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A MUC5B Gene Polymorphism, rs35705950-T, Confers Protective Effects Against COVID-19 Hospitalization but Not Severe Disease or Mortality

Rationale A common MUC5B gene polymorphism, rs35705950-T, is associated with idiopathic pulmonary fibrosis (IPF), but its role in severe acute respiratory syndrome coronavirus 2 infection and disease severity is unclear. Objectives To assess whether rs35705950-T confers differential risk for clinical outcomes associated with coronavirus disease (COVID-19) infection among participants in the Million Veteran Program (MVP). Methods The MUC5B rs35705950-T allele was directly genotyped among MVP participants; clinical events and comorbidities were extracted from the electronic health records. Associations between the incidence or severity of COVID-19 and rs35705950-T were analyzed within each ancestry group in the MVP followed by transancestry meta-analysis. Replication and joint meta-analysis were conducted using summary statistics from the COVID-19 Host Genetics Initiative (HGI). Sensitivity analyses with adjustment for additional covariates (body mass index, Charlson comorbidity index, smoking, asbestosis, rheumatoid arthritis with interstitial lung disease, and IPF) and associations with post–COVID-19 pneumonia were performed in MVP subjects. Measurements and Main Results The rs35705950-T allele was associated with fewer COVID-19 hospitalizations in transancestry meta-analyses within the MVP (Ncases = 4,325; Ncontrols = 507,640; OR = 0.89 [0.82–0.97]; P = 6.86 × 10−3) and joint meta-analyses with the HGI (Ncases = 13,320; Ncontrols = 1,508,841; OR, 0.90 [0.86–0.95]; P = 8.99 × 10−5). The rs35705950-T allele was not associated with reduced COVID-19 positivity in transancestry meta-analysis within the MVP (Ncases = 19,168/Ncontrols = 492,854; OR, 0.98 [0.95–1.01]; P = 0.06) but was nominally significant (P < 0.05) in the joint meta-analysis with the HGI (Ncases = 44,820; Ncontrols = 1,775,827; OR, 0.97 [0.95–1.00]; P = 0.03). Associations were not observed with severe outcomes or mortality. Among individuals of European ancestry in the MVP, rs35705950-T was associated with fewer post–COVID-19 pneumonia events (OR, 0.82 [0.72–0.93]; P = 0.001). Conclusions The MUC5B variant rs35705950-T may confer protection in COVID-19 hospitalizations.

A respiratory disease caused by a novel coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported near the end of 2019. Despite massive public health measures and vaccination initiatives, the coronavirus disease (COVID-19) pandemic remains a major global health threat. By February 2022, the COVID-19 pandemic had caused more than 424 million confirmed infections, resulting in more than 5.8 million deaths worldwide (1).
Parenchymal fibrosis is a late complication of severe respiratory infections due to COVID-19 (2)(3)(4). Among chronic lung diseases, idiopathic pulmonary fibrosis (IPF) (5), a disorder characterized by progressive pulmonary scarring that is associated with a median survival of 2-3 years in the absence of lung transplantation (6), shares several risk factors with those for severe COVID-19 disease, including advanced age (7), cardiovascular disease, diabetes, and a history of smoking (5). Thus, common pathological processes may be shared between the fibrotic response toward COVID-19 infection and those underlying IPF.
IPF likely develops from a multifaceted interaction between genetic and environmental factors, age-related mechanisms, and epigenetic profibrotic reprogramming (8,9). One of the most robust genetic risk factors identified for IPF susceptibility is rs35705950-T, a common G ! T transversion located approximately 3 kb upstream of the mucin 5B, oligomeric mucus/gel-forming (MUC5B) gene (10,11). Laboratory evidence supports that rs35705950-T: 1) is a functional variant located within an enhancer subject to epigenetic programming and 2) contributes to pathologic misexpression in IPF (12). Notably, although rs35705950-T has been robustly associated with increased susceptibility, the same allele has also been associated with decreased mortality in IPF (13), though whether this paradoxical effect is attributable to pleiotropy or index event bias remains controversial (14).
Given the high minor allele frequency (MAF) of rs35705950-T (11% among individuals of European ancestry) and possible shared pathophysiological pathways between IPF and severe COVID-19 disease, we examined the association between rs35705950-T and the clinical outcomes of COVID-19 infection in the Million Veteran Program (MVP), a multiancestry cohort of over 650,000 U.S. veterans with detailed electronic health record (EHR) and genotyping data (15). After our primary analysis in the MVP, we validated our results with a comparable analysis conducted in the Host Genetics Initiative (HGI), a global collaboration of over 160 genetic studies assembled to facilitate rapid discovery and dissemination of COVID-19-related science (16).

