Molecular Alteration Analysis of Human Gut Microbial Composition in Graves' disease Patients

Hafiz Muhammad Ishaq, Imran Shair Mohammad, Muhammad Shahzad, Chaofeng Ma, Muhammad Asif Raza, Xiaokang Wu, Hui Guo, Peijie Shi, Jiru Xu, Hafiz Muhammad Ishaq, Imran Shair Mohammad, Muhammad Shahzad, Chaofeng Ma, Muhammad Asif Raza, Xiaokang Wu, Hui Guo, Peijie Shi, Jiru Xu

Abstract

The gut microbial association with host co-existence is critical for body homeostasis and pathogenicity. Graves' disease (GD) is an autoimmune disease manifested with hyperthyroidism and ophthalmopathy. However, we hypothesized that gut bacteria could affect an important role in GD pathogenicity. The current study aim was to characterize and investigate the intestinal bacterial composition of GD qualitatively and quantitatively. 27 GD and 11 healthy controls were enrolled for fecal sample collection. The PCR-DGGE of 16S rRNA gene by targeting V3 region and Real-time PCR for Lactobacillus, Bifidobacterium, Bacteroides vulgatus and Clostridium leptum, were performed. High-throughput sequencing of 16S rRNA gene with the V3+V4 site was perormed on Hiseq2500 platform on randomly 20 selected samples. The relative analysis of richness indices and diversity illustrated lesser diversification of intestinal bacteria in GD patients in contrast to controls. The data statistics shows the alteration in phyla of GD as compared to control. At the family taxonomic level, the relative abundance of Prevotellaceae and Pasteurellaceae were significantly higher in patients, while Enterobacteriaceae, Veillonellaceae, and Rikenellaceae were significantly lower in the diseased group as compared to control. At the genus level, a significant raised in genera count of the diseased group were Prevotella_9 and Haemophilus, while significantly decreased in the genera of the GD group were Alistipes and Faecalibacterium. The modulation in intestinal bacterial composition was checked at species level particularly H. parainfluenza abundance was raised in GD. The outcomes of the current study are aligned with the proposed hypothesis of gut microbial dysbiosis in GD. Statistically, alpha indices and differential abundance analyses of each intestinal bacterial community were significantly changed in GD. Therefore, the current study may provide a new insight into the GD pathogenesis and, in turn, explore its contribution in possible treatments.

Keywords: DGGE; GD; Gut microbiota; High-throughput sequencing; Hyperthyroidism; Ophthalmopathy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
A. DGGE gel profile constructed by linker primer of 16S rRNA gene with V3 region, B. Cluster analysis of GD (G1-G15) and healthy control group (C1-C5) by computing a Dice's coefficient and the UPGMA, C. DGGE gel profile constructed by linker primer of 16S rRNA gene with V3 region, D. Cluster analysis of GD (G16-G27) and control group (C7-C11) by applying the UPGMA and Dice's coefficient.
Figure 2
Figure 2
GD observations for OTUs and Tag number versus in contrast to control OTUs and Tag number with 97 % similarity level.
Figure 3
Figure 3
Between-sample diversity, sample G1-G10 from GD patients and C1-C10 controls. UPGMA based on weighted UniFrac distances.
Figure 4
Figure 4
Gut microbial composition differences between GD and C at the phylum level.
Figure 5
Figure 5
10 most prevalent families with relative abundance in GD and healthy controls. The relative abundance of Prevotellaceae and Pasteurellaceae were significantly higher in patients, While Enterobacteriaceae, Veillonellaceae, and Rikenellaceae were significantly lower in the diseased group as compared to control. Where (* P

Figure 6

10 most existed genera with…

Figure 6

10 most existed genera with relative abundance observed in GD and healthy controls.…

Figure 6
10 most existed genera with relative abundance observed in GD and healthy controls. Significant increased genera in GD were Prevotella_9 and Haemophilus, while significantly decreased genera of diseased group were Alistipes and Faecalibacterium. Where (* P<0.05).
Figure 6
Figure 6
10 most existed genera with relative abundance observed in GD and healthy controls. Significant increased genera in GD were Prevotella_9 and Haemophilus, while significantly decreased genera of diseased group were Alistipes and Faecalibacterium. Where (* P<0.05).

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