Infantile Onset Intractable Inflammatory Bowel Disease Due to Novel Heterozygous Mutations in TNFAIP3 (A20)

Abstract

Background

Mutations in tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a key player in the negative feedback regulation of nuclear factor-κB signaling, have recently been recognized as leading to early onset autoinflammatory and autoimmune syndrome. Here, we have reported the phenotypes of 3 infantile onset intractable inflammatory bowel disease (IBD) patients with TNFAIP3 mutations and reviewed previously reported cases to establish phenotypic features associated with TNFAIP3 monogenicity.

Methods

From January 1, 2015, to December 31, 2017, we recruited 58 infantile-onset IBD patients. Targeted sequencing and whole-exome sequencing were performed. Sanger sequencing confirmed the variants and determined the parental origin. We followed all the patients with TNFAIP3 mutations in our cohort and analyzed their clinical data.

Results

Genetic screening in all 58 patients with infantile-onset IBD revealed 44 (75.9%) cases of monogenic disorders, and 3 de novo TNFAIP3 mutations were identified, including 1 nonsense and 2 frame shift mutations. All the mutations resulted in premature stop codon. All 3 patients had multiple systemic involvements, with predominant gastrointestinal diseases.

Conclusions

Most infantile-onset IBD was associated with monogenetic mutation, and in addition to the 50 reported genes, other rare genetic variants need to be determined. TNFAIP3 may be an important candidate gene. The treatment of TNFAIP3-associated infantile-onset-IBD was challenging.

infantile-onset inflammatory bowel disease, TNFAIP3 (A20), monogenic disease

INTRODUCTION

Increasing understanding of age-specific characteristics has led to changes in the classification of pediatric inflammatory bowel disease (IBD). Very early-onset inflammatory bowel disease (VEOIBD) refers to children with IBD diagnosis established before the sixth year of life.¹ Due to the specificity of VEOIBD, the Paris consensus proposed a specific classification for the subgroup of patients with infantile onset IBD² (IOIBD, onset of IBD before 2 years old). An extremely early subgroup, neonatal IBD, has been described with manifestations during the first 27 days of life.³ VEOIBD has an estimated incidence of 4.37/100,000 children and a prevalence of 14/100,000 children,⁴ which constitutes 4% to 10% of pediatric IBD.^5,6

VEOIBD includes conventional/standard IBD as well as monogenic IBD-like diseases.⁷ While standard IBDs are common, monogenic disorders are very rare.⁸ For patients with VEOIBD and infantile IBD in particular, environmental exposure time is extremely limited, and higher rates of affected first-degree family relatives indicate an increased genetic component. Ye et al⁹ recently reported that 71.1% of infantile-onset IBD cases were associated with monogenic mutations. Approximately 50 genetic variants associated with IBD-like intestinal inflammation were reported.¹⁰ For patients with genetic disorders, symptoms may be more serious and associated with higher mortality, and a different treatment strategy than conventional IBD is often required.^7,11 It is therefore paramount to establish effective diagnostic and management strategies early in the disease course. Even though whole exome sequencing (WES) has been proven useful in helping to properly diagnose new variants of these genetic defects, a correct diagnosis is frequently challenging and often delayed for the broad spectrum of these extremely rare diseases.

A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), plays a critical role in the negative feedback regulation of nuclear factor-kB signaling in response to multiple stimuli.¹² More specifically related to IBD, a recent genome-wide association study identified A20 as a Crohn disease (CD) susceptibility gene,¹³ and mice lacking A20 specifically in dendritic cells spontaneously developed lymphocyte-dependent colitis, arthritis, and enteritis, conditions stereotypical of human IBD.¹⁴ Our previous work found that there is an excessive inflammatory response but insufficient upregulation of A20 expression in IBD patients,¹⁵ and A20 plays an important role in inhibiting lipopolysaccharide-induced inflammation in enterocytes.¹⁶ Although many basic studies have confirmed that A20 is a central gatekeeper in inflammation and immunity, the first human disease caused by a high penetrance pathogenic mutation of A20 was not reported until 2016 by Zhou et al,¹⁷ followed by reports of several cases of TNFAIP3 deficiency.^18–21 Herein, we describe 3 cases of infantile onset intractable IBD caused by high penetrance heterozygous mutations of A20. These children were diagnosed with infantile-onset IBD based on the Porto criteria.²² We know that several monogenic IBD-like diseases were reported, but among the 50 genes, A20 was not included. Our reports may contribute to the understanding of IBD related to A20 gene deficiency, enhancing our knowledge about monogenic-associated IBD-like diseases.

