Study of Helicobacter pylori Isolated from a High-Gastric-Cancer-Risk Population: Unveiling the Comprehensive Analysis of Virulence-Associated Genes including Secretion Systems, and Genome-Wide Association Study
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Study Population and Sampling
2.2. H. pylori Isolation and Histological Evaluation
2.3. Genome Sequencing, De Novo Assembly, and Annotation
2.4. Identification of Virulence-Associated Genes and T4SS Genes
2.5. Characterization and Genotyping of Well-Known Virulence-Associated Genes
2.6. Population Structure and GWAS
2.7. Statistical Analysis
3. Results
3.1. Characteristics of Clinical Isolates H. pylori, and Their Population Structure
3.2. Diversity of OMP and Virulence Factor Genes Associated with Gastric Cancer and Other Diseases
3.3. Diversity of ICEHptfs in Gastric Cancer and Other Diseases
3.4. Risk Estimation of Significant Genes and Genotypes between Gastric Cancer and Other Diseases
3.5. Identification of SNPs and Motifs Associated with Gastric Cancer and Other Diseases by GWAS
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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H. pylori. Populations | Non-GC Group | GC group (n = 16) | OR a (95% CI) | p b | ||
---|---|---|---|---|---|---|
NAG (n = 120) | PUD (n = 9) | AG/IM (n = 78) | ||||
Sg1 (n = 158) | 73 (60.8%) | 8 (88.9%) | 62 (79.5%) | 15 (93.8%) | 6.7 (0.9–51.9) | 0.044 |
Other (n = 65) | 47 (39.2%) | 1 (11.1%) | 16 (20.5%) | 1 (6.2%) |
Genes | Genotypes | NAG (n = 120) | PUD (n = 9) | AG/IM (n = 78) | GC (n = 16) | Total (n = 223) | p a |
---|---|---|---|---|---|---|---|
cagA | AB 1 | 4 (3.3%) | 0 | 0 | 0 | 4 (1.8%) | 0.006 |
ABC 2 | 49 (40.8%) | 3 (33.3%) | 43 (55.1%) | 8 (50.0%) | 103 (46.2%) | ||
ABCn 3 | 25 (20.8%) | 4 (44.4%) | 19 (24.4%) | 7 (43.8%) | 55 (24.7%) | ||
ABD 4 | 2 (1.7%) | 1 (11.1%) | 5 (6.4%) | 0 | 8 (3.6%) | ||
Negative | 40 (33.3%) | 1 (11.1%) b | 11 (14.1%) b | 1 (6.3%) b | 53 (23.8%) | ||
vacA | s1i1m1 | 52 (43.3%) | 6 (66.7%) b | 53 (67.9%) b | 13 (81.3%) b | 124 (55.6%) | 0.014 |
s1i1m2 | 12 (10.0%) | 1 (11.1%) | 4 (5.1%) | 1 (6.3%) | 18 (8.1%) | ||
s1i3m2 | 19 (15.8%) | 1 (11.1%) | 10 (12.8%) | 1 (6.3%) | 31 (13.9%) | ||
s2i3m2 | 31 (25.8%) | 0 b | 7 (9.0%) b | 1 (6.3%) b | 39 (17.5%) | ||
other | 6 (5.0%) | 1 (11.1%) | 4 (5.1%) | 0 | 11 (4.9%) | ||
htrA | S variant | 96 (80.0%) | 9 (100%) | 69 (88.5%) | 14 (87.5%) | 188 (84.3%) | 0.118 |
L variant | 24 (20.0%) | 0 | 9 (11.5%) | 2 (12.5%) | 35 (15.7%) | ||
iceA | Type 1 | 57 (47.5%) | 7 (77.8%) | 41 (52.6%) | 4 (25.0%) | 109 (48.9%) | 0.055 |
Type 2A | 2 (1.7%) | 0 | 4 (5.1%) | 2 (12.5%) | 8 (3.6%) | ||