Data Sources
Data from the MVP, a multiancestry genetic biobank sponsored by the U.S. Department of Veterans Affairs (VA), were analyzed (15). All protocols were approved by the VA Central Institutional Review Board, and all participants provided written informed consent. Genotyping was performed using a custom Thermo Fisher Axiom genotyping platform (MVP 1.0), which included direct genotyping of rs35705950-T. Ancestry was defined using harmonized ancestry, race, and ethnicity derived from self-report and genetic ancestry data (17). Individuals from three major ancestry groups-European (EUR), African (AFR), and Hispanic (HIS)-were included. Demographic and preexisting comorbidity data were collected from questionnaires and the VA EHR; "Pre-COVID" data were from the time of enrollment into the MVP to September 30, 2019. The cohort demographics and a description of the clinical conditions for all genotyped MVP participants and COVID-19-positive MVP participants (18) that were evaluated in this study are shown in Table 1.

COVID-19 outcome definitions.
COVID-19 infection status from February 2020 to April 2021 was assessed by either self-report (if testing was performed outside the VA) or by a positive PCR-based test (19,20) performed within the VA health system. The index date was defined as a COVID-19 diagnosis date (i.e., specimen date); for a hospitalized patient, it was defined as the admission date up to 15 days before the COVID-19 diagnosis date.
Our primary analyses used definitions that were harmonized with those used by the COVID-19 HGI to maximize sample sizes, examine consistent endpoints, and facilitate meta-analyses. In accordance with the HGI phenotype definitions, the following three analyses were performed: 1) COVID-19 susceptibility: individuals who tested positive for COVID-19 2); COVID-19 hospitalization: individuals who were hospitalized for symptoms of COVID-19 3); and COVID-19 severe: individuals who either were hospitalized and required respiratory support beyond nasal cannula oxygen or died. For each phenotype, population controls were selected; i.e., all the MVP participants who were not defined as a case were assigned as control. Controls also included individuals for whom status of exposure to COVID-19 was unknown. Additionally, in the study of hospitalization for COVID-19, a fourth analysis was conducted in which individuals who tested positive and were hospitalized for COVID-19 were compared with COVID-19-positive individuals who were not hospitalized.
Separate sensitivity analyses were conducted within the MVP cohort to evaluate the effect of possible confounding variables that were not evaluated in the HGI. To ensure complete capture of relevant clinical information, we included only individuals who were tested for COVID-19 at one of the VA sites; individuals who did not have COVID testing within the VA were excluded. The four phenotypes evaluated included COVID-19 testing positive rate, COVID-19 hospitalization, severe COVID-19 infection or death, and COVID-19 death; modified from the WHO Working Group criteria (21) in the online supplement). All models were adjusted for additional possible confounders, including body mass index (BMI), Charlson comorbidity index (CCI), smoking history, asbestosis (22), rheumatoid arthritis with interstitial lung disease (RA-ILD) (23) and IPF diagnosis (24). These variables were derived from the data within 2 years before the index date of diagnosis (Table E1).

Postindex pneumonia definition.
Postindex pneumonia was defined from codes from the International Classification of Diseases (10th revision; ICD) within 60 days after the index date. Association with COVID-19 pneumonia events (within 60 days of infection; pneumonia60d) was performed among patients who received COVID-19 PCR testing at VA sites (Table E2). The ICD codes used to define pneumonia events within 60 days of COVID-19 infection (pneumonia60d) are presented in Table E3.

