3.1 SNPs Selection for Association Study
To the best of our knowledge, this is the first human association study with focus on the candidate gene TMEM176B. Using the “Ensembl” portal we identified 15 SNPs localized within the coding region (Fig. 1a, Table 2).
Taking into account the minor allele frequency (MAF > 10-20 %) in European and African population (from “Ensembl” database and 1000genome Project), the possible functional effect, and Linkage Disequilibrium data (Fig. 1b), we selected 4 SNPs out of 15: rs11546674 C>A (5’UTR), rs3173833 A>T (p.Ser94Cys), rs2072443 C>T (p.Ala134Thr), and rs2302480 G>A (3’UTR).
According to eQTL analysis (from “GTEx" Portal), rs2302480 and rs3173833 are associated with an increased TMEM176B expression, whereas rs2072443 and rs11546674 with a diminished gene expression (Table 2). A previous gene expression analysis followed by eQTL evaluation realized in a case/control cohort of multiple sclerosis reported that rs3173833 and rs2072443 were associated with the expression of TMEM176B in the blood however any significant difference in SNPs distribution has been detected (Nickles, 2013) .
As no data exist about TMEM176B variants in the Brazilian population, we first analyzed the SNPs distribution in a representative cohort of healthy donors. The observed MAF did not significantly differ from the expected ones, calculated as a mean between European and African MAF (Fisher test p > 0.05). Moreover, genotypes distribution resulted in Hardy-Weinberg equilibrium (p > 0.05) (Table 3).
3.2 Protective role of TMEM176B rs2072443 variant in CRC prognosis
TMEM176B SNPs were genotyped in a cohort of 187 unrelated CRC patients.
Before performing the association study, a linear regression analysis was executed to identify confounder factors for each principal variable (lethality, relapse, survival). As a result, sex, age at diagnosis, TNM stage and CEA level before surgery were included as correction variables in the subsequent multivariate analysis.
The unique SNP that resulted significantly associated with CRC prognosis was the rs2072443 C>T (p.Ala134Thr) (Table 4, Supplementary Table 1). Patients carrying this variant in a dominant model of inheritance for the minor T allele appear to be more protected against CRC-related death (pad j = 0.009: ORadj = 0.19) and less prone to tumor relapse after surgery (padj = 0.026: ORadj = 0.22) than non-carriers. Accordingly, rs2072443 carriers showed increased survival rate (padj = 0.036).
When survival analysis was performed by Mantel-Cox test, again the rs2072443 SNP resulted significantly associated with increased survival rate after 120 months from surgery, according to a recessive model for the minor T allele (p = 0.038) (Fig. 1c).
Haplotype’s analysis did not report any significant results (data not shown).
Altogether these data demonstrated for the first time the significant and protective association of the variant rs2072443 in TMEM176B gene with CRC prognosis.
3.3 TMEM176B rs2072443 variant is associated to increased IL-1ß release
The rs2072443 SNP is located in exon 5 of TMEM176B gene (Fig. 1a) and lead to an amino acid change (p.Ala134Thr) which is considered to be “tolerated” or “probably damaging” according to the tool used for the prediction of effect on protein function (SIFT or PolyPhen) (from the “Ensembl'' website). The substitution affects a highly conserved residue (Fig. 2a) localized in the third transmembrane domain of the channel, possibly affecting the helix conformation of the region and maybe the correct folding of the protein. Unfortunately, no resolved structure is available yet to infer a conformational change caused by the variant (from “UniProtKB/Swiss-Prot” database).
According to the “GTEx” portal TMEM176B appears to be expressed in colon as well as in whole blood (Fig. 2b). The eQTL analysis (from the “GTEx” portal) reports a significant lower expression level of TMEM176B gene both in whole blood and colon from individuals carrying the T/T genotype of rs2072443 SNP compared to non-carriers (Fig. 2c-d), suggesting that the SNP negatively affects TMEM176B, at least at transcriptional level.
Analysis performed using data from the “TCGA” public database showed that high levels of TMEM176B expression are associated with a poor 5-year survival (p = 0.012) (Fig. 2e). Moreover, TMEM176B expression is incremented in disseminated CRC tumors (TNM III and IV) compared to localized tumors (TNM I and II) (p = 0.043) (Fig. 2f).
Therefore, either the progression and the severity of CRC appeared to be associated with an augmented TMEM176B expression, confirming the possible eQLT effect of rs2072443 and the previously published data by Segovia and colleagues (Segovia, 2019) .
To elucidate the meaning of TMEM176B association with CRC in the context of NLRP3 inflammasome activation (Fig. 2g), we then tried to characterize the effect of rs2072443 SNP on the complex.
TMEM176B was first described in the myeloid compartment specifically in murine dendritic cells (Condamine, 2010) . However, the activation of NLRP3 inflammasome has been reported in myeloid as well as lymphoid cells in humans (Shelbi, 2020, Ali, 2017, Arbore, 2016) [18,13-14]. To depict the effect of the variant on NLRP3 inflammasome in distinct types of leukocytes, we performed a genotype-guided assay in monocytes, MDM and MDDC, CD4+ T and CD19+ lymphocytes from HD. The cells were stimulated with classical stimuli for NLRP3 inflammasome activation: LPS followed by ATP for monocytes, MDM and MDDC (Gattorno, 2007, Dos Reis, 2019, Souza, 2020) [10-12], ß-glucan for B lymphocytes (Ali, 2017)  polyclonal stimulation with anti (𝝰)-CD3 and 𝝰-CD28 for CD4+ T lymphocytes (Arbore, 2016) . Main findings are included in Fig. 2 (complete results in Supplementary Figure 1).
Monocytes and MDM from HD carrying the rs2072443 variant displayed significantly increased IL-1ß release in response to LPS and ATP (Fig. 2h-i). It is worth highlighting that we were not able to detect significant amounts of IL-18 in stimulated monocytes supernatants and for MDM we observed reasonable release of that cytokine however without significant difference among genotypes.
No significant differences were observed for other conditions or cells (Supplementary Figure 1), suggesting a cell-specific effect of this SNP, possibly depending on the expression of TMEM176B or either on the function played in different leukocytes. As expected, rs2072443 did not affect the secretion of a caspase-1-independent cytokine, such as TNF-α Supplementary Figure 1).
As TMEM176B has been recently described as a negative regulator of NLRP3/caspase-1-dependent IL-1ß release in mice (Segovia, 2019) , our results fit well with the eQTL data in humans: the less TMEM176B is expressed, the less it can inhibit NLRP3 inflammasome and IL-1ß release.