In this study, we have reported a novel variant, c.929A>G, within the SLCO2A1 gene. This variant was identified in a patient presenting with both CEAS and PHO. Notably, the c.929A>G variant is characterized by a single nucleotide substitution in exon 7, resulting in abnormal mRNA splicing and the deletion of 16 base pairs. This aberrant splicing is likely to activate nonsense-mediated mRNA decay (NMD). Intriguingly, despite the variant’s location within an exon, it does not manifest as a missense mutation, which typically leads to amino acid substitution as predicted by software. Instead, it exerts its effect on splicing, as demonstrated in our step-by step verification process. It is also important to note that prior reports have documented several variants located within exons of SLCO2A1 in CEAS patients [7, 8, 10,11,12], which were classified solely based on software prediction as missense or nonsense mutations. Our findings underscore the necessity of concrete verification to avoid potential misinterpretation. In summary, our research has uncovered the novel exonic splicing variant, c.929A>G, shedding light on its potential role in the underlying pathogenic mechanisms of the disease.

The patient featured in our study initially presented with PHO and later developed gastrointestinal symptoms. His PHO diagnosis was confirmed based on typical clinical manifestations and genetic testing. However, unlike the classical reports of CEAS [10, 11, 13], he did not exhibit evident intestinal lesions. Nevertheless, the presence of significantly thickened gastric folds and duodenum indicated involvement of the upper GI tract, aligning with CEAS characteristics. Additionally, the mucosal lesions in the patients’ colon and rectum closely resembled those typically observed in ulcerative colitis. This resemblance was further confirmed by the positive response to mesalazine treatment. As a result, we considered the concurrent presence of UC as a comorbidity alongside CEAS and PHO in this patient.

Our investigation has delved into how this novel variant affects splicing. Next-generation sequencing identified an SLCO2A1 variant, with Sanger sequencing confirming its homozygosity and maternal inheritance. Minigene analysis demonstrated abnormal in vitro transcription compared to the healthy control (Fig. 5a). qPCR analysis revealed reduced transcript levels and shortened transcript lengths in the patient, indicating that the variant diminishes both the quantity and length of the transcripts. Sequencing of the qPCR products allowed for the identification of the specific deleted region, suggesting potential changes in splicing sites (Fig. 6). In vivo analysis between the gastric and colonic tissues of the patient and HC showed more substantial differences in transcript levels, suggesting transcriptional disparities between in vitro and in vivo environments (Fig. 7). Furthermore, T-A cloning confirmed in vivo NMD, offering an explanation for the observed differences in mRNA levels (Fig. 8).

The precise mechanisms by which SLCO2A1 variants lead to CEAS remain elusive. It is plausible that abnormal SLCO2A1 expression disrupts the transmembrane transport and metabolism of PGE2, consequently causing an imbalance of PGE2 [1, 11]. However, the mechanisms responsible for the diverse clinical presentations and varying severities observed among patients with different variants remain poorly understood. Some patients exhibit both PHO and CEAS, while others manifest only one of these conditions. Additionally, CEAS patients present with varying involvement of the GI tract, as well as exhibit varying degrees of symptom severity [4, 7, 11]. Pathogenic variants in SLCO2A1 can disrupt its function at multiple levels, encompassing transcription, translation, membrane localization, and PGE2 transport. These distinct mechanisms may contribute to the heterogeneous clinical manifestations and severities observed in patients. For instance, specific variants may lead to the production of truncated proteins, while others result in nearly complete proteins with single amino acid substitutions [4]. Investigating the correlation between the types of variants and their clinical manifestations warrants further study.

Our study has several limitations. Firstly, in both lanes of the qPCR gel (Fig. 7), two bands were observed in both the patient and the healthy control. The brighter bands were considered as target bands, and their identities were further confirmed by Sanger Sequencing. However, the dimmer bands were not recycled and sequenced individually. Although the specific sequences of the cDNA from the dimmer bands cannot be definitively identified, we conducted T-A cloning to explore the transcripts from the gastric and colonic tissues. This revealed several different transcripts beyond the target ones. The presence of these extra bands suggested the possibility of abnormal transcripts. Secondly, we were unable to identify the variants in the patient’s father due to his early death. Consequently, the source of the patient’s variant on paternal allele could not be definitively verified. However, both WES and Sanger Sequencing supported the homozygosity of the variant, providing partial support for a paternal source.