Title: Genomic analysis of Canadian children with food allergies points to the immunoglobulin heavy chain gene locusAnne-Marie Madore, PhD,1,2 Marie-Ève Lavoie, MSc,1,2,4 Anne-Marie Boucher-Lafleur, MSc,1,2,4 Frédérique Gagnon-Brassard, BSc,2,4 Noémie Gilbert, BSc,2,4Philippe Bégin, MD, PhD,3 Sarah Lavoie, MD,2,4 Cloé Rochefort-Beaudoin, MD,2,4 Claudia Nuncio-Naud, MD,2,4Guy Parizeault, MD,2,4 Charles Morin, MD,2,4Catherine Laprise, PhD1,2,4*1 Département des sciences fondamentales, Université du Québec à Chicoutimi, Saguenay, Canada2 Centre intersectoriel en santé durable, Université du Québec à Chicoutimi, Saguenay, Canada3 Division d’immunologie clinique, de rhumatologie et d’allergie, Département de pédiatrie, Centre hospitalier universitaire Sainte-Justine, Montréal, Canada4 Zéro allergie research clinic of the Université du Québec à Chicoutimi and the Centre intégré universitaire de santé et de services sociaux du Saguenay–Lac-Saint-Jean, Saguenay, Canada*Corresponding authorCatherine Laprise, Ph.D.Université du Québec à Chicoutimi555 Boulevard de l’Université, Saguenay, QC G7H 2B1, CanadaPhone: 418 545-5011 ext. 5659E-mail: claprise@uqac.caRuning title: IGH locus is linked to food allergyWord count: 1156Number of tables: 1Number of figures: 1Material in the electronic repository: Method description in the Supporting informationTo the editor,Food allergy is a major public health concern, affecting about 8% of children in Western countries, 40% of whom are polyallergic.1 The standard approach to managing food allergy is complete avoidance of food allergens. However, avoidance is complicated by the broad presence of the most common allergens. Since symptoms vary from mild to life-threatening anaphylactic shock,2 it is crucial to better understand the biology underlying the development, diversification and severity of food allergy. To address this need, a pediatric research clinic was established in Saguenay‒Lac-Saint-Jean, a region in northeastern Quebec, Canada. The aim of this initiative is to facilitate access to oral immunotherapy, an emerging treatment for food allergy, and to develop the Zéro allergie cohort.3 This cohort was designed to study the genetic, epigenetic, metabolomic, and microbial diversity associated with food allergy and oral immunotherapy outcomes. This study aimed to analyze the genomic profile of 100 children from the Zéro allergie cohort to identify genetic variants associated with food allergy in a Canadian pediatric population.Each allergic child recruited in the cohort was selected to undergo oral immunotherapy at the research clinic for one to three food allergens (Table 1 ). Non-allergic siblings were also recruited as controls. Children of all sexes, ages (from 6 months to 17 years), and ethnicities were included. Significant differences between the food allergy and control groups were observed for the median age (3.57±2.54 years vs 5.15±2.62 years, p = 0.027), mean birth order (1.82±0.84 vs 1.33±0.49, p = 0.017), the number of children who were breastfed (69 children (86%) vs 11 children (61%), p = 0.020), duration of breastfeeding (31.66±24.80 weeks vs 21.83±33.65 weeks, p = 0.026), and the prevalence of atopic dermatitis (69 children (86%) vs 10 children (56%), p = 0.006). Sex, number of siblings at home, prevalence of preterm birth, caesarean section, and asthma were similar between the two groups (p > 0.05). The number of food allergies among allergic children ranged from one to seven, with a median of two. The number of food allergens for which children were desensitized ranged from one to four, with a median of one. One child underwent desensitization for more than three allergens because a fourth allergy was identified during the desensitization protocol. In the control group, one child had a resolved egg allergy, and another had a resolved milk allergy.After quality control, whole-genome sequencing data were available for 80 allergic children and 18 non-allergic siblings (seeSupporting information for the complete method description). Among the phenotypic variables that differed significantly between groups, age at sampling, birth order, and breastfeeding status were included in the analyses as covariates. Duration of breastfeeding was not considered due to its strong association with the binary breastfeeding phenotype (p-value = 3.32x10-11), in order to avoid redundancy in statistical models. The proportion of atopic dermatitis was not included in the analysis models because of its strong link to the prevalence of food allergy through the atopic march concept.4 A first analysis was conducted for food allergy, regardless of the specific food allergen, including the first two principal components as fixed covariates and a matrix of estimated relatedness as a random effect (see Supporting information for details on analyses). No association reached the genome-wide significance threshold of 5x10-8, but suggestive associations (p < 1x10-5) were identified for 100 variants. Compared with the analyses that included the additional selected covariates, four loci