Ryusuke Nambu; Takeo Naito; Mei Haruyama; Junichi Hosokawa; Hirotaka Shimizu; Ichiro Takeuchi; Shin-ichiro Hagiwara; Tatsuki Mizuochi; Yugo Takaki; Takashi Ishige; Takuya Nishizawa; Takahiro Kudo; Natsuki Ito; Yosuke Kawai; Yoichi Kakuta; Masao Nagasaki; Toshiaki Shimizu; Itaru Iwama; Katsuhiro Arai

doi:10.5217/ir.2025.00109

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Original Article Orofacial granulomatosis in pediatric Crohn’s disease: clinical outcomes and genetic background in the era of biologics: a retrospective study in Japan: Ryusuke Nambu^1,*, Takeo Naito^2,*, Mei Haruyama¹, Junichi Hosokawa³, Hirotaka Shimizu⁴, Ichiro Takeuchi⁴, Shin-ichiro Hagiwara⁵, Tatsuki Mizuochi⁶, Yugo Takaki⁷, Takashi Ishige⁸, Takuya Nishizawa⁸, Takahiro Kudo⁹, Natsuki Ito⁹, Yosuke Kawai², Yoichi Kakuta², Masao Nagasaki^10,11, Toshiaki Shimizu⁹, Itaru Iwama¹, Katsuhiro Arai⁴; DOI: https://doi.org/10.5217/ir.2025.00109
Published online: January 2, 2026

¹Center for Pediatric Inflammatory Bowel Disease, Division of Gastroenterology and Hepatology, Saitama Children’s Medical Center, Saitama, Japan

²Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan

³Kazusa DNA Research Institute, Kisarazu, Japan

⁴Center for Pediatric Inflammatory Bowel Disease, Division of Gastroenterology, National Center for Child Health and Development, Tokyo, Japan

⁵Department of Gastroenterology, Nutrition and Endocrinology, Osaka Women’s and Children’s Hospital, Izumi, Japan

⁶Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan

⁷Department of Pediatric Gastroenterology and Hepatology, Japanese Red Cross Kumamoto Hospital, Kumamoto, Japan

⁸Department of Pediatrics, Gunma University Graduate School of Medicine, Maebashi, Japan

⁹Department of Pediatrics, Juntendo University Faculty of Medicine, Tokyo, Japan

¹⁰Division of Biomedical Information Analysis, Medical Research Center for High Depth Omics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

¹¹Center for Genomic Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Correspondence to Ryusuke Nambu, Division of Gastroenterology and Hepatology, Saitama Children’s Medical Center, 1-2 Shintoshin, Chuo-ku, Saitama 3308777, Japan. E-mail: nambee1231@gmail.com

*These authors contributed equally to this work.

• Received: June 18, 2025 • Revised: August 25, 2025 • Accepted: September 1, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
NOTES
Supplementary Material
REFERENCES

Abstract

Background/Aims
Occasionally, pediatric Crohn’s disease (CD) may develop after diagnosis of orofacial granulomatosis (OFG), which is characterized by chronic granulomatous lesions of the oral mucosa, lips, and perioral area. This study aimed to clarify clinical characteristics, treatment responses, and potential genetic contributors in pediatric patients with CD complicating OFG.
Methods
We studied pediatric patients with CD complicating OFG who were treated from 2013 to 2022 at 7 Japanese institutions specializing in pediatric inflammatory bowel disease. Their clinical courses were analyzed retrospectively, and analyses of 71 genes associated with monogenic inflammatory bowel disease were performed.
Results
Among 13 patients, 8 were girls. Median ages at diagnosis of OFG and CD were 9.2 (3.8–15.3) and 10.3 (6.4–15.3) years old, respectively. Upper gastrointestinal lesions were frequent in 8 cases (62%), while perianal lesions were present in 7 (54%). OFG failed to improve or relapsed despite remission of intestinal lesions in about half of the patients (n = 7, 54%). During follow-up, OFG went into remission in 7 patients, including 6 of the 9 who were treated with biologics (66%) and 1 of the 4 who were not (25%). In 8 patients, the NCF1 p.Arg90His allele was detected by genetic analysis; 7 were heterozygous and 1 homozygous, a higher prevalence than in the general Japanese population.
Conclusions
Clinical features of OFG associated with pediatric CD are diverse, and biologic agents were beneficial for OFG patients. NCF1 p.Arg90His mutation may contribute to the pathogenesis of pediatric CD complicating OFG.
Keywords: Inflammatory bowel diseases; NCF1; Pediatrics; Genetics

INTRODUCTION

Crohn’s disease (CD) is an inflammatory bowel disease (IBD) that manifests as chronic and often refractory intestinal inflammation. Incidence and prevalence of CD in children are both increasing worldwide [1]. Orofacial granulomatosis (OFG) is a granulomatous disease characterized by swelling of the lips, corners of the mouth, and oral cavity. About 30% of patients with OFG subsequently develop CD [2]. Corticosteroids show efficacy in only about 50% of OFG patients, reflecting the overall difficulty of managing this disease. After the onset of CD, the clinical course of OFG reportedly resembles that of the intestinal lesions [3]. However, some recent case studies found that biologic agents were effective against refractory OFG lesions in patients whose intestinal lesions were already in remission [4,5]. Thus, much remains to be learned about clinical characteristics and effective treatment of OFG lesions in CD.

OFG that develops in childhood is more likely to be complicated by CD than OFG occurring in adulthood [6]. This aspect suggests that the origin of pediatric OFG related to CD might be related more to genetic factors than to environmental factors. Few genetic studies have considered the pathogenesis of pediatric CD related to OFG. According to one report, variants in the NOD2 gene, which exerts a strong effect on CD in Caucasian populations, also contributed to the OFG phenotype in CD [7]. However, in Japanese and other East Asian populations, NOD2 variants have neither been polymorphic nor associated with CD [8,9]. Clearly, genes other than NOD2 must be considered to understand the genetic basis of OFG in Japanese populations. Considering the early age of onset, the low frequency of CD cases associated with OFG, and the chronic granulomatous nature of both conditions, a detailed search for rare monogenic disorders exhibiting IBD and IBD-like inflammation (monogenic IBD) particularly those responsible for chronic granulomatous diseases–should help identify relevant genetic factors.