MATERIALS AND METHODS

Patients

From January 1, 2015, to December 31, 2017, we recruited 58 infantile-onset IBD patients and performed WES and Sanger sequencing. These children were diagnosed with infantile-onset IBD based on the Porto criteria.²²

Ethics and Informed Consent

Informed consent for participation and blood sample collection were obtained from parents, with approval from the research ethics board at the Children’s Hospital of Fudan University.

Genetic Sequencing

The methods of genetic sequencing were described previously by Ye et al.⁹

RESULTS

Among 58 patients with infantile-onset IBD, 75.9% (44/58) were determined to have monogenetic mutations, including interleukin 10 receptor subunit alpha (IL10RA) causal mutations in 36/58 (62.1%) patients, TNFAIP3 causal mutations in 3/58 (5.2%), forkhead box P3 (FOXP3) causal mutations in 2/58 (3.4%), epithelial cell adhesion molecule (EPCAM) causal mutations in 1/58 (1.7%), lipopolysaccharide-responsive and beige-like anchor protein (LRBA) causal mutations in 1/58 (1.7%),and WAS causal mutations in 1/58(1.7%). In the remaining 14 cases, we did not identify known disease-causing mutations. Among the 36 patients with IL10RA mutations, 24 cases were reported by Ye et al,⁹ and 2 cases were reported by Huang et al²³ (case 21 and 28). Two patients with FOXP3 mutations, 1 patient with EPCAM mutation, and 1 patient with LRBA mutation were also described by Ye et al.⁹ The genetic information of the 10 previously unreported patients with IL10RA mutations is displayed in Supplementary Table 1.

For these 3 patients with TNFAIP3 mutations, clinical features and genetic information are summarized in Tables 1, 2, and 3. In our study, WES and Sanger sequencing (Supplementary Fig. 1) were performed, and 3 new mutations were detected, including 1 nonsense mutation, 1 deletion mutation, and 1 insertion mutation. All the mutations resulted in premature codon stop. To increase our understanding of the disease caused by mutations in the A20 gene, we searched the literature in the PubMed and HGMD databases from January 1, 2016, to December 31, 2017. The total number of A20 mutations that we found from this search was 23 (all patients were genetically defined), and the clinical manifestations and genetic information of each are summarized in Table 4.

Table 1:

Clinical Characteristics of the 3 Patients with TNFAIP3 Mutations

Patient	Gender	Admission Age	Onset Age	Height at Admission	Weight at Admission	Initial Symptoms	Affected Intestinal Tract	Arthritis	Oral Ulcers	Genital Ulcers	Skin Rash	Ophtho	Other Phenotypes
P1*	F	3 y, 4 m	2 m	96 cm	12.6 kg	Diarrhea and fever	Whole gastrointestinal tract	Yes	Yes	No	Yes	No	Hepatitis
P2	M	5 y, 4 m	1 m	98 cm	15.4 kg	Diarrhea	Whole gastrointestinal tract	Yes	Yes	No	No	No	Hepatosplenomegaly
P3	M	2 y, 8 mo	6 m	83 cm	10.4 kg	Diarrhea, fever, and oral ulcers	Duodenum and colon (small intestine was not examined)	No	Yes	No	Yes	No	Perianal abscess

*Patient1 was previously described by Ye et al.⁹

Table 2:

Clinical Characteristics and Treatment Response of the 3 Patients with TNFAIP3 Mutations