Statistical Analysis
Meta-analysis with HGI. The analysis recommendations from the COVID-19 HGI were utilized to test for associations between the rs35705950-T allele and COVID-19 outcomes. First, we conducted analyses within MVP by each ancestry group using plink2a. Inverse variance weighted metaanalyses were then performed with summary statistics from Release 5 (01/18/2021) of the HGI using GWAMA (18) (Additional details are given in the online supplement).
Sensitivity analysis. Firth logistic regression, as implemented in the R (v3.6.1) package "brglm2" (version 0.7.1) was used in the sensitivity analyses, as it provides a bias-reduced estimate in the setting of small sample sizes and is most powerful for analyses of genetic mutations. This was relevant because adjustment for multiple variables can lead to small sample sizes within covariate categories. Associations between COVID-19 outcomes and rs35705950-T were performed separately by ancestry, with adjustment for age, age 2 , sex, BMI, CCI, smoking history, asbestosis, RA-ILD, and IPF (Table E1), and the first 20 principal components. The meta-analyses were performed using random-effects models in "metafor" (version 2.4-0).
Post-index pneumonia. Interactions between COVID-19 infection status and rs35705950-T on the outcome of COVID-19 pneumonia within 60 days (pneumonia60d) were assessed using a multiplicative interaction term followed by stratified analyses by COVID-19 infection status. The firth logistic model for interaction included the independent variables of interest: COVID-19 infection status, rs35705950-T, and their multiplicative interaction term, adjusted for preindex pneumonia (yes/no, within 2 years preindex), age, age 2 , sex, BMI, CCI, smoking history, asbestosis, RA-ILD and IPF diagnosis, and first 20 ancestry-specific principal components (PC1-20). The SNP rs35705950-T was modeled as a continuous variable (additive genetic model with values 0, 1, and 2) so that the interaction odds ratio (OR) is equal for heterozygous versus homozygous wild-type and homozygous mutation versus heterozygous mutation. The additive interaction of COVID-19 and rs35705950-T (for every increase in one allele; i.e., 0 to 1, or 1 to 2 copies) was also assessed with the estimate of excess risk due to interaction (RERI) and the 95% confidence interval estimated with the delta method.
Phenome-wide and laboratory-wide association studies (PheWAS and LabWAS, respectively). Associations between rs35705950-T and preexisting comorbid conditions and laboratory values were

Association between rs35705950-T with COVID-19 Positivity and Outcome Severity
Our study included 19,168 COVID-19positive patients from three major ancestry groups (EUR, AFR, and HIS). The minor allele frequency for rs35705950-T was 9% among the entire MVP participants and 8% among COVID-19-positive individuals (Table 1); among these, 14.4% (n = 2,758) and 0.7% (n = 138) were carriers of one copy and two copies of MUC5B rs35705950-T, respectively (Table 1; Figure E1). Associations between rs35705950-T and clinical outcomes by ancestry group within the MVP, the transancestry meta-analysis within the MVP, and the joint meta-analysis with HGI are shown in Figure 1 and Table 2. In the joint meta-analysis, the most significant association was between rs35705950-T and fewer hospitalization events compared with population controls (OR, 0.90 [95% CI, 0.86-0.93], P = 8.993 10 25 ; Figure 1 and Table 2). The MUC5B rs35705950-T allele was not associated with reduced COVID-19 positivity in transancestry meta-analysis within the MVP (OR, 0.98 [0.95-1.01], P = 0.06) but was nominally significant (P , 0.05) in the joint meta-analysis with HGI (OR, 0.97 [0.95-1]; P = 0.02). The MUC5B rs35705950-T allele was not associated with severe COVID-19 disease (critically ill).
To further evaluate the robustness of our findings from the primary analyses, we performed sensitivity analyses among MVP participants from three ancestry groups (EUR, AFR, and HIS) who had COVID-19 testing performed within the VA (n = 136,164). Associations between rs35705950-T and COVID-19 positivity and outcomes with adjustment for additional covariates, including BMI, smoking status, CCI, asbestosis, RA-ILD, and IPF diagnosis, which were not available in HGI, were performed in MVP participants. We observed similar effect sizes as the primary analyses (Table E4) [7.36, 9.61], and 6.47 [4.93, 8.48] in patients with zero, one, and two copies of MUC5B rs35705950-T respectively among COVID-19-positive relative to COVID-19-negative subjects (P , 0.0001; Table 3). This differential effect of an additional MUC5B rs35705950-T allele on postindex pneumonia in COVID-19-positive versus COVID-19negative patients was statistically significant (multiplicative scale interaction OR, 0.77 [0.66, 0.89], P = 0.0004) in EUR individuals (Table 3). There was also evidence of a negative additive interaction, with the RERI corresponding to an increase of one copy of rs35705950-T allele estimated as 22.07 (95% CI = 23.24, 20.9). This suggests that the relative risk for pneumonia in COVID-19positive patients is 22.07 less for each increase in one copy of MUC5B rs35705950-T allele than if there were no interaction between COVID-19 and MUC5B rs35705950-T. The number of post-COVID-19 pneumonia events in HIS or AFR individuals was too low to permit further analysis.