Type 2B | 16 (13.3%) | 0 | 7 (9.0%) | 0 | 23 (10.3%) | ||
Type 2C | 20 (16.7%) | 1 (11.1%) | 9 (11.5%) | 1 (6.3%) | 31 (13.9%) | ||
Type 2D | 24 (20.0%) | 1 (11.1%) b | 17 (21.8%) | 9 (56.3%) b | 51 (22.9%) | ||
Negative | 1 (0.8%) | 0 | 0 | 0 | 1 (0.4%) | ||
babA | AD2 | 2 (1.7%) | 0 | 1 (1.3%) | 0 | 3 (1.3%) | 0.417 |
AD3 | 36 (30.0%) | 5 (55.6%) | 25 (32.1%) | 4 (25.0%) | 70 (31.4%) | ||
AD4 | 77 (64.2%) | 4 (44.4%) | 52 (66.7%) | 11 (68.8%) | 144 (64.6%) | ||
Negative | 5 (4.2%) | 0 | 0 | 1 (6.3%) | 6 (2.7%) | ||
babB | BD1 | 22 (18.3%) | 2 (22.2%) | 19 (24.4%) | 5 (31.3%) | 48 (21.5%) | 0.516 |
BD2 | 46 (38.3%) | 4 (44.4%) | 30 (38.5%) | 9 (56.3%) | 89 (39.9%) | ||
BD3 | 19 (15.8%) | 2 (22.2%) | 10 (12.8%) | 1 (6.3%) | 32 (14.3%) | ||
unclassified | 21 (17.5%) | 1 (11.1%) | 10 (12.8%) | 1 (6.3%) | 33 (14.8%) | ||
Negative | 12 (10.0%) | 0 | 9 (11.5%) | 0 | 21 (9.4%) | ||
hopQ | Type I | 74 (61.7%) | 8 (88.9%) b | 65 (83.3%) b | 15 (93.8%) b | 162 (72.6%) | <0.001 |
Type II | 42 (35.0%) | 0 b | 9 (11.5%) b | 1 (6.3%) b | 52 (23.3%) | ||
Type I/II | 4 (3.3%) | 1 (11.1%) | 4 (5.1%) | 0 | 9 (4.0%) | ||
hopZ | Type I | 97 (80.8%) | 8 (88.9%) | 73 (93.6%) | 14 (87.5%) | 192 (86.1%) | 0.121 |
Type II | 23 (19.2%) | 1 (11.1%) | 5 (6.4%) | 2 (12.5%) | 31 (13.9%) | ||
sabA | Present | 107 (89.2%) | 7 (77.8%) | 66 (84.6%) | 14 (87.5%) | 194 (87.0%) | 0.688 |
Negative | 13 (10.8%) | 2 (22.2%) | 12 (15.4%) | 2 (12.5%) | 29 (13.0%) | ||
sabB | Present | 58 (48.3%) | 7 (77.8%) b | 55 (70.5%) b | 14 (87.5%) b | 134 (60.1%) | 0.001 |
Negative | 62 (51.7%) | 2 (22.2%) b | 23 (29.5%) b | 2 (12.5%) b | 89 (39.9%) | ||
homA/B | homA | 67 (55.8%) | 3 (33.3%) b | 32 (41.0%) b | 3 (18.8%) b | 105 (47.1%) | 0.115 |
homB | 45 (37.5%) | 6 (66.7%) | 41 (52.6%) | 10 (62.5%) | 102 (45.7%) | ||
homA/B | 5 (4.2%) | 0 | 3 (3.8%) | 2 (12.5%) | 10 (4.5%) | ||
homI | 3 (2.5%) | 0 | 2 (2.6%) | 1 (6.3%) | 6 (2.7%) |
T4SS | Genotypes | NAG (n = 120) | PUD (n = 9) | AG/IM (n = 78) | GC (n = 16) | Total (n = 223) | p a | ||
---|---|---|---|---|---|---|---|---|---|
cagPAI | Presence § | Y | 80 (66.7%) | 8 (88.9%) b | 68 (87.2%) b | 16 (100.0%) b | 172 (77.1%) | <0.001 | |
N | 40 (33.3%) | 1 (11.1%) b | 10 (12.8%) b | 0 (0.0%) b | 51 (22.9%) | ||||
Completeness §§ | Y | 79 (65.8%) | 8 (88.9%) b | 67 (85.9%) b | 15 (93.8%) b | 169 (75.8%) | <0.001 | ||
N | 1 (0.8%) | 0 b | 1 (1.3%) b | 1 (6.3%) b | 3 (1.3%) | ||||
cagA presence | Y | 80 (66.7%) | 8 (88.9%) b | 67 (85.9%) b | 15 (93.7%) b | 170 (98.8%) | 0.274 | ||
N | 40 (33.3%) | 1 (11.1%) b | 11 (14.1%) b | 1 (6.3%) b | 2 (1.2%) | ||||
comB | Presence § and Completeness §§ | Y | 120 (100%) | 9 (100%) | 78 (100%) | 16 (100%) | 223 (100%) | - | |