Patient	Gastroduodenoscopy	Capsule Endoscopy	Colonoscopy	Histopathology	Calprotectin at Admission	ESR at Admission (mm/h)	CRP at Admission (mg/L)	Medication
P1	Antral ulcer and duodenal inflammation	Small intestine inflammation and ulcers	Rectum, total colonic ulcers, (predominantly small, superficial ulcers), terminal ileitis;	Crypt abscesses, partial gland atrophy, lymphocytes, and plasma cell infiltrate	>1800 μg/g	42	33	MSLZ, CS, IFX, and thalidomide
P2	Cardiac ulcer and hemorrhagic gastritis	Small intestine inflammation and ulcers	Rectum, total colonic, terminal ileum ulcers (predominantly small, superficial ulcers)	Focal chronic inflammation, transmural inflammatory infiltrate， crypt atrophy, and crypt architecture	>1800 μg/g	64	25	MSLZ, MTX, CS, IFX, and Recombinant Human Tumor Necrosis Factor-Ⅱ
P3	Duodenal ulcer, gastritis and esophagitis	NA	Colon and terminal ileum ulcers	Focal epithelial cells gathered, mucosal active inflammation, cryptitis, and crypt atrophy	997 μg/g	79	132	MSLZ, CS and thalidomide

CRP indicates C-reactive protein; CS, corticosteroids; ESR, erythrocyte sedimentation rate; IFX, infliximab; MTX, methotrexate; MSLZ, mesalazine; NA, not available.

Table 3:

Genetic Information of the 3 Patients with TNFAIP3 Mutations

Patient	Ancestry	Inheritance	Nucleotide Alteration	Exon	cDNA Alteration	Amino Acid Alteration	Domain
P1	Chinese Han	De novo	chr6:138199687delC	7	c.1108het_delC NM_006290	p.Q370Rfs*16	OUT-ZnF1 link region
P2	Chinese Han	De novo	chr6:138199897_138199898insA	7	c.1315_1316insA NM_006290	p.R439Qfs*6	ZnF1-ZnF2 link region
P3	Chinese Han	De novo	Chr6:138192618 G>A	2	c.254G>A NM_006290	p.W85*	OTU

Table 4:

Clinical Manifestations of Individuals Reported with Mutations in TNFAIP3

Ancestry	Mutation Location	Domain	Sex	Onset Age	Mainly Manifestations	References
Canadian (family 1)	chr6:138197178 T>A p.L227* c.680T>A	OTU	F	11 y	Oral and genital ulcers, polyarthritis	Zhou et al17
			F	7 m	Oral ulcers, uveitis, tendonitis
			F	2 y	Oral and genital ulcers, bilateral anterior uveitis, headache, polyarthritis
American (family 2)	chr6:138197169 delT p.F224Sfs*4 c.671delT	OTU	F	5 y	Oral and genital ulcers, skin rash, polyarthritis, periodic fever, hemolytic anemia, asthma
			F	6 y	Oral and genital ulcers, skin rash, polyarthritis, periodic fever
			F	10 y	Oral and genital ulcers, skin rash, polyarthritis, retinal vasculitis, anterior uveitis, CNS vasculitis, chorea, migraine, oropharynx and colon ulcers, idiopathic thrombocytopenic purpura
Turkish (family 3)	chr6:138198218 C>T p.R271* c.811 C>T	OTU	M	13 y	Oral and genital ulcers, arthralgia
			M	7 y	Oral and genital ulcers, terminal ileum ulcers and colitis, |pericarditis in infancy
American (family 4)	chr6:138200391delG p.T604Rfs*93 c.1809 delG	ZnF4	F	2 y	Oral and genital ulcers, anorexia, weight loss
Dutch (family 5)	chr6:138198325 C>G p.Y306* c.918 C>G	OTU	F	16 y	Oral and genital ulcers, skin rash
			F	5 y	Oral and genital ulcers, mild undifferentiated colitis
Turkish (family 6)	chr6:138197297delG p.P268Lfs*19 c.799 delG	OTU	F	29 y	Oral and genital ulcers, mild fever, skin rash
			F	15 y	NA
			F	13 y	NA
Britain (family 7)	chr6:138200048_138200049delTG p.V489Afs*7 c.1466_1467delTG	ZnF2	M	10 y	Insulin-dependent diabetes, cytopenias, hepatitis, enteropathy, interstitial lung disease	Duncan et al18,20
Japan (family 8)	chr6: 138192616delC p.W85Gfs*11 c.252delC	OTU	F	11 y	Fever, polyarthritis, palpebral conjunctiva ulcer, stomatitis, genital ulcer, colon ulcers, aquagenic acrokeratoderma	Ohnishi et al19
			F	9 m	Recurrent hematemesis, stomatitis, duodenal ulcers, genital ulcers
Japan (family 9)	chr6: 138197226G>A p.C243Y c.728G>A	OTU	M	9 y	Oral ulcers, fever, nephrotic syndrome, perianal ulcer, skin rash, pseudofolliculitis	Shigemura et al20
			F	8 y	Oral and genital ulcers, intestinal ulcers, skin rash, pseudofolliculitis,
			F	8 y	Oral and genital ulcers, fever, skin rash, pseudofolliculitis,
			F	10 y	Oral and genital ulcers, skin rash, pseudofolliculitis
			F	12 y	Oral and genital ulcers, skin rash, pseudofolliculitis
Japan (family 10)	chr6: 138199826_138199831delAAAC p.K417Sfs*4 c.1245_1247delAAAC	ZnF1-ZnF2 link region	M	7 m	Fever, liver dysfunction, lymph node swelling, skin rash, hepatosplenomegaly	Takagi et al21

