Novel NR4A1 Arg293Ser Mutation in Patients With Familial Crohn’s Disease

Abstract

Background/Aim: The underlying etiology of Crohn’s disease remains unknown. The aim of this study was to identify genomic alterations associated with the development of Crohn’s disease in one Japanese family with a family history of Crohn’s disease. Materials and Methods: We performed whole-exome sequence and pedigree analysis of a Japanese family in which both sisters developed Crohn’s disease. Whole-exome sequencing was performed using the Ion Torrent Proton™ system. Data from the Proton runs were initially processed using the Ion Torrent platform-specific pipeline software Ion Reporter. An autosomal dominant mode of inheritance was assumed, and stringent selection criteria were applied. Results: A substitution in the NR4A1 gene at codon 293 resulting in an amino acid change from arginine to serine was identified only in the affected sisters. Conclusion: The impaired DNA-binding capacity of the NR4A1 protein due to an NR4A1 germline mutation may be a possible cause of Crohn’s disease.

Crohn’s disease (CD) (OMIM 266600) is an inflammatory bowel disease (IBD) that forms discontinuous erosions and ulcers throughout the digestive tract. Various genetic and environmental factors (1) and several risk loci associated with susceptibility to CD have been reported (2,3). However, the definitive etiology remains elusive.

In this study, we used whole-exome sequencing and pedigree analysis of a Japanese family with CD to evaluate which germline alterations affected the onset of CD.

Materials and Methods

Written informed consent for this study and publication were obtained from all study participants and their parents. The study was approved by the Research Ethics Committee of the Institute of Biomedical Research and Innovation (No. 16-02) and conducted in accordance with the ethical principles of the Declaration of Helsinki.

Patients and family members. Patients III-6 and III-7 were diagnosed with CD at the ages of 18 and 20, respectively. Their initial symptoms were abdominal pain, diarrhea, and bloody stool. Their unaffected father (II-3) has a history of sarcoidosis at the age of 27. He was diagnosed with a chest computed tomography (CT) and his condition had regressed without treatment.

Mutation detection: Whole-exome sequencing. Whole-exome sequencing was performed using the Ion Torrent Proton™ system (Thermo Fisher Scientific, Waltham, MA, USA) in conjunction with the Ion AmpliSeq™ Exome RDY Kit PIV3 (Thermo Fisher Scientific).

Ion Torrent library preparation and sequencing. An Ion Torrent adapter-ligated library was generated following the Ion AmpliSeq™ Exome RDY Kit Library Preparation manufacturer’s protocol (Thermo Fisher Scientific, MAN0010084). Briefly, 100 ng of gDNA was used as the starting material in the AmpliSeq exome amplification step. The amplicons were end-repaired, and the Ion Torrent P1 adapters and amplicons were ligated using DNA ligase. Barcoding was performed using the Ion DNA Barcoding kit (Thermo Fisher Scientific). Following AMPure bead purification (Beckman Coulter, CA, USA), the concentrations and sizes of the library components were determined using the Life Technologies StepOne™ system (Thermo Fisher Scientific) and Ion Library TaqMan^® Quantitation Assay Kit (Thermo Fisher Scientific). Sample emulsion PCR, emulsion breaking, and enrichment were performed using the Ion PI™ Hi-Q Chef Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. An input concentration of one DNA template copy/ion sphere particle (ISP) was added to the emulsion PCR master mix, and the emulsion was generated using the Ion Chef™ system (Thermo Fisher Scientific). Ion PI chips (version 3) and the Ion Torrent Proton system were used to enrich template-positive ISPs and to perform the sequencing.

Variant calling. Data from the Proton runs were initially processed using the Ion Torrent platform-specific pipeline software Ion Reporter (version 5.0) to generate sequence reads, trim adapter sequences, and filter and to remove poor signal-profile reads using the optimized parameters provided by the manufacturer of the AmpliSeq exome. The other filter employed was visual examination of the mutations using CLC Genomics Workbench version 9.0.1 (Qiagen, Hilden, Germany) in addition to the removal of possible strand-specific errors (i.e., a mutation detected only in either the ‘‘plus’’ or ‘‘minus’’ strand but not in both strands). A limitation of this analysis is that structural variants such as large deletions or exon skipping variants could not be detected.

Results

Whole-exome sequencing was performed with DNA samples from patients III-6 and III-7, their unaffected father (II-3), and their unaffected mother (II-4) (Figure 1A). A mutation at codon 293 resulting in an amino acid substitution from arginine to serine, Arg293Ser, was identified in NR4A1 in patient III-6, patient III-7, and their unaffected father (II-3) (Figure 1B). An autosomal dominant mode of inheritance, which is speculative, was assumed, and stringent selection criteria were applied (Table I and Table II). The variant did not exist in the data of 10 non-consanguineous, unrelated persons whose library was created using AmpliSeq Exome Kit and sequenced by Ion Torrent System as in the present study.

Figure 1. Pedigree plotting and sequencing results. A). Pedigree of the study family and the disease status of each member. Squares denote male family members, circles denote female members, shaded symbols denote affected members, and slashes denote deceased members. We performed whole-exome sequencing using DNA from peripheral blood samples from the following four family members: II-1, II-3, III-1, and III-2. B). Sequence chromatogram and direct sequence chart of the mutant genome. The NR4A1 germline mutation c.877C>T, a missense substitution of cysteine for arginine at position 293 (encoding p.Arg293Cys; position chr12:52449814 T/C), was detected with Ion Torrent technology.

Table I. Filtering steps for homozygous variants.

CADD: Combined annotation dependent depletion; FATHMM: functional analysis through Hidden Markov Models (v2.3); PolyPhen: polymorphism phenotyping; SIFT: sorting intolerant from tolerant; ToMMo: Tohoku Medical Megabank Organization.