We therefore conducted a multicenter retrospective study of children with comorbid OFG-CD to clarify clinical features, natural history, and response to treatments including biologic agents of both CD and OFG. We included genetic analyses and pathogenetic considerations.

METHODS

1. Study Design

We retrospectively analyzed patients with CD complicating OFG diagnosed at less than 18 years of age who were treated at 7 institutions specializing in pediatric IBD between April 2012 and January 2022. Clinical course of OFG and CD, relationship between OFG and intestinal lesions during the course of illness, the effect of treatment for CD on the course of OFG, and patients’ genetic background were investigated. Diagnosis of CD was made at each institution based on the revised Porto criteria [3]. OFG was diagnosed by dermatologists, oral surgeons or attending certified pediatric gastroenterologists based on established criteria [10].

2. Clinical Information

Patient information was obtained with respect to sex, family history (second-degree relatives or closer), age at OFG diagnosis, age at CD diagnosis, and intervals between OFG and CD diagnoses. For CD, we determined disease type at diagnosis, severity of disease, presence of upper gastrointestinal lesions, presence of extraintestinal symptoms, and observation period following CD diagnosis. The weighted Pediatric Crohn’s Disease Activity Index (wPCDAI) was used to assess CD severity at diagnosis, and the Paris classification was used to determine disease type [11]. For OFG, we collected information about phenotypes, subjective OFG symptoms, involvement of dermatologists, oral surgeons, and pathologists, and whether granulomas were detected in lesions. Response of OFG to treatment of intestinal lesions or OFG, presence or absence of relapse of OFG at the time of intestinal relapse, and presence or absence of intestinal relapse at the time of OFG relapse were also determined. Response to OFG treatment was rated as good, partial, or poor response, based on the attending certified pediatric gastroenterologists’ examination findings and subjective symptoms of affected children. Each relapse was defined as a necessity for change of or addition to treatment.

3. Exome Sequencing

The study design for genetic analysis is summarized in Fig. 1. Exome sequencing using a custom gene panel targeting immune-related genes was performed as described in a previous report [12]. Briefly, DNA was extracted from peripheral blood samples. Paired-end exome sequencing was performed for the 12 patients based on the Illumina platform. Reading alignment to the human reference genome GRCh37 was performed using Burrows-Wheeler alignment. Variant calling was performed based on the Genetic Analysis Toolkit best practices. Individual variants with genotyping quality below 65, call rate of 1.0, or reading depth below10 were removed from consideration. All quality controls were performed using Hail (Hail Team. Hail 0.2.115-10932c754edb. https://github.com/hail-is/hail/commit/10932c754edb).

4. Monogenic IBD Variant Extraction

Monogenic IBD variants were annotated by ANNOVAR software and Combined Annotation Dependent Depletion (CADD) score version GRCh37, version 1.6 [13]. We extracted nonsynonymous exonic variants located within 71 monogenic IBD-related genes [14]. We included only variants present in at least 2 patients, considering the homogenous phenotype. Finally, we excluded variants with CADD scores below 10. This filtering yielded a total of 33 variants within 25 genes in the 12 patients.

5. NCF1-Specific Sequencing

Because of the presence of NCF1B and NCF1C, genotype of a NCF1 variant detected in our study (rs201802880) was validated by NCF1-specific sequencing (Supplementary Fig. 1) [15]. An NCF1-specific fragment was amplified using Takara Gflex (Takara, Japan) by targeting the GTGT sequence in exon 2 of NCF1. These polymerase chain reaction (PCR) products of approximately 6 k bp were analyzed by a next-generation Illumina sequencer. However, since pseudogenes might be amplified with this primer, digital PCR (dPCR) was performed to verify this possibility.

6. Digital PCR

Genotypes of single-nucleotide polymorphisms were determined by dPCR (QuantStudio Absolute Digital PCR System, Applied Biosystems, Waltham, MA, USA). The customized primer/probe sets used are given in Supplementary Fig. 2. For PCR, DNA samples were added to a reaction mixture containing Absolute DNA Digital PCR Master Mix (Applied Biosystems) and TaqMan probes. PCR conditions consisted of initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, and annealing at 60°C for 60 seconds.

7. Statistical Analysis

Among patient characteristics, categorical variables were indicated as number of patients showing the variable and corresponding percentage within the cohort, while quantitative variables were given as medians with range.

To investigate differences of minor allele frequency (MAF) between Japanese individuals without IBD and the OFG patients in our cohort, we used MAF data from the Tohoku Medical Megabank Organization (ToMMo) cohort as a reference (https://jmorp.megabank.tohoku.ac.jp/downloads/tommo-54kjpn-20230626-af_snvindelall) [16]. The ToMMo cohort, one of the largest Japanese biobanks holding whole genome sequencing data from 54,000 Japanese individuals, is considered ideal for MAF analysis in the Japanese population. The Fisher exact test was used to compare the difference in MAF between our OFG cohort and the ToMMo cohort. A P-value < 1.51e-3 (0.05/33) was considered statistically significant. All statistical analyses were performed with R software, version 4.1.3 (http://www.r-project.org/; R Foundation for Statistical Computing, Vienna, Austria).

8. Ethical Matters

This study, conducted in accordance with the Declaration of Helsinki, was approved by the Ethics Committee at Saitama Children’s Medical Center (Nos. 2021-02-041 and 2021-06-028). As for use of clinical information, consent was obtained using the study website, which included provisions for opting out. As for genetic analysis, informed consent was obtained from all parents or caregivers. Written assents were obtained from children when appropriate.

RESULTS

1. Study Subjects and Clinical Characteristics

This study included 13 patients (Table 1). None had a family history of IBD. The median age at diagnosis of CD was 10.3 years, and the median observation period for the course of CD was 6.1 years (range, 1.0–8.2 years) from diagnosis. In 7 of 13 patients (54%), OFG symptoms preceded CD diagnosis; in the other 6 they were concurrent with CD onset. The median time from OFG diagnosis to CD diagnosis among the 7 cases was 2.8 years (range, 1.0–6.1 years). Severity of CD at diagnosis was mild to moderate (wPCDAI <57.5) in 9 patients, and severe (wPCDAI ≥ 57.5) in 4. All 13 patients had small intestinal involvement at the time of CD diagnosis. Upper gastrointestinal lesions (sites ranging from esophagus to duodenum) were present in 8 of the 13 patients (61.5%). Perianal lesions were present in 7 (53.8%).

