Compared to those in the healthy control population, C10X polymorphic gene carriers were more prevalent in our cohort of meningitis patients. A slightly greater percentage of female patients were found to be polymorphic gene carriers than male patients, with percentages of 43% and 39%, respectively.
The present study showed that fever, headache, neck stiffness and vomiting were the dominant clinical characteristics. Moreover, there was statistically significant difference in manifestation of these clinical symptoms among heterozygous gene carriers compared to wild-type gene carriers, which indicates the strong association heterozygote carriership with development of the above-mentioned clinical manifestations. The odds of fever, headache nausea, vomiting, cough, high respiratory rate, altered cautiousness and altered mental status were greater among homozygous, CARD8 (C10X), gene carriers than wild-type gene carriers, which indicates the potential association of homozygote carriership with the mentioned clinical manifestations, which agreed with a previous study [22].
The development of clinical manifestations is mediated by induction of pro-inflammatory cytokines which is regulated by NLRP3 inflammasomes which activates caspase 1 (CASP1) through caspase recruitment domains (CARD). The clinical manifestation in turn is influenced by the genetic make-up of these components of the host [27, 28], which might explain the enhanced clinical manifestation in polymorphic gene carrier observed in this study.
Other studies indicated that single nucleotide polymorphisms (SNPs) in different components, or regulators, of this pathway have been associated with enhanced clinical manifestations in different infective conditions including hepatitis C, TB and other bacterial infections. [22, 27].
The implication that a loss-of-function mutation in the CARD 8 gene, which makes it unable to inhibit caspase-1 activation, could be associated with increased NLRP3 inflammasome activity [29] was observed in the present study. The wild-type gene was found to be protective against bacterial meningitis in the present study. Our finding agrees with a study conducted by Dennis et al. to identify the effect of genetic variation on susceptibility to bacterial infections, which showed overrepresentation of the wild-type allele in healthy controls [30] in vitro. On the other hand, some findings have not been robustly replicated, suggesting that the effect of relatively frequent C10X polymorphisms might be modest or that associations with additional factors are needed to significantly drive disease outcomes [17].
The data also showed that the heterozygote type of the C10X polymorphism was more dominant among meningitis patients and blood donors, followed by homozygous wild-type gene carriers. The homozygote polymorphic gene carriers were found to be more susceptible to bacterial meningitis than the heterozygote gene carriers and healthy controls. This finding is supported by a previous report by A. Idosa et al., who reported that homozygote carriers are more susceptible to infectious disease than heterozygote carriers are, indicating that healthy C10X homozygous individuals might have a greater risk of developing an infection when exposed to infectious agents [22].
The current study also demonstrated that the proportion of C10X gene polymorphisms was relatively greater among patients with identified pathogens than among clinically diagnosed patients and healthy controls. Furthermore, C10X-heterozygote carriers were more prevalent among culture-positive meningitis (67%) than those with suspected nonpathogen-identified meningitis (37%) and healthy controls (47%), with odds ratios of 4 and 3.1, respectively. Previously, Idosa et al. reported that the polymorphism of C10X in the CARD8 gene was significantly more prevalent in patients with bacteremia than in healthy controls and that the prevalence of heterozygote gene distribution was greater among bacteremia patients than among those with negative blood cultures and healthy controls [22].
The C10X polymorphism causes a nonsense allele, resulting in reduced expression of functional CARD8, which in turn is suggested to result in a loss of its inhibitory effect on caspase 1[29, 30]. There are also reports on the polymorphism of CARD8 (C10X) that has been associated with an increased risk of inflammation. Polymorphism of CARD8 contributes to the development of some noninfectious diseases, such as inflammatory bowel disease, gout, rheumatoid arthritis and Alzheimer’s disease, and contributes to susceptibility to and poor disease outcomes in patients with infectious diseases [22, 31]. A loss-of-function mutation in the CARD8 gene is reported to render CARD8 unable to inhibit caspase-1 activation, which could also be associated with increased NLRP3 inflammasome activity [23]. Sarkar A et al reported that caspase-1-deficient mice are protected from diseases involving excessive inflammation, such as bacterial-induced sepsis, and are markedly resistant to the lethal effects of endotoxin [32]. The C10X genotype has been found to be associated with chronic consequences on human health by resulting in severe autoimmune and autoinflammatory disease, probably due to the long-term effect of an excessive caspase-1 response [33].
The functional consequences of the C10X polymorphism in infectious diseases do not seem to clear way of mechanism with the involvement of different gene variants. G.V. Paramel et al. reported the discovery of possible isoforms of the CARD8 gene, such as T47, in which its transcription begins downstream of C10X; hence, not affecting the isoform could affect the functional consequences of the C10X polymorphism [18]. As the review stated, the high rate of homozygous patients with loss-of-function polymorphisms, which appear to be human knockouts, might reflect partial rescue of CARD8 function by alternative splicing, leading to an almost functional full-length protein. Patients who are homozygous for the C10X genotype might therefore have a functional CARD8 protein due to the presence of the T47 isoform [15]. Therefore, to clarify the effect of C10X polymorphisms and gene variants during bacterial meningitis, or other infectious diseases a detailed study on the mechanistic effect of polymorphic genes and the impact of possible isoforms present is needed. The limitations of this study were the small number of laboratory-confirmed meningitis patients which influenced the statistical analysis and limit to do sub-group analysis and it was not managed to measure IL-1β due to logistical and resource constraints.