F indicates female; M, male; OUT, ovarian tumor domain; NA, not available.

As far as we know, 26 patients (including our 3 patients) with A20 mutations were identified, including 19 females and 7 males. These 26 patients came from 13 families, and different families had different mutation sites. The mutations occurred de novo in 6/13 of the probands. In the 13 mutations, 5 were base substitution and 8 were frame shift mutations caused by base (or bases) deletion (7/8) or insertion (1/8). Among the 13 mutations, 92.3% (12/13) resulted in premature codon termination, and 61.5% mutations were located in the OTU domain (8/13). Figure 3 provides the location of the mutations. Among the 26 patients, 24 had detailed clinical information. For these 24 patients, most had oral ulcers (83.3%, 20/24), 70.8% had genital ulcers (17/24), 54.2% had skin rash (13/24), 41.7% had gastrointestinal ulcers or colitis (10/24), and 37.5% of cases involved the joints (9/24).

The detailed information of our 3 patients with TNFAIP3 mutations:

• P1: The patient is a girl who presented at 2 months with blood and mucus in stool (the stool frequency was 5 to 6 times/d) and intermittent fever of unknown origin. She experienced polyarthritis (nondeformation, predominantly large joints, such as knees, ankles, and hips) at 1.5 years old. Recurrent oral ulcers occurred at 2 years old, and in the course of the disease, there were intermittent various erythema nodosum-like lesions on the skin. She was referred to our hospital at 40 months of age, and gastroscopy, capsule endoscopy, and colonoscopy revealed disseminated multiple ulcers involving the entire gastrointestinal tract (Fig. 1A–B). Biopsy detected mucosal inflammation with a large number of lymphocytes and plasma cell infiltration, crypt abscess, and partial glandular atrophy. Computed tomography (CT) examination found that the intestinal wall was thickened (Fig. 2B–C). Fecal calprotectin was more than 1800 μg/g. The child was treated with mesalazine and corticosteroids (steroid was used for a total of 29 weeks) for a presumptive diagnosis of CD, but with limited success. After application of steroids for 1 month, anti-tumor necrosis factor-α treatment (infliximab) was initiated. There was no significant improvement in clinical symptoms after infliximab was administered 3 times, so thalidomide was attempted, and the clinical symptoms were partially alleviated.