2. Phenotype of OFG

Twelve of the 13 patients were examined by a dermatologist or oral surgeon. Biopsy specimens were obtained from the lips in 8, showing granulomas in 5 patients (62.5%). Lip swelling was the most common finding, present in all patients followed by gingivitis in 8 cases (61.5%), angular cheilitis in 7 (53.8%), buccal mucositis in 5 (38.5%), oral ulcers in 4 (30.8%), and glossitis in 1 (7.7%) (Fig. 2, Supplementary Table 1). Subjective symptoms included pain in most cases (n=7, 53.8%). Fewer patients experienced dysphonia or difficulty in chewing. Six patients had no subjective symptoms of OFG.

3. Correlation between the Clinical Course of OFG and Intestinal Lesions

Remissions and relapses of OFG and intestinal lesions during follow-up were categorized into 3 patterns (Fig. 3): (1) concordant remission and relapse of both OFG and intestinal lesions (n=6), observed in 5 patients who received biologic agents immediately after diagnosis; (2) remission of intestinal lesions without improvement of OFG (n=4); and (3) remission of intestinal lesions with relapse of OFG (n=3). Two of these 3 cases showed improvement in OFG following a change to a different biologic or an increase in biologic dose. As mentioned above, courses of OFG and gastrointestinal lesions did not coincide in 7 of the 13 cases. During follow-up, OFG was judged to be in complete remission in only 7 of the 13 patients, with 3 showing partial remission and 3 others showing unchanged or worsening OFG. Biologics were used for 9 patients, with 6 attaining remission of OFG (66%); among the 4 patients for whom they were not used, OFG showed remission in only 1 (25%) (Table 2).

4. Genetic Analysis

Twelve of 13 consenting patients were studied with a genetic testing panel related to inborn errors of immunity, seeking genes associated with monogenic IBD (Table 3). NCF1 p.Arg90His, which has a general allele frequency of 1% in the Japanese population, was heterozygous in 7 of 12 patients; 1 patient was homozygous. The allele frequency in our study population was 37.5%, significantly higher than in the Japanese database. All 8 patients with the NCF1 p.Arg90His variant required biologic agents sometime during clinical observation. Results of dihydrorhodamine (DHR) testing were normal in patient 7 who was homozygous, establishing that she did not have chronic granulomatosis disease (CGD).

We needed to consider that NCF1 is located within a region that is difficult to sequence due to adjacent homologous pseudogenes (NCF1B and NCF1C). We therefore carried out longread PCR, which confirmed the presence of the variant in all 8 patients (Supplementary Fig. 3). To determine the copy number of NCF1B/C and NCF1, we performed a dPCR assay containing probes specific for either the GTGT in NCF1 or the ΔGT sequence in NCF1B/C (Supplementary Fig. 2) [17]. Since a total of 6 copies of NCF1/NCF1B/NCF1C would be expected, our cases were predicted to have 2 GTGT copies vs. 4 ΔGT copies, expressed as 2/6. The ratio of GTGT/(GTGT+ΔGT) in the group of 8 patients was 2/6, corresponding to the expected 2 GTGT copies and 4 ΔGT copies (Supplementary Table 2). These tests confirmed that the variants detected were NCF1 p.Arg90His rather than pseudogenes.

DISCUSSION

In our case analysis of pediatric CD associated with OFG, upper gastrointestinal and perianal lesions were more common. Clinical courses of OFG and intestinal Crohn’s lesions frequently differed. Biologic agents were more effective in treating OFG than exclusive enteral nutrition or thiopurines. The NCF1 p.Arg90His allele was present in 8 of 12 patients, with an allele frequency of 37.5% in the study population. This suggests that NCF1 p.Arg90His may contribute to the pathogenesis of pediatric CD complicating OFG.

We found upper gastrointestinal and perianal lesions to be frequent in OFG-related CD. Dupuy et al. [18] similarly reported that upper gastrointestinal and perianal lesions were particularly frequent in CD with oral lesions (50% and 88%, respectively). Harty et al. [19] found a significant difference in frequency of perianal lesions between CD with and without oral lesions. Malmquist et al. [20] noted that 64% of 25 CD patients with OFG had perianal complications during a 5-year period, stressing that the development of perianal lesions should be anticipated in CD with OFG. On the other hand, upper gastrointestinal and perianal lesions in pediatric patients with CD have been reported to be more common in Asia (both approximately 40%–50%) than in the West [21-23]. These epidemiologic differences suggest that the high frequency of perianal lesions observed in the present study may not necessarily be specific to CD patients with OFG.

In our cohort of CD patients with OFG, the clinical course of OFG did not parallel that of their intestinal lesions. Hussey et al. [24] reported that OFG gradually disappears, unlike intestinal lesions. Citing such observations, the revised Porto criteria stated that OFG is likely to gradually diminish. However, a study by Vernier-Massouille et al. [25] concluded that OFG lesions in CD may be persistent and refractory. In our study, Patients 1 and 5 had relapses of OFG lesions during biologic agent treatment despite remission of intestinal lesions. In the era of biologic agents, then, courses of a patient’s OFG and intestinal lesions do not necessarily parallel each other, as is occasionally observed with perianal lesions [26]. In addition to cosmetic problems, OFG may cause pain and other symptoms in about half of patients, compromising nutrition and quality of life. Given these findings, refractory OFG associated with CD might be considered a potential indication for biologic therapy, much like refractory intestinal or perianal disease.

Our results suggest that biologic agents may be more effective treatments for OFG in CD patients than exclusive enteral nutrition or 5-aminosalicylic acid. While Mutalib et al. [27] and Cameron and Middleton [28] found dietary therapy such as exclusive enteral nutrition to be effective against OFG in many cases. Malmquist et al. [20] reported that most OFG in CD is refractory, with 9 of 25 patients (38%) showing no improvement during a 5-year follow-up period. While Phillips et al. [29] reported a response rate to tumor necrosis factor-alpha agents of only about 32%, successful responses to relatively new biologic agents such as vedolizumab and ustekinumab in CD with OFG have also been reported [4,5]. As is true for luminal lesions, changes in therapy, such as new drugs or higher doses may successfully treat OFG in CD.

