Abstract
The incidence of gastric cancer in Okinawa is lowest in Japan. Some previous reports using small number of strains suggested that the high prevalence of Helicobacter pylori with Western-type cagA in Okinawa compared to other areas in Japan might contribute to the low incidence of gastric cancer. It has still not been confirmed why the prevalence of Western-type cagA strains is high in Okinawa. We examined the association between the virulence factors of H. pylori and gastroduodenal diseases in Okinawa. The genotypes of cagA and vacA of 337 H. pylori strains were determined by PCR and gene sequencing. The genealogy of these Western-type cagA strains in Okinawa was analyzed by multilocus sequence typing (MLST). Overall, 86.4% of the strains possessed cagA: 70.3% were East-Asian type and 16.0% were Western type. After adjustment by age and sex, the presence of East-Asian-type cagA/vacA s1m1 genotypes was significantly associated with gastric cancer compared to gastritis (odds ratio = 6.68, 95% confidence interval = 1.73 to 25.8). The structure of Western-type CagA in Okinawa was different from that of typical Western-type CagA found in Western countries. Intriguingly, MLST analysis revealed that the majority of Western-type cagA strains formed individual clusters but not hpEurope. Overall, low prevalence of gastric cancer in Okinawa may result from the high prevalence of non-East-Asian-type cagA strains. The origin of Western-type cagA strains in Okinawa may be different from those of Western countries.
INTRODUCTION
Helicobacter pylori infection is now accepted as the major cause of chronic gastritis. Several epidemiological studies have shown that H. pylori infection is also linked to severe gastritis-associated diseases, including peptic ulcer and gastric cancer (33). Although gastric cancer is one of the most common cancers, only a minority of individuals with H. pylori infection ever develops it. One possible reason for the various outcomes of H. pylori infection relates to differences in the virulence of H. pylori strains in addition to host, environmental, and dietary factors. Several H. pylori virulence factors, including cagA and vacA, have been reported to be associated with the severe outcomes (4, 8, 45).
cagA, which encodes a highly immunogenic protein (CagA), is the most extensively studied H. pylori virulence factor (8, 40). cagA is a polymorphic gene; there are different numbers of repeat sequences located in the 3′ region of cagA of different H. pylori strains (46, 48, 51). The repeat regions were initially classified into two types; the first repeat and the second repeat (48). The sequence of the second repeat region was found to differ considerably between East-Asian strains (East-Asian-type cagA) and Western strains (Western-type cagA) (46, 48, 51). Each repeat region of the CagA protein contains Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs (48). It has now become more common to name the first repeat region as EPIYA-A and EPIYA-B segments and the second repeat region as EPIYA-C segment for Western-type strains or EPIYA-D segment for East-Asian-type strains (45). As such, each CagA is assigned to a sequence type consisting of the names of the EPIYA segments in its sequence (that is, ABC, ABCC, or ABCCC for Western-type CagA and ABD for East-Asian-type CagA). East-Asian-type CagA has a higher binding affinity for the Src homology-2 domain-containing phosphatase 2 (SHP2) than Western-type CagA (16). Some reports showed that individuals infected with East-Asian-type cagA strains have an increased risk of peptic ulcer and/or gastric cancer compared to those infected with non-East-Asian-type cagA strains (18, 42). Recent report using Korean population showed that individuals infected with East-Asian-type cagA strains have an increased risk of gastric cancer compared to those infected with non-East-Asian-type cagA strains (18). In addition, in Western countries, the incidence of gastric cancer is higher in patients infected with strains carrying multiple EPIYA-C repeats compared to those infected with strains of a single repeat (3, 6, 44, 46, 48). The CagA multimerization (CM) sequence (FPLxRxxxVxDLS KVG) was also different between East-Asian strains and Western strains although the functional difference has not been cleared (28). The CM sequence is located within the EPIYA-C segment, but is just downstream of the EPIYA-D segment. The same region was reported to be responsible for the interaction of CagA with activated c-Met and named as the conserved repeat responsible for phosphorylation-independent activity (CRPIA) (37).
VacA is another extensively studied H. pylori virulence factor. VacA can induce vacuolation and multiple cellular activities, including membrane-channel formation, cytochrome c release from mitochondria leading to apoptosis, and binding to cell-membrane receptors, followed by initiation of a proinflammatory response (5, 9, 20). There are variations in the vacuolating activity of different H. pylori strains (10, 21), primarily due to differences in vacA structure in the signal (s) regions (s1 and s2) and the middle (m) regions (m1 and m2) (4). The status of vacA can be classified into four subtypes by the combination of the s and m regions. In vitro experiments showed that s1m1 strains are the most cytotoxic, followed by s1m2 strains, whereas s2m2 strains have no cytotoxic activity, and s2m1 strains are rare (4). In agreement with in vitro data, many studies showed that individuals infected with vacA s1 or m1 H. pylori strains have an increased risk of peptic ulcer and/or gastric cancer compared to those with s2 or m2 strains in Western countries (4, 10, 34, 35).