• P2: This patient is a 5-year-old boy who suffered from diarrhea after birth and experienced joint pain from 4 years old. He was diagnosed with juvenile idiopathic arthritis in the local hospital, and recombinant human tumor necrosis factor receptorⅡand methotrexate were given. The symptoms of joint swelling and pain were improved, but there was no significant improvement in diarrhea, which was accompanied by abdominal pain and recurrent oral ulcers. Gastroscopy, capsule endoscopy, and colonoscopy revealed extensive ulcers involving the entire gastrointestinal tract (Fig. 1C). Histology of biopsy samples showed focal chronic inflammation, transmural inflammatory infiltrate，crypt atrophy, and crypt architecture. Magnetic resonance imaging (MRI) found joint capsule effusion with abnormal enhancement of the wall (Fig. 2A). Abdominal CT revealed patchy liver lesions and splenomegaly (Fig. 2D). Fecal calprotectin was more than 1800 μg/g. The patient was treated with methotrexate and infliximab. After 4 doses of infliximab (5 mg/kg, at 0, 2, 6, and 14 weeks), the effect was still not satisfactory.

• P3: This patient is a boy who presented with bloody, mucous diarrhea and intermittent fever in the first 6 months of life (the stool frequency was more than 10 times/d), and colonoscopy detected multiple ulcers in the colon. Perianal abscess occurred at 8 months of age, and he suffered from oral ulcers and skin rash since 2 years old. He was referred to our hospital at 32 months of age, and endoscopies revealed ulcers involving the upper gastrointestinal tract, colon, and terminal ileum. After administration of corticosteroids for 3 months, the clinical symptoms and endoscopic performance did not improve. Then, thalidomide was attempted.

FIGURE 1.

Endoscopic appearance of patients with TNFAIP3 mutations. A, Deep ulcerations of the small intestine detected by capsule endoscopy. B, Small, superficial ulcers in the colon. C, Large, deep ulcers in the colon.

FIGURE 2.

Radiological images from patients with TNFAIP3 mutations. A, Left knee joint synovitis. B and C, Colon wall thickening with abnormal enhancement. D, Hepatosplenomegaly.

FIGURE 3.

Schematic of TNFAIP3/A20 protein domains. The N-terminal ovarian tumor domain (OTU) is essential for deubiquitinase activity, and C-terminal zinc finger (ZF1-7) domains mediate E3 ubiquitin-ligase activity. The locations of the mutations were indicated with arrows (our 3 patients were marked using red arrows).

DISCUSSION

With the development of molecular diagnostic techniques, an increasing number of monogenetic associated IBD-like intestinal inflammation diseases have been discovered, and VEOIBD has been described in a number of hyperinflammatory, autoinflammatory, and immunodeficiency disorders.^10,24,25 For example, approximately 40% of patients with chronic granulomatous inflammation develop CD-like intestinal inflammation,^26–28 and at least 20% of patients with X-linked inhibitor of apoptosis protein defects develop a CD-like immunopathology with severe fistulizing perianal phenotype.^29–33 Differences in the prognosis and medical management of these diseases argue that a genetic diagnosis should not be missed. As mentioned by Ye et al,⁹ at our center focusing on pediatric IBD diagnosis and treatment, we have developed a standardized protocol for patients with infantile-onset IBD. In recent years, genetic sequencing has been performed for every patient diagnosed with infantile-onset IBD in our center. This cohort included 58 cases of infantile-onset IBD, and 75.9% were associated with monogenic disorders, except for IL-10RA, TNFAIP3, FOXP3, EPCAM, LRBA, and WAS were also established.

