study comprising children of general population. All participated adolescents have been followed since their birth, before they developed PFAS. Study size was inevitably determined by the number of the participants of this cohort study who joined medical check up and answered the questionnaire. They represent the general population of Tokyo metropolitan area in Japan. Table 2. Questionnaire items about oral allergy syndrome for fruits and vegetables.
A questionnaire was given to caregivers to evaluate OAS for children and fruits at 13 years of age.
The survey included the following questions: 1) Has your child ever had an itchy mouth or redness around his/her mouth after eating fruits and vegetables? Those who chose "Yes" in 1) answered after 2).
2) What did your child eat (multiple answers allowed)?
3) What were the symptoms (multiple answers allowed)?
Itching in the mouth and throat, lips, swelling of eyelids, redness/urticaria (face), redness/urticaria (body/limbs), cough/zeize, vomiting/abdominal pain/diarrhea, and others. 4) Dose your child eat the above fruits and vegetables raw (without heating) in his/her daily life?
• He/she took without any particular restrictions.
• He/she took some restrictions.
• He/she has completely removed it and therefore did not take it.
• He/she hasn't had a chance to take it in the last 12 months.

Statistical analysis
The target population in this study was children born as singletons and followed until they reached age 13 years and who had no missing variables for blood tests. Descriptive statistics were performed for all outcomes. The missing values were not imputed. Statistics were performed using JMP version 15 (SAS Institute, Inc., Cary, NC, USA).

Characteristics of patients with PFAS
The characteristics of patients with PFAS are shown in Table 6. Of 59 adolescents with PFAS, 18 (30.5%) reported ever having atopic dermatitis, 21 (35.6%) ever had asthma, and 10 (16.9%) had recent immediate symptoms related to foods.

Symptoms in patients with PFAS
Clinical symptoms of adolescents with PFAS are shown in Table 7. The most common symptom was itching in the mouth and throat (83.1%); this was followed by swelling of the lips and

Sensitization status of PFAS
We examined association between causal foods and sensitization status. The most common allergen associated with sensitization among adolescents with in PFAS in this study was Cry j 1 (93.2%), shown in Table 4). ImmunoCAP ISAC includes four components (Cora1.0101, Cora1.0401, Betv1, Alng1) as PR-10 proteins of trees/grass/weeds, and four components (Betv2, Hevb8, Mera1, Phlp12) as profilin of trees/grass/weeds. The sensitization status of PR-10 protein, profilin, and Cry j 1 of trees, grass, and weeds for each food causing PFAS are shown in Table 9.

Discussion
To our best knowledge, this was the first report of the epidemiological signatures of PFAS confirmed by a clinical history of pollen allergy and OAS and component sensitization, using data of a general population of adolescents from a birth cohort in Japan. This study revealed a high prevalence of pollen allergy and PFAS among adolescents. Common causal foods of PFAS were kiwi, pineapple, peach, and apple. A past history of allergy such as atopic dermatitis and asthma was not very common among adolescents with PFAS. The high prevalence of PFAS in adolescents found in this study revealed the possibility that PFAS is becoming more common in adolescents than previously thought.

Pollen allergy
Japanese cedar is widely distributed throughout Japan, including in Tokyo. A questionnaire survey by Okuda et al. [23] showed that the prevalence of hay fever caused by Japanese cedar was approximately 20% in children age 10-19 years across Japan. The present study results coincided with those previous findings, as the most common sensitization among adolescents with pollen allergy was to Japanese cedar. Birch is a common tree found in parts of Hokkaido and Nagano, but not in Tokyo. However, alder, which is a type of birch tree, is common in parks of Tokyo. At least 36% of adolescents with pollen allergy showed Bet v 1 sensitization. A review by Biedermann et al. [24] found that birch pollen sensitization ranged from 8% to 16% in the general population of Europe. From our previous study, Bet v 1 sensitization was identified in 13.9% of a general population of children age 9 years [12]. The population of Japan might have similar rates of sensitization to birch and alder as those in Europe.
In our present study, we observed relatively few participants with sensitization to grasses and weeds, such as ragweed and timothy grass. We speculate that there were few grasses and weeds near participants' residential area as the adolescents included in this study lived around Tokyo.