Approximately 70% of patients with OFG have NCF1 p.Arg90His variants, which may be important to understanding the pathogenesis of pediatric CD complicated by OFG. Biallelic loss-offunction variants of NCF1, NCF2, and NCF4 cause CGD. Dysregulation of p47phox, a cytoplasmic protein encoded by NCF1, results in impaired NADPH oxidase activity and reduced production of reactive oxygen species. In CGD, 30% to 80% of patients develop granulomatous colitis that resembles IBD [30]. However, we know of no reports of NCF1 p.Arg90His causing CGD, and the homozygous patient in this study (patient 7) did not develop CGD. In contrast, NCF1 p.Arg90His has been associated with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and psoriasis [15,17,31-35]. NCF1 p.Arg90His has been identified as a functionally relevant variant in VEO-IBD, with heterozygous carriers showing a reduced oxidative burst without overt immunodeficiency [36]. Hypomorphic variants in the NOX2 NADPH oxidase complex can contribute to IBD susceptibility rather than severe immunodeficiency. Consistently, we found a higher prevalence of this variant in CD patients with OFG, suggesting that it predisposes to intestinal inflammation via impaired innate immunity rather than producing classical CGD-like defects. In an animal study of altered immunity, Li et al. [31] found that NCF1 p.Arg90His-expressing mice had hyperactivated macrophages, altered keratinocytes, and dramatically increased numbers of γδT17 cells with upregulated interleukin-17A, interleukin-23, and tumor necrosis factor-alpha. In addition, the JAK1/STAT3 signaling pathway was upregulated in the psoriatic skin of NCF1 p.Arg90His mice. This may suggest that ustekinumab, risankizumab and JAK inhibitors may be effective in pediatric CD complicated with OFG.

Two pseudogenes, NCF1B and NCF1C, are very similar in genetic structure to NCF1, creating difficulty in studying the NCF1 gene using only short-read exome sequencing or Sanger sequencing. In our study, validation by nested PCR and Taq-Man assays was therefore highly important. The reported allele frequency of NCF1 p.Arg90His is 2% to 19% depending on race and ethnicity. Yokoyama et al. [34] reported an allele frequency of NCF1 p.Arg90His in healthy Japanese controls of 19.6% (344/1,752) according to nested PCR and TaqMan assays. Patients in our present study had an NCF1 p.Arg90His allele frequency of 37.5%. Even relative to the allele frequency of healthy controls (344/1,752) reported by Yokoyama et al. [34], this variant remains significantly more frequent in the OFG group (P=0.0386). Thus, this mutation appears to be associated with the development of childhood CD that includes OFG.

This study elucidated the clinical course and genetic background of our pediatric CD patients with OFG. Analysis of their genetic background is a particular strength of this study. However, several limitations should be noted. Despite a multicenter design, the sample size is small. A larger prospective, observational study in the biologic agent era would be desirable. The study showed a high proportion of NCF1 p.Arg90His in childhood-onset CD with OFG, but no comparison was made with childhood-onset CD without OFG. As NCF1 p.Arg90His is difficult to detect using conventional genome-wide association studies or whole-exome sequencing methods, future studies of pediatric CD need to incorporate long-read sequencing or nested PCR. Another limitation is that the analysis was performed in a single ethnic group. Genetic analysis in a multiethnic population is important to assess the general significance of our present findings.

In this study, during the era of biologics, we should note that OFG lesions did not necessarily progress in parallel with intestinal lesions. In addition, since biologics have proven effective in treating refractory OFG in pediatric CD, the presence of OFG may be a strong indication for biologics in CD patients. Finally, NCF1 p.Arg90His may contribute to the pathogenesis of pediatric CD, especially when it complicates OFG.

NOTES

Funding Source

This work was supported in part by the MEXT Cooperative Research Project Program, Medical Research Center Initiative for High Depth Omics, and CURE:JPMXP1323015486 for MIB, Kyushu University. This work was partly performed in the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University.

Conflict of Interest

Nambu R received speaker’s fees from AbbVie GK., Takeda Pharmaceutical Co., Ltd., and Mitsubishi Tanabe Pharma Corp. Shimizu H received speaker’s fees from AbbVie GK., Takeda Pharmaceutical Co., Ltd., and Covidien Japan Inc. Hagiwara S received speaker’s fees from AbbVie GK., Mitsubishi Tanabe Pharma Corp., Takeda Pharmaceutical Co., Ltd., and Alfresa Pharma Co., Ltd. Mizuochi T received lecture fees from AbbVie GK, Takeda Pharmaceutical Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., EA Pharma Co., Ltd., Eisai Co., Ltd., Kyorin Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Nobelpharma Co., Ltd., Sekisui Medical Co., Ltd., and Nippon Kayaku Co., Ltd., and consulting fees from AbbVie GK and Takeda Pharmaceutical Co., Ltd. Takaki Y received speaker’s fees from AbbVie GK, Janssen Pharmaceutical K.K., Kyorin Pharmaceutical Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., Mochida Pharmaceutical Co., Ltd., and Takeda Pharmaceutical Co., Ltd. Ishige T received speaker’s fees from Mitsubishi Tanabe Pharma Corp., EA Pharma Co., AbbVie GK, Janssen Pharmaceutical K.K., Nippon Kayaku Co., Ltd., Alfresa Pharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., JIMRO Co., Ltd., Eisai Co., Ltd., Sandoz K.K. and Miyarisan Pharmaceutical Co., Ltd. Kudo T received speaker’s fees from AbbVie GK, Takeda Pharmaceutical Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., EA Pharma Co., Ltd., Kyorin Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Sandoz K.K., Janssen Pharmaceutical K.K., and Mochida pharmaceutical Co., Ltd. Shimizu T received a research grant from JCR Pharma and the Food Science Institute Foundation (Ryosyoku-kenkyukai), and consulting fees from Eli Lilly Japan K.K. and Pfizer Inc. Iwama I received speaker’s fees from AbbVie GK. and Mitsubishi Tanabe Pharma Corp. Arai K received research grant and/or speaker’s fee from AbbVie GK, Janssen Pharmaceutical K.K., Takeda Phartaceutical CO., Ltd., EA Pharma Co., Ltd., Eli Lilly Japan K.K., Pfizer Inc., Zeria Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corp., Kissei Pharmaceutical Co., Ltd., Miyarisan Pharmaceutical Co., Ltd., Kyorin Pharmaceutical Co., Ltd., Mochida pharmaceutical Co., Ltd., and Bristol Myers Squibb Company. All other authors declare no conflict of interest.