N | 0 | 0 | 0 | 0 | 0 | ||||
tfs3 | Presence § | Y | 84 (70.0%) | 7 (77.8%) | 57 (73.1%) | 11 (68.8%) | 159 (71.3%) | 0.927 | |
N | 36 (30.0%) | 2 (22.2%) | 21 (26.9%) | 5 (31.3%) | 64 (28.7%) | ||||
Completeness §§ | Y | 28 (23.3%) | 1 (11.1%) | 12 (15.4%) | 1 (6.3%) | 42 (18.8%) | 0.132 | ||
N | 56 (46.7%) | 6 (66.7%) | 45 (57.7%) | 10 (62.5%) | 117 (52.5%) | ||||
ctkA | Y | 43 (35.8%) | 5 (55.6%) | 31 (39.7%) | 4 (25.0%) | 83 (37.2%) | 0.451 | ||
N | 77 (64.2%) | 4 (44.4%) | 47 (60.3%) | 12 (75.0%) | 140 (62.8%) | ||||
tfs4 | Presence § | Y | 101 (84.2%) | 8 (88.9%) | 74 (94.9%) | 16 (100.0%) b | 199 (89.2%) | 0.022 | |
N | 19 (15.8%) | 1 (11.1%) | 4 (5.1%) | 0 b | 24 (10.8%) | ||||
Completeness §§ | Y | 42 (35.0%) | 1 (11.1%) | 16 (20.5%) b | 2 (12.5%) b | 61 (27.4%) | 0.005 | ||
N | 59 (49.2%) | 7 (77.8%) | 58 (74.4%) b | 14 (87.5%) b | 138 (61.9%) | ||||
dupA | Y | 37 (30.8%) | 2 (22.2%) | 15 (19.2%) | 2 (12.5%) | 56 (25.1%) | 0.163 | ||
N | 83 (69.2%) | 7 (77.8%) | 63 (80.8%) | 14 (87.5%) | 167 (74.9%) | ||||
Type | tfs4a | 7 (5.8%) | 0 | 1 (1.3%) | 1 (6.3%) | 9 (4.0%) | 0.012 | ||
tfs4b | 31 (25.8%) | 1 (11.1%) | 12 (15.4%) | 1 (6.3%) | 45 (20.2%) | ||||
tfs4c | 1 (0.8%) | 0 | 2 (2.6%) | 1 (6.3%) | 4 (1.8%) | ||||
tfs4-other §§§ | 62 (51.7%) | 7 (77.8%) b | 59 (75.6%) b | 13 (81.3%) b | 141 (63.2%) | ||||
Combinations | cagPAI+tfs3 | 69 (57.5%) | 6 (66.7%) | 51 (62.4%) | 11 (68.8%) | 137 (61.4%) | 0.224 | ||
cagPAI+tfs4 | 72 (60.0%) | 7 (77.8%) | 64 (82.1%) | 16 (100%) | 159 (71.3%) | 0.229 | |||
tfs3+tfs4 | 75 (62.5%) | 6 (66.7%) | 54 (69.2%) | 11 (68.8%) | 146 (65.5%) | 0.313 | |||
tfs3+tfs4a | 2 (1.7%) | 0 | 1 (1.3%) | 0 | 3 (1.3%) | 0.386 | |||
tfs3+tfs4b | 19 (15.8%) | 0 | 10 (12.8%) | 1 (6.3%) | 30 (7.4%) | ||||
tfs3+tfs4c | 0 | 0 | 0 | 0 | 0 | ||||
All T4SS | 62 (51.7%) | 5 (55.6%) | 48 (61.5%) | 11 (68.8%) | 126 (56.5%) | 0.398 |
Univariate Logistic Regression | |||
---|---|---|---|
OR a | OR 95% CI | p | |
cagA presence/oipA “on” vs. cagA absence/oipA “off” status | 5.0 | 0.6–39.0 | 0.122 |
vacA s1i1m1 vs. vacA other alleles | 3.7 | 1.0–13.5 | 0.044 |
hopQ-I vs. II or I/II alleles | 6.1 | 0.8–47.4 | 0.083 |
sabB presence vs. absence | 5.1 | 1.1–22.9 | 0.035 |
sabB “on” vs. “off” status | 2.9 | 1.0–8.1 | 0.042 |
tfs4 complete vs. incomplete | 0.4 | 0.1–1.6 | 0.183 |
tfs4b vs. tfs4 other types | 0.3 | 0.0–1.9 | 0.182 |
ceuE presence vs. absence | 0.14 | 0.0–1.1 | 0.062 |
hopD presence vs. absence | 0.27 | 0.1–0.8 | 0.014 |
frpB2 presence vs. absence | 4.3 | 0.6–33.3 | 0.164 |
Iron regulated OMP genes (numbers) many vs. less | 2.3 | 0.6–8.9 | 0.240 |