PFAS
In this study, we revealed that 11.7% of adolescents in our study had PFAS, and 22.9% with pollen allergy had PFAS. Unfortunately, the actual prevalence of PFAS among children outside of Japan remains unclear as there are no reports regarding PFAS in these populations. From case series, the prevalence of PFAS among study participants with pollen allergy was 33.6% in one European report [25], 41.7% in a Korean study [26] including children and adults, and 12.1% among Australian children [27]. As mentioned earlier, PFAS prevalence differs according to region.
In our study, PFAS accounted for 22.9% of adolescents with pollen allergy. The percentage of PFAS among our participants with pollen allergy was lower than that of the Korean population. The reason may be that Japanese cedar is the most common allergen associated with pollen allergy in Japan. According to age-independent reports from Japan, the prevalence of PFAS in Japanese patients with cedar pollinosis is approximately 10%-13% [9,23]. Furthermore, Cry j 1 sensitization was most common among trees, grasses, and weeds in adolescents with pollen allergy in our study, as compared with a report from South Korea where birch and Table 9. Causal foods and sensitization (PR-10, profilin, and Cry j 1) in PFAS patients.  alder sensitization were most common [26]. As the prevalence of pollen allergy caused by Japanese cedar is highest in Japan, this study showed that the prevalence of PFAS in pollen allergy is relatively low. According to our study findings, 59.3% of adolescents with PFAS did not know whether their symptoms would disappear if they ate causal foods that were heated or processed. There are several possible reasons for this result. First, awareness among the general public about PFAS may still be low. In other words, most Japanese people have no knowledge that heating and processing causal foods can reduce the symptoms of PFAS. Second, eating habits among Japanese people involve less processing and cooking at home compared with populations in Europe and the United States. Nonetheless, as PFAS becomes more common, public awareness about how to manage PFAS should be improved.

Sensitization status of PFAS in adolescents
The most common allergen resulting in sensitization was Cry j 1 (93.2%) among adolescents with PFAS in this study. Regarding the PR-10 protein in foods, sensitization to the PR-10 protein in apples and peaches was highly positive in those with PFAS symptoms after consuming apples or peaches, which was consistent with a past report by Shirasaki et al. [28] in Japan. However, for PFAS symptoms caused by kiwi, the positive rate of sensitization to the PR-10 protein related to kiwi was not higher than that of apple and peach. It is suggested that the degree of involvement of the PR-10 protein varies depending on the food that causes PFAS.
In this study, participants who had PFAS symptoms with foods in the Rosaceae family (e.g., apples, peaches, pears, cherries, plums) were more sensitized to the birch family PR-10 protein. This result is consistent with a past report of Bet v 1 cross-reactivity with allergens of the Rosaceae family [29].
Using a questionnaire survey, Osawa et al. [30] demonstrated that melons, kiwis, and pineapples were the causative foods of OAS symptoms among Japanese children age 10 years and older. In this study, the top causative foods in PFAS were kiwi and pineapple, and a high percentage of sensitization to Japanese cedar (Cry j 1) was observed. We speculate that certain individuals who have OAS symptoms with pineapple and kiwi may be affected by cross-reactivity with Japanese cedar pollinosis. However, another theory has been raised, namely, it is also possible that some adolescents who complain of OAS symptoms with pineapple or kiwi may have non-allergic mechanisms owing to proteolytic enzymes such as bromelain and actinidin.
In this study, few people complained of PFAS symptoms with tomatoes, despite the high percentage of Cry j 1 sensitization observed. Cry j 2 has been pointed out as a cedar allergen that is largely involved in tomato PFAS [31]. Therefore, we considered the possibility that adolescents with PFAS might be less sensitized to Cry j 2. It has been pointed out in a previous report that because Cry j 1 is abundant on the outer wall surface of Japanese cedar pollen and the outer layer of orbicles, the moulting phenomenon is not always an essential condition when acting as an allergen, but Cry j 2 is released as an allergen only after Japanese cedar pollen has molted. Sensitization patterns might differ between Cry j 1 and Cry j 2 [32].

Limitations
This study has some limitations. First, although the background was similar between the 726 participants with completed questionnaires and the 506 participants who also had ISAC measurements, ISAC measurement could not be performed in all participants at age 13 years. In addition, information about a history of rhinitis and history of OAS with fruits and vegetables was based on questionnaire responses given by children's caregivers and not a doctor's diagnosis. From the above, it is possible that the prevalence of pollen allergy and PFAS was underestimated or overestimated. Second, because this survey was conducted in Tokyo, the area of residence of participating children was limited. As has been pointed out, regional differences exist in pollen allergy; therefore, further investigation of regional differences in PFAS is necessary. Unfortunately, this study may make it difficult to take a closer look at the association between allergens and the season of the rhinitis symptoms because the various pollen allergens in each season in Japan (e.g., Japanese cedar, cypress, birch, and alder in spring) commonly lead to multiple allergen IgE sensitizations. Therefore, it is impossible to specify the specific pollen allergen for the pollen allergy in each child.

Conclusions
Our study findings revealed a high prevalence of PFAS among adolescents in Tokyo metropolitan area in Japan. These results suggest that PFAS is common in adolescents; however, most adolescents with PFAS in this study had poor knowledge about PFAS. Early intervention with respect to allergenic foods can prevent immediate food allergy to peanut and hen's egg, although interventional strategies against PFAS remains to be developed. The issue of PFAS has been neglected, and further investigation is needed to explore interventional strategies against the global PFAS epidemic. Furthermore, public awareness about PFAS should be encouraged.