Exploring Shared Pathobiology with the MUC5B rs35705950-T through PheWAS and LabWAS Analyses
To explore clinical conditions and biomarkers associated with the MUC5B rs35705950-T allele that may impact the susceptibility and severity of COVID-19, PheWAS and LabWAS analyses using pre-COVID-era data (through September 2019) were performed. The sample sizes for MVP participants included in PheWAS and COVID-19 association studies are shown in Table 1 ( Figure E1). The results of the PheWAS analyses are shown in Figure 2, with full summary statistics presented in Table E5.
In PheWAS analyses between rs35705950-T and PheCodes with .200 cases, significant associations were identified exclusively with pulmonary processes. Increased risk for "other alveolar and parietoalveolar pneumonopathy" (Phecode 504; OR = 2.64 [2.50 -2.78], P = 7.07 3 10 2289 ) and "postinflammatory pulmonary fibrosis" (Phecode 502; OR = 2.85 [2.65-3.05], P = 8.90 3 10 2186 ) were significantly associated (P Bonferroni , 1.2 3 10 25 ) with rs35705950-T in all three ancestry groups (EUR, AFR, and HIS). Among the EUR and AFR groups, rs35705950-T was also associated with increased risk for idiopathic fibrosing alveolitis (Phecode 504.1). Notably, there were no significant associations identified between rs35705950-T and influenza infection (Phecode 481) or bacterial pneumonia (Phecode 480.1) in any of the ancestry groups. The power to detect a difference with these conditions was .95%, as there were 4,728 cases of influenza and 10,579 cases of bacterial pneumonia in the EUR cohort. There were also no significant associations between rs35705950-T and non-pulmonary conditions potentially relevant to COVID-19 outcomes, including pulmonary embolism/deep vein, systemic inflammatory syndrome/sepsis, or acute renal failure ( Figure E2 and Table E5).
The LabWAS of MUC5B rs35705950-T with median values of pre-COVID clinical laboratory tests is shown in Table E6 and Figure E3. Among EUR participants, 10 laboratory tests were significant after Bonferroni adjustment, with the majority belonging to white blood cell counts or fractions, with an increase in monocyte count also significant in the HIS subgroup. There were no significant associations among individuals in the AFR subgroup.

Discussion
Our study supports that the "T" allele of rs35705950 in MUC5B, which has been associated with an increased risk for the development of IPF, confers a decreased risk for COVID-related hospitalization among MVP participants; among participants of European descent, a decreased incidence of pneumonia after COVID-19 infection was also observed. The protective effect of the rs35705950-T may appear to contradict previous work demonstrating an increased risk for acute respiratory distress syndrome (ARDS) in the pre-COVID era (27) as well as the increased risk of severe COVID-19 disease observed for other well-established causal variants of IPF located in the TERC, DEPTOR, and FAM13A (28) genes. However, our findings are consistent with previous studies conducted in European cohorts (29) and in the HGI (28), which support that pathophysiological changes due to rs35705950-T may result in distinct interactions with SARS-CoV-2, which confers the differential risks observed.
The rs35705950 polymorphism is located within an enhancer region of the MUC5B gene, the protein product of which is a major gel-forming mucin in the lung that plays a key role in mucociliary clearance and host defense (30)(31)(32)(33). Consistent with this, mouse knockout models for MUC5B demonstrate increased susceptibility toward bacterial infections of the respiratory tract and persistent inflammation. In contrast to the loss-of-function of knockout models, the "T" allele of rs35705950 results in a gainof-function and is associated with enhanced expression of the MUC5B transcript in lung tissue from human subjects (24) without clinical IPF. Notably, although excess MUC5B protein is observed in the epithelial cells of respiratory bronchioles and honeycomb cysts (31,32,34) of individuals with IPF, differential expression in lung tissue by rs35705950 genotype has not been consistently observed in patients with advanced disease and may be attributable to the universally elevated MUC5B protein levels in IPF patients, regardless of their rs35705950 allelic configuration (24,32,34).
The functional impact of increased MUC5B expression on clinical outcomes in populations both with and without IPF remains incompletely understood. In a study that antedated the COVID-19 pandemic, a modestly increased risk for the development of ARDS, a major feature of severe SARS-CoV-2 infection, was observed among homozygotes for rs35705950-T who were .50 years old, none of whom had clinical or radiographic evidence of IPF on post hoc chart reviews (27). It should be noted, however, that the underlying etiologies for ARDS in the population examined were heterogeneous, with over half of the cases attributed to either trauma or non-pneumonia-related sepsis. In contrast to the increased risk for pre-COVID-era ARDS, the rs35705950-T allele was associated with decreased risk for acute respiratory exacerbation events among non-Hispanic White ever-smokers with interstitial lung abnormalities (ILAs), defined as nondependent parenchymal infiltrates that are frequently considered subclinical precursors to clinically apparent ILD, in the COPDGene study (35). Whether increased mucin production confers protection against viral infections (36), which are believed to contribute to a substantial proportion of acute respiratory exacerbation events in chronic lung disease (37), should be explored in future studies.
Our analyses suggest that the decreasing risk of postindex pneumonia associated with rs35705950-T may be specific to COVID-19. First, to our knowledge, there have been no previously published reports of differential susceptibility or clinical outcomes in non-COVID-19 respiratory viral or bacterial infections by rs35705950-T genotype. Within our own MVP data, there were no associations between prepandemic influenza infection or bacterial pneumonia and rs35705950-T allele. Second, although rs35705950-T carrier status or number of allele copies did not impact testing rates for COVID-19 (which were 22.2%, 22.3%, and 22.4% for individuals of European descent with zero, one, and two copies of rs35705950-T, respectively), individuals with the "T" allele demonstrated a trend toward decreasing rates of testing positive in MVP, supporting that rs35705950-T may modulate an individual's susceptibility to infection by SARS-CoV-2. Third, in addition to attenuating the risk for postindex pneumonia exclusively among COVID-19-positive individuals, differential risk by rs35705950-T copy number was observed, supporting a dose-response relationship for this outcome.
We did not observe associations between rs35705950 and severe COVID-19 illness or mortality. This may be due to the low incidence and/or multifactorial causes of these severe outcomes, which may include nonrespiratory disorders such as shock and multisystem organ failure. This is supported by the PheWAS analysis, which found nearly exclusive associations between rs35705950-T with pulmonary processes, with no evidence of association (with .80% power to detect differential effects) with other processes potentially relevant to severe COVID-19 disease, including thromboembolic disease, septic shock, and acute renal failure.
The COVID-19 pandemic amplified healthcare disparities due to socioeconomic factors and likely contributed to differences in outcomes (38). However, heterogeneity in the magnitude of disparities between healthcare systems exists, with numerous studies demonstrating reduced levels of disparities and relatively equitable access to care among VA healthcare users (relative to non-VA healthcare users) (39-41); thus, differences in outcomes attributable to socioeconomic factors within the VA may be less than those in the general population. Consistent with this, a study by Trivedi and colleagues (42) reported minimal changes in 30-day mortality rates for heart failure and pneumonia hospitalizations after adjustment for a comprehensive panel of socioeconomic factors, including poverty, housing, education, and rurality. Within our cohort, access to care was uniformly high, with .98% of individuals having >1 primary care visit within the preceding 18 months and comparable numbers of patients with >1 clinical encounter or admission in the preceding year. However, because of the complexities of socioeconomic factors and their potential impact on and interactions with rs35705950-T, we acknowledge that future studies in this area are needed.

