Alterations of Gut Microbiota in Patients With Graves' Disease

Abstract

Graves' disease (GD) is a systemic autoimmune disease characterized by hyperthyroidism. Evidence suggests that alterations to the gut microbiota may be involved in the development of autoimmune disorders. The aim of this study was to characterize the composition of gut microbiota in GD patients. Fecal samples were collected from 55 GD patients and 48 healthy controls. Using 16S rRNA gene amplification and sequencing, the overall bacterial richness and diversity were found to be similar between GD patients and healthy controls. However, principal coordinate analysis and partial least squares-discriminant analysis showed that the overall gut microbiota composition was significantly different (ANOSIM; p < 0.001). The linear discriminant analysis effect size revealed that Firmicutes phylum decreased in GD patients, with a corresponding increase in Bacteroidetes phylum compared to healthy controls. In addition, the families Prevotellaceae, and Veillonellaceae and the genus Prevotella_9 were closely associated with GD patients, while the families Lachnospiraceae and Ruminococcaceae and the genera Faecalibacterium, Lachnospira, and Lachnospiraceae NK4A136 were associated with healthy controls. Metagenomic profiles analysis yielded 22 statistically significant bacterial taxa: 18 taxa were increased and 4 taxa were decreased. Key bacterial taxa with different abundances between the two groups were strongly correlated with GD-associated clinical parameters using Spearman's correlation analysis. Importantly, the discriminant model based on predominant microbiota could effectively distinguish GD patients from healthy controls (AUC = 0.825). Thus, the gut microbiota composition between GD patients and healthy controls is significantly difference, indicating that gut microbiota may play a role in the pathogenesis of GD. Further studies are needed to fully elucidate the role of gut microbiota in the development of GD.

Keywords: 16S rRNA; Graves’ disease; clinical parameters; gut microbiota; next-generation sequencing.

Figure 1

The gut microbiota of GD patients differs from that of healthy controls. Rank-abundance curves were used to explain species richness and evenness (A). Rarefaction curves were used to evaluate the species richness. The bacterial communities of the healthy controls (HC) and GD patients exhibited similar patterns (B). The relative abundances of the top 10 microbial phyla and genera are represented. Only phyla and genera present at relative abundances >1% are shown. Taxa with lower abundances are grouped as “other” (C, D). Principal coordinates analysis (PCOA) based on the distance matrix of Bray-Curtis dissimilarity at the OTU level showed that the gut microbiota of GD patients was separated clearly from those of healthy controls (E). The PLS-DA (Partial Least Squares Discriminant Analysis) showed a significant separation between GD and healthy controls (F).

Figure 2

Taxonomic cladogram plotted from linear discriminant analysis (LDA) effect size (LEfSe) analysis and statistical analysis of metagenomic profiles (STAMP) analysis. Linear discriminant analysis effect size (LEfSe) analysis shows bacterial taxa significantly enriched in the GD (red) or healthy controls (HC, green) groups. Taxonomic cladogram and linear discriminant analysis (LDA) scores show differences among taxa between GD and healthy controls. Only taxa meeting a significant LDA threshold value of >4 are shown (A, B). Differentially abundant taxa from the phylum to genus level were further analyzed by STAMP analysis using Welch’s t-test (p < 0.05, q < 0.05) (C).

Figure 3

Spearman correlation analysis of the gut microbiota with clinical parameters. Heatmap showing the relationships of altered 29 gut microbiota and 5 clinical parameters in healthy controls and GD patients. Color intensity indicates the magnitude of correlation. Red = positive correlation; blue = negative correlation. ⁺p < 0.05, ⁺⁺p < 0.01. BMI, body mass index; FT4, free thyroxine; TSH, thyrotropin; TPOAb, thyroperoxidase antibody (A). Heatmap analyses of the relative abundant of top 15 taxa from 29 gut microbiota. The heatmap plot depicts the relative abundance of each bacterial taxa (vertical axis) within each group (horizontal axis). The color of the spot in the right panel corresponds to the relative values of the taxa in each group (B). Random forest analysis was used to classify GD patients and healthy controls based on the top 15 most abundant taxa. The performance of the random forest analysis was assessed with the AUC of the ROC curve. The light pink area indicates the 95% confidence interval (C).