A20 is a cytoplasmic zinc finger protein that was originally identified as a TNF-inducible protein and which has been characterized as a dual inhibitor of NF-κB activation and cell death.³⁴ A20-deficient mice (A20^-/^-) display persistent NF-κB activation, spontaneous multi-organ inflammation, and early lethality.³⁵ Enterocyte-specific A20 deficiency sensitizes to experimental colitis.³⁶ It is well known that polymorphisms of A20 gene are related to multiple human diseases, including rheumatoid arthritis, systemic lupus erythematosus, psoriasis, type 1 diabetes, and inflammatory bowel disease.³⁷ Although there were several basic and correlation analysis studies on A20, the first clinical case caused by A20 mutations was not reported until 2016.¹⁷ Later, some other cases were identified and reported.^18–21 For the 24 patients with A20 mutations, all of them had multisystem involvement, most of them had oral ulcers (83.3%), genital ulcers (70.8%), and gastrointestinal ulcers or colitis (41.7%), and arthritis (37.5%) was also very common. In our cohort, IL10 axis deficiency-associated IOIBD was more common. Among the 58 cases with infantile-onset IBD, 62.1% of cases were due to the IL10RA mutation. With increasing reported cases, the phenotypic features of IL10-associated IBD-like diseases were well documented. Huang²³ et al recently reported the largest cohort of 42 patients with IL10R mutations based on a multicenter study in China. As reported by Huang et al, the incidence of oral ulcers was 47.6%, and the incidence of perianal complications was at least 88%. No joint involvement was detected. Furthermore, typical endoscopic lesions of patients with IL10 axis deficiency were deep cobblestone ulcerations. However, in our 3 patients with TNFAIP3 mutations, the lesions were predominantly extensive superficial ulcers with some large deep ulcers. If perianal lesions are a hallmark of IL10-related IBD, oral and (or) genital ulcers, arthritis would be the characteristic of A20-related disease, but the number of cases is small. To establish a phenotype associated with A20 genotype, more cases need to be identified.

The study also underlines the notion that IOIBD represents a distinct and heterogeneous group of patients, a significant fraction of whom may bear monogenetic defects underlying their disease. In this study, we can see that the occurrence of diarrhea was very early, occurring at the age of 2 months in one case, during the neonatal period in another, and within 6 months of life in the third case. The gastrointestinal ulcers were extensive, diffuse ulcers involving the entire gastrointestinal tract. Polyarthritis is very common and early, including the large and small joints, and predominantly asymmetrical nondeforming polyarthritis. Inflammatory indexes, such as C-reactive protein, erythrocyte sedimentation rate, and fecal calprotectin were elevated significantly. The treatment is very challenging, as corticoids, immunosuppressive agents, and even biological agents were ineffective. As reported, the severity of the disease and the response to the treatment were varied, with some patients even requiring stem cell transplantation.^17–21 These may be associated with different mutation sites.

There are limitations in this study. Although in previous studies, several disease-causing mutations in TNFAIP3 have been functionally analyzed and multiple nonsense and frame shift variants associated with impaired function of TNFAIP3 have been identified,¹⁷ in this study, all 3 mutations resulted in premature stop signs. In addition, these 3 patients had obvious and similar clinical symptoms, and genetic testing detected TNFAIP3 mutations without identifying other genetic mutations that can explain their clinical symptoms. Therefore, we speculate that the clinical manifestations were due to mutations in TNFAIP3. However, functional verification was not conducted.

In conclusion, this study represents the first cases due to TNFAIP3 gene mutation reported in Chinese populations. The very early onset IBD-like illness described here represents a novel manifestation of this genetic syndrome. The onset symptom of our 3 patients was diarrhea, and the gastrointestinal involvement was predominant. Awareness of this disease is warranted for the gastroenterologist to establish effective diagnostic and management strategies early in the disease course and offer genetic counseling for family members of the patients. In addition, ongoing genetic and functional studies will further elucidate the molecular etiology of A20 gene mutation-associated disease and open avenues for personalized therapeutic approaches in the future.

ACKNOWLEDGMENTS

The authors express our gratitude to Drs Huijun Wang, Bingbing Wu, and Ping Zhang from the Shanghai Key Laboratory of Birth Defects of the Translational Medicine Center of Child Development and Disease of Fudan University, Children’s Hospital of Fudan University, who performed WES and bioinformatic analysis. The authors thank all the patients and families who participated in the study.

The authors have no conflicts of interest to disclose.

This work was supported by the National Natural Science Foundation of China (81300291) and Jiujiu Charitable Trust-PIBD China.