Table II. Characteristics of the variants identified in the affected members of the studied family.

CADD: Combined annotation dependent depletion; ICK: intestinal cell kinase; FATHMM: functional analysis through Hidden Markov Models (v2.3); mCSM-NA: effects of mutations on protein-nucleic acids interactions; N/A: not applicable; PhyloP: phylogenetic p-values; PolyPhen: polymorphism phenotyping; SIFT: sorting intolerant from tolerant. *http://biosig.unimelb.edu.au/mcsm_na/prediction.

Discussion

NR4A1 was initially known as a nerve growth factor (4), and was also identified as a member of the “orphan” nuclear steroid receptor superfamily (5). NR4A1 is transcriptionally active in many types of cells (6,7) under different culture conditions. Furthermore, the suppression of NR4A1 can inhibit apoptosis (8). Moreover, NR4A1 plays a significant role in the functioning of macrophages (9). The loss of NR4A1 in myeloid cells leads to the acceleration of leukocyte infiltration to the central nervous system (10). The loss of NR4A1 also exacerbates organ fibrosis in the experimental mouse model by dysregulating transforming growth factor-β pathway (11). Thus, NR4A1 is involved in the regulation of fundamental cellular functions, including inflammation and cell survival (12,13).

The NR4A1 gene contains seven exons. Exon2 encodes an N-terminal transactivation domain, exon3 and 4 encode a DNA-binding domain, and dimerization and ligand-binding domains are encoded by exons 5 to 7 (14). The DNA-binding domain is a highly conserved amino acid sequence also found in other DNA-binding proteins such as the retinoid X receptor (RXR), estrogen receptor 2, steroid receptor, and androgen receptor (Figure 2B). This cysteine-rich region appears to fold into two zinc fingers. The DNA-binding domain interacts with cis-acting elements of the regulated genes (15). In the case of the androgen receptor, a mutation within the DNA-binding domain (Arg585Lys) is linked to a positive receptor-binding form that exhibits androgen insensitivity (16). The identified Arg293Ser variant (Figure 2A and B), which is equivalent to Arg585Lys identified in androgen insensitivity syndromes, is located in the DNA-binding domain in exon 4 (Figure 2B) and results in a severely impaired DNA-binding capacity. This Arg293Ser mutation is extremely rare in the general population, and was not documented in the Database for Genome Aggregation.

Figure 2. Structure of mutated NR4A1. A). Crystal structure of the DNA-binding domain of the Arg293-containing protein. B). DNA-binding domain structures of human proteins and the conservation of the Cys293 position across human proteins. The cysteine at this position in the strand is responsible for binding to the promoter region of target genes.

There have been two reports on experimental colitis using the NR4A1^–/– mouse model. In the first report, Hamers et al. defined the role of NR4A1 in IBD in induced mouse IBD models. Inflammation was increased in NR4A1^–/– mice and the mediated invasion of T-cells and monocytes into the colon was more pronounced. The expression of MCP-1, TNFα and IL-6 was increased and the transcription of Foxp3 was decreased. Moreover, the over-expression of NR4A1 in activated RAW macrophages contributed to the upregulation of IL-10 and the down-regulation of TNFα, MIF-1, and MCP-1 transcription via NFĸB repression. The authors concluded that NR4A1 has a protective function in the induced colitis model (17).

In the second report, Wu et al. found genetic variants of NR4A1 associated with susceptibility to both ulcerative colitis (UC) and CD in the most recent GWASs on UC and CD. They confirmed that NR4A1 expression was suppressed in the colon tissues of UC or CD patients. They also revealed that deficiency of NR4A1 increased vulnerability to colitis, whereas treatment with an NR4A1 agonist significantly reduced the induced colitis in the NR4A1^–/– mouse model. The authors concluded that NR4A1 was a key regulator of the TRAF6/TLR/IL-1R-initiated inflammatory pathway in IBD (18).

In an in vitro model, Nr4a1 deletion in B2 cells increased atherosclerosis and was associated with increased T follicular helper cell-Germinal Center response and NR4A1 expression in Marginal Zone B cells regulating PD-L1 expression, limiting the T follicular helper cell-Germinal Center response and protecting from atherosclerosis (19). The NR4A1 expression was elevated in human osteoarthritis cartilage and in an in vitro osteoarthritis model, which could be blocked by the NF-ĸB signal inhibitor JSH23 (20).

Recently, Klepsh et al. identified that the loss of NR2F6 induced spontaneous late-onset colitis in the NR2F6^–/– mouse model (21). They suggested that agonists of NR2F6 might be a potential treatment strategy of human IBD. Onuki et al. reported that the activation of RXR/peroxisome proliferator-activated receptor δ and NR4A1/RXR heterodimers diminished the monocyte-mediated inflammatory response in the gut (22). Liu et al. also reported that NR4A1 is an important regulator of T cell function and a potential target for tumor immunotherapy (23). These observations also support our findings.

In conclusion, we speculate that the onset of CD in this family may be due to the impaired DNA-binding capacity of the NR4A1 protein caused by the NR4A1 Arg293Ser germline mutation. As treatment with an agonist for NR4A1 has been reported to attenuate excessive inflammation in an experimental mouse colitis model (18), NR4A1 protein impairment may be a possible cause of IBD. NR4A1 agonists may be future targets for preventing and treating IBD, and screening for NR4A1 germline mutations will provide important information that can help elucidate the etiology of IBD in the future.

Conflicts of Interest

The Authors have no conflicts of interest to declare regarding this study.

Authors’ Contributions

KM: project development, manuscript writing, study conceptualization and design, data collection and abstraction, and statistical analyses; SF: study conceptualization and design, and manuscript preparation and revision; All Authors read and approved the final version of the manuscript.