Data Availability Statement

Data analyzed in this study are available from the corresponding author upon reasonable request.

Author Contributions

Conceptualization: Nambu R, Naito T, Haruyama M, Hosokawa J, Shimizu H. Data curation: Nambu R, Takeuch I, Hagiwara S, Mizuochi T, Takaki Y, Ishige T, Nishizawa T, Kudo T, Ito N, Iwama I, Arai K. Formal analysis: Nambu R, Naito T, Haruyama M, Hosokawa J, Kawai Y, Kakuta Y, Nagasaki M. Investigation: Nambu R, Naito T, Haruyama M, Hosokawa J, Shimizu H, Takeuch I, Hagiwara S, Mizuochi T, Takaki Y, Kawai Y, Kakuta Y, Nagasaki M, Arai K. Methodology: Nambu R, Naito T, Hosokawa J, Ishige T, Nishizawa T, Kudo T, Nagasaki M, Iwama I, Arai K. Resources: Nagasaki M. Software: Naito T, Hosokawa J, Ito N. Supervision: Shimizu T, Arai K. Visualization: Nambu R, Naito T. Writing - original draft: Nambu R, Naito T, Haruyama M. Writing - review & editing: Hosokawa J, Shimizu H, Takeuch I, Hagiwara S, Mizuochi T, Takaki Y, Ishige T, Nishizawa T, Kudo T, Ito N, Kawai Y, Kakuta Y, Nagasaki M, Shimizu T, Iwama I, Arai K. Approval of final manuscript: all authors.

Additional Contributions

The authors thank all participating patients, their families, and physicians for collaborating in data collection. The authors also thank Prof. Atushi Masamune for his careful guidance. The infrastructure of Omics Science Center Secure Information Analysis System, Medical Institute of Bioregulation at Kyushu University provides the (part of) computational resource (https://sis.bioreg.kyushu-u.ac.jp/).

Supplementary Material

Supplementary materials are available at the Intestinal Research website (https://www.irjournal.org).

Supplementary Table 1.

Clinical Features of Orofacial Granulomatosis in 13 Patients

ir-2025-00109-Supplementary-Table-1.pdf

Supplementary Table 2.

Digital PCR Analyses of NCF1 in Genomic DNA from 8 Cases

ir-2025-00109-Supplementary-Table-2.pdf

Supplementary Fig. 1.

NCF1 long-read polymerase chain reaction (PCR) conditions.

ir-2025-00109-Supplementary-Fig-1.pdf

Supplementary Fig. 2.

NCF1 digital polymerase chain reaction (PCR) conditions.

ir-2025-00109-Supplementary-Fig-2.pdf

Supplementary Fig. 3.

Results of NCF1 long-read polymerase chain reaction (PCR).

ir-2025-00109-Supplementary-Fig-3.pdf

Fig. 1.

Genetic analysis workflow. OFG, orofacial granulomatosis; CADD, Combined Annotation Dependent Depletion; IBD, inflammatory bowel disease; ToMMo, Tohoku Medical Megabank Organization; MAF, minor allele frequency; PCR, polymerase chain reaction.

Fig. 2.

Physical and histopathologic findings of orofacial granulomatosis in this study. (A) Lip swelling in patient 1. (B) Oral ulcer in patient 2. (C) Angular cheilitis in patient 3. (D) Gingivitis in patient 11. (E) Noncaseating epithelioid cell granuloma in a lip biopsy specimen from patient 5 (hematoxylin and eosin staining, ×400).

Fig. 3.

Disease course of OFG and intestinal lesions. (A) Parallel course pattern for OFG and intestinal lesions (n=6). (B). Pattern in which intestinal lesions improved but OFG did not (n=4). (C). Pattern in which intestinal lesions were in remission but OFG lesions flared up (n=3). OFG, orofacial granulomatosis.

Table 1.

Clinical Characteristics of CD in 13 Patients

Characteristic	Value (n = 13)
Sex (female: male)	9:4
Family history^a	0
Age at diagnosis of OFG (yr)	9.2 (3.8–15.3)
Age at diagnosis of CD (yr)	10.3 (6.4–15.3)
OFG preceded CD (yr)	2.8 (1.0–6.1)
Timing of OFG onset
Prior to CD	7 (53.8)
Same time as CD	6 (46.2)
wPCDAI at diagnosis
Mild (wPCDAI ≤ 30)	6 (46.2)
Moderate (30 < wPCDAI ≤ 57.5)	3 (23.1)
Severe (wPCDAI > 57.5)	4 (30.8)
Paris classification
L1	3 (23.1)
L2	1 (7.7)
L3	11 (84.6)
L4a	7 (53.8)
L4b	6 (46.2)
Stricture	1 (7.7)
Perianal disease	7 (53.8)
Upper GI lesion	8 (61.5)
Extraintestinal manifestation
Dermatitis	5 (38.5)
Arthritis	1 (7.7)
Observation after CD diagnosis (yr)	6.1 (1.0–8.2)

Values are presented as median (range) or number (%).

^a Within 2 generations.

CD, Crohn’s disease; OFG, orofacial granulomatosis; wPCDAI, weighted Pediatric Crohn’s Disease Activity Index; GI, gastrointestinal.

Table 2.

