Prospective virome analyses in young children at increased genetic risk for type 1 diabetes

Abstract

Viruses are implicated in autoimmune destruction of pancreatic islet β cells, which results in insulin deficiency and type 1 diabetes (T1D)^1-4. Certain enteroviruses can infect β cells in vitro⁵, have been detected in the pancreatic islets of patients with T1D⁶ and have shown an association with T1D in meta-analyses⁴. However, establishing consistency in findings across studies has proven difficult. Obstacles to convincingly linking RNA viruses to islet autoimmunity may be attributed to rapid viral mutation rates, the cyclical periodicity of viruses⁷ and the selection of variants with altered pathogenicity and ability to spread in populations. β cells strongly express cell-surface coxsackie and adenovirus receptor (CXADR) genes, which can facilitate enterovirus infection⁸. Studies of human pancreata and cultured islets have shown significant variation in enteroviral virulence to β cells between serotypes and within the same serotype^9,10. In this large-scale study of known eukaryotic DNA and RNA viruses in stools from children, we evaluated fecally shed viruses in relation to islet autoimmunity and T1D. This study showed that prolonged enterovirus B rather than independent, short-duration enterovirus B infections may be involved in the development of islet autoimmunity, but not T1D, in some young children. Furthermore, we found that fewer early-life human mastadenovirus C infections, as well as CXADR rs6517774, independently correlated with islet autoimmunity.

Extended Data Fig. 1. Percentage of stool samples at age of first appearance of Enterovirus B

Extended Data Figure 1 (Panels a-d). Percentage of stool samples at age of first appearance of Enterovirus B. Panel a shows sample positivity and Panel b sample consecutive positivity. Panels c and d show months prior to autoantibody seroconversion of Enterovirus B for sample positivity (c) and sample consecutive positivity (d) by autoantibody case status (n=383 matched pair children). Blue line represents control samples and red line represents case samples. The timing of the first appearance of an Enterovirus B infection from enrollment (3 months of age) or months prior to islet autoimmunity showed no obvious trend by age of child.

Extended Data Fig. 2. Common human viruses related to type 1 diabetes (T1D)

Extended Data Figure 2 (Panels a-c). Common human viruses related to type 1 diabetes. The three forest plots (a-c) show how common human viruses relate to the odds of children being diagnosed with T1D. The results were shown as odds ratios (OR, circle) and 95% confidence intervals (CI, bars) and were calculated using conditional logistic regression models with adjustment for HLA-DR-DQ genotype. OR>1 indicated a positive correlation between virus pattern and diagnosis with T1D, OR<1 indicated an inverse correlation. Plot (a) examined if an increase in the number of samples positive for virus was correlated with T1D (n=112 matched pair children). Plot (b) examined if children positive for the virus between 3 and 6 months of age were related to T1D (n=103 matched pair children). Plot (c) examined if children positive for the common virus in at least two consecutive samples (yes versus no) were related to T1D (n=112 matched pair children). Black circles and CI bars represent non-significant associations. Red circles and CI bars represent significant association with T1D. The number of positive stool samples for Enterovirus B was lower among T1D cases compared to matched controls. Human mastadenovirus C, similar to islet autoimmunity cases, was less likely to be detected in early stool samples (3–6 months of age) compared to the matched control for T1D cases. All p-values were two-sided.

Extended Data Fig. 3. Heatmaps of contig alignments of successive stools (n=6 children)

Extended Data Figure 3. Heatmaps of contig alignments of successive stools (n=6 children). Heatmaps showing percent homology of alignments of enterovirus contigs isolated from successive stools from the same child. Stool collection date (successive days in the study) are shown, the serotype for the enterovirus aligned, all are aligned to an enterovirus genome map with scale of nucleotides at the bottom. Heatmap color is assigned on ~7 nt/pixel, heatmap color scale of percent homology is shown at the top.

Extended Data Fig. 4. Children consecutive positive for Enterovirus B with prolonged shedding of same serotype

Extended Data Figure 4. Children consecutive positive for Enterovirus B with prolonged shedding of same serotype. Categorical months of shedding by number of children for islet autoimmunity cases (n=45) and controls (n=25). Red bars denote cases and blue bars denote controls. Length of prolonged shedding period (duration) was not associated with case status in the children with consecutive positive Enterovirus B. Conditional logistic regression was used to evaluate significance; test was two-sided.

Figure 1 (a-f).. Stool virome composition up to 36 months of age and common human viruses related to islet autoimmunity.