Strengths and Limitations
The strengths of our study include a large, ethnically and geographically diverse cohort with harmonized prospective outcomes data, directly genotyped rs35705950 data, and the use of robust statistical approaches. In addition to interrogating the relationships between rs35705950-T and clinical outcomes, the availability of rich clinical phenotyping data permitted exploration of the underlying pathobiological mechanisms through PheWAS and LabWAS and support that the differential risks observed by rs35705950-T are likely mediated by pulmonary-specific processes. Despite these strengths, we acknowledge the following limitations. First, although the MVP is one of the largest and most diverse genomic medicine databases established to date, participants are predominantly male and of European ancestry; this, and the lower minor allele frequency of rs35705950-T among non-European populations, may have impacted our power to detect associations between rs35705950-T genotype and clinical outcomes among females and racial/ethnic minorities. Second, because of the timing of the data freeze for our analysis, only shortterm outcomes were captured. Future studies with extended follow-up to explore the potential relationship between rs35705950-T and post-COVID syndromes, including lung fibrosis, are needed. Third, although our analyses were adjusted for critical variables, residual confounding and bias may still exist. Given the association between rs35705950-T and an increased incidence of subclinical ILA and respiratory symptoms (43), individuals harboring the MUC5B variant may exhibit behavior modifications (increased testing or more stringent self-isolation) that could have introduced biases that cannot be captured or adjusted for in our analyses (28). Fourth, viral subtypes, vaccination status, and treatment approaches evolved with time, which may have impacted severity outcomes. Fifth, we acknowledge that the effect estimates associated with rs35705950-T in our study are modest and may limit its application in clinical or predictive algorithms. However, despite these limitations, we assert that our results, which identify a potentially protective role of rs35705950-T in COVID-19 outcomes, support that this functional polymorphism confers pleiotropic effects that may be modulated by epistasis and environmental interactions; longer term follow-up and future analyses into these complex relationships are warranted.