Disease Type, Treatment Response, Course, and Presence of NCF1 Variants in 13 Cases

No.	Sex	Age at CD diagnosis	Paris classification	wPCDAI at diagnosis	Treatment response of OFG								Remission of OFG during the observation period	NCF1 p.Arg90His
No.	Sex	Age at CD diagnosis	Paris classification	wPCDAI at diagnosis	EEN	5-ASA	PSL	AZA/6-MP	IFX	ADL	UST	Other treatments effective for OFG	Remission of OFG during the observation period	NCF1 p.Arg90His
1	M	9 yr 2 mo	L3, B1, P0	42.5	Poor	Poor	Good	Poor	Good		Good	Zinc: good	Yes	Heterozygous
2	F	13 yr 9 mo	L3+L4a, B1, P1	70.0	Poor	Poor	Good	Poor	Partial	Partial	Good		Yes	Heterozygous
3	F	15 yr 3 mo	L3+L4ab, B1, P1	12.5	Partial					Good	Good		Yes	Heterozygous
4	M	9 yr 10 mo	L3+L4ab, B1, P1	52.5	Partial	Poor		Partial		Good	Good		Yes	Heterozygous
5	F	11 yr 2 mo	L1, B1, P1	65.0	Good	Partial		Poor		Good			Yes	Negative
6	F	10 yr 2 mo	L3+L4ab, B1, P1	65.0	Partial		Partial	Partial	Partial	Partial	Partial		Yes	Heterozygous
7	F	11 yr 4 mo	L1, B1, P1	7.5	Partial	Partial		Partial		Partial			No	Homozygous
8	M	13 yr 11 mo	L3+L4b, B1, P1	17.5	Poor	Poor				Poor		Topical PSL: partial	No	Negative
8	M	13 yr 11 mo	L3+L4b, B1, P1	17.5	Poor	Poor				Poor		Topical Tac: partial	No	Negative
9	F	13 yr 7 mo	L3+L4a, B1, P0	52.5	Partial				Partial				No	Heterozygous
10	F	6 yr 5 mo	L2+L4a, B2, P0	80.0	Good		Good	Partial					No	NA
11	F	10 yr 3 mo	L3+L4b, B1, P0	7.5	Poor	Poor						Topical PSL: partial	No	Heterozygous
11	F	10 yr 3 mo	L3+L4b, B1, P0	7.5	Poor	Poor						Topical Tac: partial	No	Heterozygous
12	M	8 yr 1 mo	L3, B1, P0	7.5		Poor						Topical PSL: good	No	Negative
12	M	8 yr 1 mo	L3, B1, P0	7.5		Poor						DXS Elixir: partial	No	Negative
13	F	9 yr 9 mo	L1+L4ab, B1, P0	7.5		Partial							Yes	Negative

CD, Crohn’s disease; wPCDAI, weighted Pediatric Crohn's Disease Activity Index EEN, exclusive enteral nutrition; 5-ASA, 5-aminosalicylic acid; PSL, prednisone; AZA, azathioprine; 6-MP, 6-mercaptopurine; IFX, infliximab; ADL, adalimumab; UST, ustekinumab; OFG, orofacial granulomatosis; M, male; F, female; Tac, tacrolimus; NA, not analyzed.

Table 3.

Candidate Monogenic IBD Variants Potentially Causal for OFG in Crohn’s Disease

rsIDs	CHR	Position^a	Ref	Alt	Genes	AA change	MAF in ToMMo	MAF in OFG	P-value	Ref/Ref	Hetero	Alt/Alt	CADD score^b
rs201802880	7	74193642	G	A	NCF1	p.R90H	0.011	0.375	2.70E-12	4	7	1	23.70
rs1800470	19	41858921	G	A	TGFB1	p.P10L	0.486	0.750	1.28E-02	0	6	6	19.21
rs5956583	X	123034511	A	C	XIAP	p.Q423P	0.047	0.150	8.10E-02	9	2	1	14.32
rs2274064	1	183542387	T	C	NCF2	p.K136R	0.639	0.458	8.73E-02	2	9	1	14.86
rs80346348	16	67685694	G	A	CARMIL2	p.G845D	0.023	0.083	1.02E-01	10	2	0	22.20
rs2834167	21	34640788	A	G	IL10RB	p.K47E	0.535	0.375	1.51E-01	5	5	2	22.60
rs2273346	1	11090897	A	G	MASP2	p.V377A	0.174	0.292	1.71E-01	6	5	1	12.45
rs1054480	5	178540975	G	A	ADAMTS2	p.P1177S	0.394	0.250	2.09E-01	7	4	1	15.88
rs4673	16	88713236	A	G	CYBA	p.Y72H	0.905	0.833	2.80E-01	0	4	8	22.70
rs115303435	20	62326159	G	A	RTEL1	p.A836T	0.047	0.083	3.15E-01	10	2	0	11.97
rs2271211	5	178771082	C	T	ADAMTS2	p.V74M	0.048	0.083	3.24E-01	11	0	1	25.90
rs117304542	2	47233124	C	G	TTC7A	p.Q23E	0.053	0.083	3.69E-01	10	2	0	18.95
rs2303650	5	94826655	C	A	TTC37	p.R1296S	0.144	0.208	3.77E-01	8	3	1	23.00
rs17084873	5	94861118	G	C	TTC37	p.L437V	0.145	0.208	3.80E-01	8	3	1	18.19
rs506121	9	271638	C	T	DOCK8	p.A22V	0.368	0.458	4.00E-01	4	5	3	23.30
rs2231495	22	17669306	T	C	ADA2	p.H94R	0.174	0.083	4.15E-01	10	2	0	10.81
rs35441642	10	14976727	G	C	DCLRE1C	p.P56R	0.186	0.250	4.30E-01	7	4	1	23.00
rs2290846	4	151199080	G	A	LRBA	p.S2797L	0.291	0.208	5.01E-01	7	5	0	22.50
rs3749574	4	151207127	C	T	LRBA	p.A2692T	0.289	0.208	5.02E-01	7	5	0	19.45
rs12711521	1	11090916	C	A	MASP2	p.D371Y	0.700	0.625	5.04E-01	1	7	4	23.40
rs2304290	2	47251469	G	C	TTC7A	p.V184L	0.559	0.625	5.46E-01	1	7	4	11.34
rs12614	6	31914179	C	T	CFB	p.R32W	0.066	0.083	6.70E-01	11	0	1	15.86
rs1782360	4	151773593	G	C	LRBA	p.A1090G	0.109	0.125	7.41E-01	9	3	0	11.77
rs1805388	13	108863591	G	A	LIG4	p.T9I	0.114	0.125	7.50E-01	9	3	0	18.87
rs10279499	7	92733766	C	A	SAMD9	p.V549L	0.139	0.167	7.65E-01	8	4	0	21.80
rs2232074	20	6065729	C	T	FERMT1	p.R526K	0.633	0.667	8.34E-01	2	4	6	19.44
rs2227973	11	36597313	A	G	RAG1	p.K820R	0.600	0.583	8.39E-01	2	6	4	17.33
rs334773	3	3170792	C	T	TRNT1	p.P23L	1.000	1.000	1.00E+00	0	0	12	14.47
rs3730089	5	67588148	G	A	PIK3R1	p.M56I	0.149	0.125	1.00E+00	9	3	0	21.10
rs568040559	5	178772259	G	GGCA	ADAMTS2	p.L23_P24insL	0.340	0.333	1.00E+00	5	6	1	12.99
rs529208	9	286593	C	A	DOCK8	p.P29T	0.753	0.750	1.00E+00	1	4	7	23.40
rs2301612	9	136301982	C	G	ADAMTS13	p.Q448E	0.190	0.167	1.00E+00	9	2	1	11.74
rs12768894	10	14974905	T	C	DCLRE1C	p.H128R	0.085	0.083	1.00E+00	10	2	0	14.70