were found in both analyses (9q33.3 from positions 124,448,987 to 124,450,621; 12q24.32-24.33 from positions 127,816,100 to 128,765,615; 14q32.33 from positions 106,414,804 and 106,419,471; and 16p13.3 from positions 2,835,923 and 2,916,358,Figure 1A-1B ).Among these, locus 14q32.33 emerged as the most consistent association across the two models, with 16 genetic variants in common and the strongest association signals overall when the two analyses were considered together (p = 8.98x10-6—1.23x10-7 in the first model; p = 9.37x10-6—7.51x10-7 in the fully adjusted model). This locus lies within a non-coding region of the immunoglobulin heavy chain gene cluster and is surrounded by functional and non-functional sequences of immunoglobulin segments for the variable domain (V domain; Figure 1C ). These segments play a key role in immunity by rearranging with diversity and joining domains (D and J domains) to generate highly diverse immunoglobulins in developing B cells.5, 6 They are essential for producing a broad repertoire of B cell and T cell receptors to mount an appropriate immune response.6 Analysis of antibody repertoire from transcriptomic data showed that B cells from children with food allergen sensitization exhibit skewed V-domain usage, potentially reflecting constitutional differences in recombination mechanisms in food-allergic individuals.7 In contrast, genetic variants at this locus remain poorly documented since their study requires DNA sequencing data. Only four variants in the immunoglobulin heavy chain locus are represented on the Illumina Infinium ImmunoArray24v2, and imputation using reference sequences is hampered by the high variability and number of repeats in this region.6 Since most previous genome-wide association studies have relied on microarray and imputation data, no other genetic associations with food allergy have been reported in the literature.Even though their link with food allergy is more indirect, the three other loci are also of interest as they point to genes involved in immune response or asthma, another disease within the atopic march.4 The 9q33.3 locus is near the ADGRD2 gene, which was previously associated with white blood cell and neutrophil counts.8 The 12q24.32-24.33 and 16p13.3 loci span the TMEM132C and FLYWCH1 genes, respectively, both of which have been linked to forced expiratory volume in one second,9, 10 a proxy of asthma phenotype.This study has some limitations. The relatively small and imbalanced number of controls reduces statistical power, although this is preferable to an equal distribution with a smaller total sample size. The imbalance reflects the difficulty of recruiting non-allergic siblings as some families include only allergic children, and of obtaining parental consent for invasive blood sampling. Another limitation arises from the high variability and repetitive nature of the immunoglobulin regions, for which long-read sequencing would capture more variants.6 Still, short-read sequencing is more accurate for these regions than microarrays and imputation, and it enables the identification of an allergy-related locus highly relevant to food allergy. Recruitment for the Zero allergy cohort is ongoing. It will allow sequencing of additional samples from allergic and non-allergic siblings, as well as long-read sequencing to further analyze the immunoglobulin heavy and light chain loci, including the impact of associated non-coding variants on the complex rearrangements within these regions.This study uncovered four loci associated with food allergy, most notably within the highly variable immunoglobulin heavy chain gene region at chromosome 14q32.33. Despite its likely causal relevance, this locus remains largely unexplored due to its repetitive and complex structure. A better understanding of this region in the context of food allergy could reveal key genetic mechanisms underlying both risk and protective factors, ultimately enabling improved risk prediction and targeted interventions.Anne-Marie Madore, PhD 1,2Marie-Ève Lavoie, MSc 1,2Anne-Marie Boucher-Lafleur, MSc 1,2Frédérique Gagnon-Brassard, BSc 1,2Noémie Gilbert, BSc 1,2Philippe Bégin, MD, PhD 3Sarah Lavoie, MD 2,4Cloé Rochefort-Beaudoin, MD 2,4Claudia Nuncio-Naud, MD 2,4Guy Parizeault, MD 2,4Charles Morin, MD 2,4Catherine Laprise, PhD 1,2,4*1 Département des sciences fondamentales, Université du Québec à Chicoutimi, Saguenay, QC, Canada2 Centre intersectoriel en santé durable, Université du Québec à Chicoutimi, Saguenay, QC, Canada3 Division d’immunologie clinique, de rhumatologie et d’allergie, Département de pédiatrie, Centre hospitalier universitaire Sainte-Justine, Montréal, Canada4 Zéro allergie research clinic of the Université du Québec à Chicoutimi and the Centre intégré universitaire de santé et de services sociaux du Saguenay–Lac-Saint-Jean, Saguenay, QC, Canada*Corresponding authorCatherine Laprise, Ph.D.Université du Québec à Chicoutimi555 Boulevard de l’Université, Saguenay, QC G7H 2B1, CanadaPhone: 418 545-5011 ext. 5659Fax: 418 615-1203E-mail: catherine.laprise@uqac.ca
