We found no significant differences in allele frequencies or genotype prevalence for variants in the TNF
genes between fatal influenza cases in patients <40 years old and a nationally representative reference sample. However, among the case-patients who died, most of whom died in childhood, variants of MBL2
responsible for low production of MBL were associated with MRSA co-infection. This observation should be viewed cautiously as a hypothesis for further exploration, given the small number of case-patients with MRSA in our study (n = 8). This finding is consistent with results from previous studies that found associations between MBL insufficiency (defined by genotype) and respiratory infection in children (12–14
), severe and fatal sepsis (9,15–17
), and systemic inflammatory response syndrome in children (18
TNF is a potent proinflammatory cytokine produced early in the innate immune response to infection that promotes a wide range of immunologic responses. Excessive systemic TNF is responsible for many symptoms of clinical infection and may lead to fatal complications. Studies have demonstrated a significant genetic contribution to circulating TNF levels, with 50%–60% of variance in TNF levels genetically determined (19–21
). The most studied SNP is at position −308 (rs1800629), with the A allele associated with 20%–40% greater TNF production (22–24
) and with susceptibility to and severity of numerous infectious diseases (20,22,25,26
). Carriage of the A allele at the −238 position (rs361525) also has been associated with a variety of diseases (20,22
MBL, another key component of the innate immune system, is a soluble protein of the collectin family that binds to microbial surfaces and promotes phago-opsonization directly and indirectly by activating the lectin complement pathway. Low serum MBL levels are common and associated with an increased risk for a variety of infections and autoimmune diseases (15,27–29
), including acute respiratory infection in young children (12
). MBL levels are strongly influenced by genetic factors, with >75% of variation in MBL levels explained by a small number of polymorphisms in the MBL2
). Variant proteins are unstable and of lower oligomeric form, which decreases affinity for microbial ligands and complement-activating ability. Each variant produces significantly reduced serum MBL levels.
MBL has been shown to strongly bind S. aureus
) and susceptibility to fatal S. aureus
infection due to MBL deficiency has been convincingly demonstrated in murine models (32
). Phase I clinical trials of MBL replacement therapy indicate that this therapy is well tolerated and effective at improving MBL deficiency in healthy persons (33
). Reports of MBL replacement therapy administered to severely ill persons (34–36
) or to patients with S. aureus
) suggest that therapy can improve clinical conditions, although results of these studies were mixed, and in some cases, clinical improvements were temporary. The clinical implications of MBL replacement therapy for influenza treatment or prevention are unknown.
Among persons with fatal cases, we observed an increased risk for sudden death in carriers of the variant allele of TNF
rs1800750. We are unaware of previous literature reporting a similar association; there is no obvious biologic mechanism to explain the finding. The TNF
rs1800750 variant is in linkage disequilibrium with other TNF
), some of which (including TNF
rs361525) have been associated with increased TNF serum levels. Therefore, it is possible that the observed association may be due to linkage disequilibrium with unmeasured polymorphisms that are the causal variants, and more exhaustive analysis of TNF
variants is worthy of future study.
A strength of this study is its use of a cohort of case-patients particularly well-suited for investigation of potential host genetic risk factors—these case-patients died with active influenza infections, yet were predominantly children and young adults without severe preexisting medical conditions. In such a group, other factors associated with severe influenza are less likely to obscure possible genetic associations. An additional strength was access to postmortem lung tissue for immunohistochemistry and/or RT-PCR confirmation of influenza infection.
We recognize that this study has several limitations. Although the study cohort is, to our knowledge, the largest sample of fatal influenza cases in children and young adults, the analysis has limited statistical power to detect associations because of small sample sizes, especially when examining subsamples. We had access to limited information about racial and ethnic background of case-patients. Clinical data were obtained primarily from a US surveillance system and were not validated with medical chart review. Although we were able to infer truncated haplotypes for MBL2
, haplotype information for TNF
was unavailable. Despite these shortcomings, the possibility that specific variants of the MBL2
gene known to influence serum MBL levels appear to be associated with severe bacterial co-infection is an intriguing finding deserving of additional study, especially given the prevalence of co-infection among case-patients who died of pandemic (H1N1) 2009 virus infection (38
) and observations that children co-infected with influenza and S. aureus
may have higher case-fatality rates (39
That we observed a stronger relationship between low-producing MBL genotypes and MRSA infection than between those genotypes and S. aureus
infection in general is puzzling. We are unaware of an obvious physiologic explanation for why low MBL would predispose more strongly to infection with methicillin-resistant versus methicillin-sensitive S. aureus
. One possibility is that MRSA is a marker for other strain characteristics. For example, such an association could arise if MRSA infections were predominantly the USA300 strain while other S. aureus
infections were predominantly the USA100 strain. Unfortunately, we do not have data on S. aureus
genetic strain types. We also found that of the 4 fatal influenza cases in which patients had both MRSA co-infection and low-producing MBL genotypes, 2 patients reportedly also had asthma. It is well-established that asthma increases the risk for serious complications of influenza, and although we know of no evidence suggesting that low-producing MBL genotypes are associated with increased risk for asthma (40
), this finding may be worth further exploration in future studies.
Our findings suggest several opportunities for additional influenza-related research. An obvious next step is examination of all functional variants of the MBL2
gene in conjunction with gene expression and functional assays in a larger group of severely ill influenza case-patients with sufficiently detailed clinical data to define important phenotypes (e.g., MRSA co-infection). Interest in association studies of rare variants, the availability of new sequencing technologies that dramatically decrease the cost of sequencing, and access to reference human sequence data suggest that investigating rare variants in candidate genes (including MBL2
) and their functional effects may be a promising avenue of research. Large-scale genotyping of a sample of case-patients to look for common variants by using methods such as genomewide association studies may be possible if a network of collaborators capable of pooling a sufficient number of case-patients is developed. Recent initiatives such as the Genome-based Research and Population Health International Network (www.graphint.org/ver2
) are aimed at encouraging such networks. Given the rapid acceleration in laboratory technologies, enhancement in bioinformatics methods and capacity, and trends toward collaborative research within large consortia, exploration of the role of host genomic factors in serious illness associated with influenza and other viral pathogens is increasingly feasible. We believe that host genomics is a promising area for future research regarding who is at risk for severe complications of acute infectious diseases, including influenza.