^a Positions are based on the Genome Reference Consortium human build 37 (GRCh37).

^b CADD scores are based on CADD model GRCh37-v1.6.

IBD, inflammatory bowel disease; OFG, orofacial granulomatosis; CHR, chromosome number; Ref, reference allele; Alt, alternative allele; AA change, amino acid change caused by each variant; MAF, minor allele frequency; ToMMo, Tohoku Medical Megabank Organization; CADD, Combined Annotation Dependent Depletion.

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Orofacial granulomatosis in pediatric Crohn’s disease: clinical outcomes and genetic background in the era of biologics: a retrospective study in Japan

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Orofacial granulomatosis in pediatric Crohn’s disease: clinical outcomes and genetic background in the era of biologics: a retrospective study in Japan

Fig. 1. Genetic analysis workflow. OFG, orofacial granulomatosis; CADD, Combined Annotation Dependent Depletion; IBD, inflammatory bowel disease; ToMMo, Tohoku Medical Megabank Organization; MAF, minor allele frequency; PCR, polymerase chain reaction.

Fig. 2. Physical and histopathologic findings of orofacial granulomatosis in this study. (A) Lip swelling in patient 1. (B) Oral ulcer in patient 2. (C) Angular cheilitis in patient 3. (D) Gingivitis in patient 11. (E) Noncaseating epithelioid cell granuloma in a lip biopsy specimen from patient 5 (hematoxylin and eosin staining, ×400).

Fig. 3. Disease course of OFG and intestinal lesions. (A) Parallel course pattern for OFG and intestinal lesions (n=6). (B). Pattern in which intestinal lesions improved but OFG did not (n=4). (C). Pattern in which intestinal lesions were in remission but OFG lesions flared up (n=3). OFG, orofacial granulomatosis.

Fig. 1.

Fig. 2.

Fig. 3.

Orofacial granulomatosis in pediatric Crohn’s disease: clinical outcomes and genetic background in the era of biologics: a retrospective study in Japan

Characteristic	Value (n = 13)
Sex (female: male)	9:4
Family history^a	0
Age at diagnosis of OFG (yr)	9.2 (3.8–15.3)
Age at diagnosis of CD (yr)	10.3 (6.4–15.3)
OFG preceded CD (yr)	2.8 (1.0–6.1)
Timing of OFG onset
Prior to CD	7 (53.8)
Same time as CD	6 (46.2)
wPCDAI at diagnosis
Mild (wPCDAI ≤ 30)	6 (46.2)
Moderate (30 < wPCDAI ≤ 57.5)	3 (23.1)
Severe (wPCDAI > 57.5)	4 (30.8)
Paris classification
L1	3 (23.1)
L2	1 (7.7)
L3	11 (84.6)
L4a	7 (53.8)
L4b	6 (46.2)
Stricture	1 (7.7)
Perianal disease	7 (53.8)
Upper GI lesion	8 (61.5)
Extraintestinal manifestation
Dermatitis	5 (38.5)
Arthritis	1 (7.7)
Observation after CD diagnosis (yr)	6.1 (1.0–8.2)

No.	Sex	Age at CD diagnosis	Paris classification	wPCDAI at diagnosis	Treatment response of OFG								Remission of OFG during the observation period	NCF1 p.Arg90His
No.	Sex	Age at CD diagnosis	Paris classification	wPCDAI at diagnosis	EEN	5-ASA	PSL	AZA/6-MP	IFX	ADL	UST	Other treatments effective for OFG	Remission of OFG during the observation period	NCF1 p.Arg90His
1	M	9 yr 2 mo	L3, B1, P0	42.5	Poor	Poor	Good	Poor	Good		Good	Zinc: good	Yes	Heterozygous
2	F	13 yr 9 mo	L3+L4a, B1, P1	70.0	Poor	Poor	Good	Poor	Partial	Partial	Good		Yes	Heterozygous
3	F	15 yr 3 mo	L3+L4ab, B1, P1	12.5	Partial					Good	Good		Yes	Heterozygous
4	M	9 yr 10 mo	L3+L4ab, B1, P1	52.5	Partial	Poor		Partial		Good	Good		Yes	Heterozygous
5	F	11 yr 2 mo	L1, B1, P1	65.0	Good	Partial		Poor		Good			Yes	Negative
6	F	10 yr 2 mo	L3+L4ab, B1, P1	65.0	Partial		Partial	Partial	Partial	Partial	Partial		Yes	Heterozygous
7	F	11 yr 4 mo	L1, B1, P1	7.5	Partial	Partial		Partial		Partial			No	Homozygous
8	M	13 yr 11 mo	L3+L4b, B1, P1	17.5	Poor	Poor				Poor		Topical PSL: partial	No	Negative
8	M	13 yr 11 mo	L3+L4b, B1, P1	17.5	Poor	Poor				Poor		Topical Tac: partial	No	Negative
9	F	13 yr 7 mo	L3+L4a, B1, P0	52.5	Partial				Partial				No	Heterozygous
10	F	6 yr 5 mo	L2+L4a, B2, P0	80.0	Good		Good	Partial					No	NA
11	F	10 yr 3 mo	L3+L4b, B1, P0	7.5	Poor	Poor						Topical PSL: partial	No	Heterozygous
11	F	10 yr 3 mo	L3+L4b, B1, P0	7.5	Poor	Poor						Topical Tac: partial	No	Heterozygous
12	M	8 yr 1 mo	L3, B1, P0	7.5		Poor						Topical PSL: good	No	Negative
12	M	8 yr 1 mo	L3, B1, P0	7.5		Poor						DXS Elixir: partial	No	Negative
13	F	9 yr 9 mo	L1+L4ab, B1, P0	7.5		Partial							Yes	Negative