The pie chart (a) indicates the relative proportion of major classes of viruses found in the stool virome (n=5,725 samples, % positive overall, combined primary and cultured stools) in the first three years of life. Bar graphs indicate the estimated prevalence in the TEDDY cohort (n=6,890 children observed at risk for islet autoimmunity and type 1 diabetes up to 36 months of age) of the 20 most abundant mammalian viruses (b) and enterovirus serotypes found among the two most abundant enterovirus species (Enterovirus A, Enterovirus B) within the same stool dataset (c). The cohort prevalence were estimated by weighting the proportion of stool samples that were positive for the virus to account for how the children were selected as controls (n=495) into the two nested-matched case-control studies. The three forest plots (d-f) show how common human viruses relate to the odds of developing islet autoimmunity. The results were shown as odds ratios (OR, circle) and 95% confidence intervals (CI, bars) and were calculated using conditional logistic regression models with adjustment for HLA-DR-DQ genotype. All p-values were two-sided. OR>1 indicated a positive correlation between virus pattern and development of islet autoimmunity, OR<1 indicated an inverse correlation. Plot (d) examined if an increase in the number of samples positive for virus (n=4,327 matched pair samples) correlated with islet autoimmunity. Plot (e) examined if children positive for the virus between age 3 and 6 months (n=370 matched pair children) were related to islet autoimmunity. Plot (f) examined if children positive for the common virus in at least two consecutive samples (n=383 matched pair children) were related to islet autoimmunity. Black circles and CI bars represent non-significant associations. Red circles and CI bars represent statistical significant association for the a prior enterovirus at p<0.05 or for other viruses that showed a false discovery rate (FDR) at <0.05.

Figure 2 (Panels a-h).. The percentage of children positive for a specific virus between ages 3 and 6 months and percentage of children with consecutive positive samples before islet autoimmunity development by clinical site.

Panels a-d show the percentage positive for virus between ages 3 and 6 months (Panel a – Enterovirus A (EV-A), Panel b – Enterovirus B (EV-B), Panel c – Human mastadenovirus C (HAdV-C), and Panel d – Human mastadenovirus F (HAdV-F)). Panels e-h show the percentage consecutive positive before islet autoimmunity (Panel e – EV-A, Panel f – EV-B, Panel g – HAdV-C, and Panel h – HAdV-F). The red and blue bars represent cases and matched controls (by clinical site, gender and family history of type 1 diabetes), respectively. The darker color show the percentage of children positive for virus pattern with matching child negative. The light blue and red bars represent percentage of children positive for virus pattern with a concordant result in the matching child. Specifically, dark blue denotes control is positive for the virus pattern and matching case is negative. Light blue denotes both control and matching case are positive for virus pattern. Dark red denotes case is positive for virus pattern and matching pair control is negative. Light red denotes both case and matching control are positive for virus pattern. Asterisk (*) denotes statistically significant difference overall of discordant cases positive for virus (dark red) compared to discordant controls positive for virus (dark blue) across clinical sites (matched pair children in US-Colorado, n=55; US-Georgia/Florida, n=28; US-Washington, n=36; Finland, n=104; Germany, n=31; and, Sweden, n=129). Significance was assessed using conditional logistic regression adjusted for HLA-DR-DQ genotype. All p-values are two-sided.

Figure 3.. Children single positive, multiple positive, consecutively positive and/or prolonged shedding for Enterovirus B (EV-B) on risk of islet autoimmunity.

The pattern groups were the following: 1) child was negative for EV-B (reference group), 2) child had a single sample positive for EV-B, 3) child had multiple, independent, non-consecutive samples positive for EV-B, 4) child had consecutive EV-B, but with unconfirmed prolonged shedding, and 5) child had multiple and prolonged shedding of the same EV-B serotype. Consecutive positive allowed for no more than one missed monthly stool sample. Odds ratios (OR, circle) and 95% confidence intervals (CI, bars) were calculated using conditional logistic regression models with adjustment for HLA-DR-DQ genotype. All p-values were two-sided.

Figure 4.. Multivariable conditional logistic regression of Enterovirus B (EV-B), Human mastadenovirus C (HAdV-C) and F (HAdV-F) on islet autoimmunity case status (n=378 matched pair children).

The model shown in figure includes HLA-DR-DQ genotype, a single nucleotide polymorphism (SNP) in PTPN22, INS, ERBB3 and SH2B3 (all previously reported associated with islet autoimmunity in the TEDDY cohort and confirmed associated with islet autoimmunity in this nested-matched case-control study), a SNP in the Coxsackie and adenovirus receptor gene (CXADR), prolonged shedding or consecutive positive samples for EV-B, HAdV-C detected in stool samples up to age 6 months and number of HAdV-F detected in stools during follow-up, on islet autoimmunity case status. In addition, the model included the first two principal components describing ancestry and a propensity score for a stool to be EV-B positive. Odds ratios (black circle), 95% confidence intervals bars, and test of significant difference between cases and matched controls are estimated from multivariable conditional logistic regression model. All p-values are two-sided.