Types | p | Gene Locus in 26,695 | Gene Name | Position in the 26,695 Genome | Position in the Gene | Safe Genotype | Risk Genotype | Frequency of Non-GC/GC, % | Effect on Amino Acid Sequence | Predicted Function |
---|---|---|---|---|---|---|---|---|---|---|
SNP | 3.62 × 10−9 | HP0763 | ftsY | 817,043 | 161 | T | C | 2.8/40.0 | Leu54Ser | Signal recognition particle-docking protein |
SNP | 3.80 × 10−8 | HP0053 | hpyAV | 52,671 | 1048 | G | A | 3.5/40.0 | Leu350Phe | R-M system type II |
SNP | 3.80 × 10−8 | 52,679 | 1039 | G | T | 3.5/40.0 | Thr347Asn | |||
SNP | 3.80 × 10−8 | 52,681 | 1041 | C | T | 3.5/40.0 | ||||
SNP | 2.57 × 10−7 | HP1222 | dld-II | 1,297,719 | 2251 | C | T | 4.2/40.0 | Val751Ile | FAD-binding and (Fe-S)-binding domain-containing protein |
SNP | 5.39 × 10−7 | HP0452 | hyp a | 469,160 | 405 | G | T | 2.8/33.3 | Glu135Asp | (McrB family protein) |
SNP | 5.39 × 10−7 | HP1280 | hyp a | 1,355,874 | 808 | C | T | 2.8/33.3 | Thr270Val | (Anthranilate phosphoribosyltransferase) |
SNP | 5.39 × 10−7 | HP1547 | leuS a | 1,625,904 | 2014 | C | T | 2.8/33.3 | Ala672Thr | Leuci-tRNA ligase |
k-mer | 3.62 × 10−9 | HP1220 | yhcG | 1,295,987–1,296,020 | 283–316 | other b | TTTTACAAGGATTTTTTTAGCGATTTTGATCCAT | 2.8/40.0 | multiple | ABC transporter ATP-binding protein |
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Saruuljavkhlan, B.; Alfaray, R.I.; Oyuntsetseg, K.; Gantuya, B.; Khangai, A.; Renchinsengee, N.; Matsumoto, T.; Akada, J.; Azzaya, D.; Davaadorj, D.; et al. Study of Helicobacter pylori Isolated from a High-Gastric-Cancer-Risk Population: Unveiling the Comprehensive Analysis of Virulence-Associated Genes including Secretion Systems, and Genome-Wide Association Study. Cancers 2023, 15, 4528. https://doi.org/10.3390/cancers15184528
Saruuljavkhlan B, Alfaray RI, Oyuntsetseg K, Gantuya B, Khangai A, Renchinsengee N, Matsumoto T, Akada J, Azzaya D, Davaadorj D, et al. Study of Helicobacter pylori Isolated from a High-Gastric-Cancer-Risk Population: Unveiling the Comprehensive Analysis of Virulence-Associated Genes including Secretion Systems, and Genome-Wide Association Study. Cancers. 2023; 15(18):4528. https://doi.org/10.3390/cancers15184528
Chicago/Turabian StyleSaruuljavkhlan, Batsaikhan, Ricky Indra Alfaray, Khasag Oyuntsetseg, Boldbaatar Gantuya, Ayush Khangai, Namsrai Renchinsengee, Takashi Matsumoto, Junko Akada, Dashdorj Azzaya, Duger Davaadorj, and et al. 2023. "Study of Helicobacter pylori Isolated from a High-Gastric-Cancer-Risk Population: Unveiling the Comprehensive Analysis of Virulence-Associated Genes including Secretion Systems, and Genome-Wide Association Study" Cancers 15, no. 18: 4528. https://doi.org/10.3390/cancers15184528