rsIDs	CHR	Position^a	Ref	Alt	Genes	AA change	MAF in ToMMo	MAF in OFG	P-value	Ref/Ref	Hetero	Alt/Alt	CADD score^b
rs201802880	7	74193642	G	A	NCF1	p.R90H	0.011	0.375	2.70E-12	4	7	1	23.70
rs1800470	19	41858921	G	A	TGFB1	p.P10L	0.486	0.750	1.28E-02	0	6	6	19.21
rs5956583	X	123034511	A	C	XIAP	p.Q423P	0.047	0.150	8.10E-02	9	2	1	14.32
rs2274064	1	183542387	T	C	NCF2	p.K136R	0.639	0.458	8.73E-02	2	9	1	14.86
rs80346348	16	67685694	G	A	CARMIL2	p.G845D	0.023	0.083	1.02E-01	10	2	0	22.20
rs2834167	21	34640788	A	G	IL10RB	p.K47E	0.535	0.375	1.51E-01	5	5	2	22.60
rs2273346	1	11090897	A	G	MASP2	p.V377A	0.174	0.292	1.71E-01	6	5	1	12.45
rs1054480	5	178540975	G	A	ADAMTS2	p.P1177S	0.394	0.250	2.09E-01	7	4	1	15.88
rs4673	16	88713236	A	G	CYBA	p.Y72H	0.905	0.833	2.80E-01	0	4	8	22.70
rs115303435	20	62326159	G	A	RTEL1	p.A836T	0.047	0.083	3.15E-01	10	2	0	11.97
rs2271211	5	178771082	C	T	ADAMTS2	p.V74M	0.048	0.083	3.24E-01	11	0	1	25.90
rs117304542	2	47233124	C	G	TTC7A	p.Q23E	0.053	0.083	3.69E-01	10	2	0	18.95
rs2303650	5	94826655	C	A	TTC37	p.R1296S	0.144	0.208	3.77E-01	8	3	1	23.00
rs17084873	5	94861118	G	C	TTC37	p.L437V	0.145	0.208	3.80E-01	8	3	1	18.19
rs506121	9	271638	C	T	DOCK8	p.A22V	0.368	0.458	4.00E-01	4	5	3	23.30
rs2231495	22	17669306	T	C	ADA2	p.H94R	0.174	0.083	4.15E-01	10	2	0	10.81
rs35441642	10	14976727	G	C	DCLRE1C	p.P56R	0.186	0.250	4.30E-01	7	4	1	23.00
rs2290846	4	151199080	G	A	LRBA	p.S2797L	0.291	0.208	5.01E-01	7	5	0	22.50
rs3749574	4	151207127	C	T	LRBA	p.A2692T	0.289	0.208	5.02E-01	7	5	0	19.45
rs12711521	1	11090916	C	A	MASP2	p.D371Y	0.700	0.625	5.04E-01	1	7	4	23.40
rs2304290	2	47251469	G	C	TTC7A	p.V184L	0.559	0.625	5.46E-01	1	7	4	11.34
rs12614	6	31914179	C	T	CFB	p.R32W	0.066	0.083	6.70E-01	11	0	1	15.86
rs1782360	4	151773593	G	C	LRBA	p.A1090G	0.109	0.125	7.41E-01	9	3	0	11.77
rs1805388	13	108863591	G	A	LIG4	p.T9I	0.114	0.125	7.50E-01	9	3	0	18.87
rs10279499	7	92733766	C	A	SAMD9	p.V549L	0.139	0.167	7.65E-01	8	4	0	21.80
rs2232074	20	6065729	C	T	FERMT1	p.R526K	0.633	0.667	8.34E-01	2	4	6	19.44
rs2227973	11	36597313	A	G	RAG1	p.K820R	0.600	0.583	8.39E-01	2	6	4	17.33
rs334773	3	3170792	C	T	TRNT1	p.P23L	1.000	1.000	1.00E+00	0	0	12	14.47
rs3730089	5	67588148	G	A	PIK3R1	p.M56I	0.149	0.125	1.00E+00	9	3	0	21.10
rs568040559	5	178772259	G	GGCA	ADAMTS2	p.L23_P24insL	0.340	0.333	1.00E+00	5	6	1	12.99
rs529208	9	286593	C	A	DOCK8	p.P29T	0.753	0.750	1.00E+00	1	4	7	23.40
rs2301612	9	136301982	C	G	ADAMTS13	p.Q448E	0.190	0.167	1.00E+00	9	2	1	11.74
rs12768894	10	14974905	T	C	DCLRE1C	p.H128R	0.085	0.083	1.00E+00	10	2	0	14.70

Table 1. Clinical Characteristics of CD in 13 Patients

Values are presented as median (range) or number (%).

Within 2 generations.

CD, Crohn’s disease; OFG, orofacial granulomatosis; wPCDAI, weighted Pediatric Crohn’s Disease Activity Index; GI, gastrointestinal.

Table 2. Disease Type, Treatment Response, Course, and Presence of NCF1 Variants in 13 Cases

Table 3. Candidate Monogenic IBD Variants Potentially Causal for OFG in Crohn’s Disease