Okinawa consists of small islands (2,276 km2) in southwestern Japan. Although the prevalence of H. pylori in Okinawa is not significantly different from other parts of Japan (17, 25), the incidence of gastric cancer (6.3 deaths/100,000 population) in Okinawa is the lowest in Japan (mean mortality rate of Japan, 11.8 deaths/100,000 population in 2009) (Center for Cancer Control and Information Services, National Cancer Center, Japan [http://www.ncc.go.jp/]). Okinawa was under the rule of the United States after World War II (WWII) until 1972, and there are still many U.S. populations (the number of U.S. residents in Okinawa, military personnel, civilian employees, and their families, was estimated to be 48,490 in 2009 [(http://www.pref.okinawa.jp/annai/index.html]). The different environmental factors and diets in Okinawa compared to mainland Japan are thought to be one reason for the lower incidence of gastric cancer (43). In addition, a few previous reports reported that different incidence of gastric cancer between Okinawa and mainland Japan might be explained by the high prevalence of Western-type cagA strains in Okinawa compared to other areas in Japan (7, 30, 52). However, the number of subjects in the previous studies was too small to confirm the association between different cagA types and clinical outcomes in Okinawa (e.g., only four strains from gastric cancer). These reports also did not examine the association between vacA, another important virulence factor of H. pylori, and gastric cancer in Okinawa (7, 30, 52). In the present study, we therefore aimed to examine the association between H. pylori cagA and vacA genotypes and gastroduodenal diseases in Okinawa.
Recently, genomic diversity within H. pylori populations was examined by the multilocus sequence typing (MLST) method using seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, and yphC) (13, 22, 23). At present, seven population types have been identified based on geographical associations and designated as follows: hpEurope, hpEastAsia, hpAfrica1, hpAfrica2, hpAsia2, hpNEAfrica, and hpSahul (13, 22, 23). Furthermore, hpEastAsia can be divided into three subgroups: hspEAsia, hspAmerind, and hspMaori. H. pylori strains with Western-type cagA are mainly isolated from European countries and belong to the bacterial population hpEurope, and strains from African countries belong to hpAfrica1 and hpNEAfrica. On the other hand, most H. pylori isolates with East-Asian-type cagA are isolated from East-Asian counties and belong to hpEastAsia (13, 22, 23). Although there is a report that the structure of Western-type cagA in Okinawa is rather different from that of native Western countries (39), there is no report that analyzed the population type of H. pylori isolate from Okinawa. In particular, the population type of Western-type cagA strains isolated from Okinawa has not been elucidated. We hypothesized that Western-type cagA strains did not derive from the U.S. populations after WWII but existed before WWII. To confirm the hypothesis, we also sought to analyze the population type of H. pylori isolated from Okinawa by MLST to investigate the genealogy of Western-type cagA strains.
MATERIALS AND METHODS
Patients and H. pylori.
H. pylori strains were obtained from the gastric mucosa of H. pylori-infected Japanese patients who underwent endoscopy at University of the Ryukyus (Okinawa, Japan) between February 1993 and March 2005. Presentations included gastritis, duodenal ulcer (DU), gastric ulcer (GU), and gastric cancer. Gastric biopsy specimens were taken from the antrum (pyloric gland area) and the corpus (fundic gland area). The biopsy specimens were fixed in 10% buffered formalin, embedded in paraffin, and cut into sequential 4-μm sections. DU, GU, and gastric cancer were identified by endoscopy, and gastric cancer was further confirmed by histopathology (29). Gastritis was defined as H. pylori gastritis in the absence of peptic ulcer or gastric malignancy. Patients with a history of partial gastric resection were excluded. Written informed consent was obtained from the all participants, and the protocol was approved by the Ethics Committee of University of the Ryukyus.
Isolation and genotyping of H. pylori.
Antral biopsy specimens were obtained for the isolation of H. pylori using standard culture methods as previously described (49). H. pylori DNA was extracted from confluent plate cultures expanded from a single colony using a commercially available kit (Qiagen, Inc., Santa Clarita, CA). The status of cagA was determined by PCR for conserved region of cagA and for direct sequencing using the primers cagTF (5′-ACC CTA GTC GGT AAT GGG-3′) and cagTR (5′-GCT TTA GCT TCT GAY ACY GC-3′ [Y = C or T]) designed in the 3′ repeat region of cagA, as described previously (51). To confirm the presence of cagA, we also constructed a new primer pair for conserved region of cagA: cagOMF (5′-AGC AAA AAG CGA CCT TGA AA-3′) and cagOMR (5′-AGT GGC TCA AGC TCG TGA AT-3′). The PCR conditions were initial denaturation for 5 min at 95°C, 35 amplification steps (95°C for 30 s, 56°C for 30 s, and 72°C for 30 s), and a final extension cycle of 7 min at 72°C, using Blend Taq DNA polymerase (Toyobo, Japan). The absence of cagA was confirmed by the presence of cagA empty site, as previously described (2). PCR products were purified using a QIAquick purification kit (Qiagen) according to the manufacturer's instructions, and the cagA genotype was confirmed by sequencing of PCR products (East-Asian type and Western type). DNA direct sequencing was performed using an AB 3130 genetic analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The amplified fragment was detected by a 1.5% agarose gel electrophoresis using an UV transilluminator.
The EPIYA segment types of CagA were defined according to the segment pattern as described previously (44). These EPIYA segment types of CagA were compared to previous data from strains in Okinawa obtained from GenBank. The CRPIA motifs from strains in Okinawa were compared to those of Western-CagA strains from GenBank by using the program WebLogo (version 3) (http://weblogo.threeplusone.com/) (11, 31).
The vacA genotyping (s1, s2, m1, and m2) were performed as described previously (4, 46). Primers for signal region yielded a fragment of 259 bp for s1 variants and that of 286 bp for s2 variants. Primers for middle region yielded a fragment of 570 bp for m1 variants and that of 645 bp for m2 variants.
Phylogenetic analysis of H. pylori strains.
MLST of the seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, and yphC) were determined by PCR-based sequencing as described previously (1). For construction of phylogenetic tree based on MLST genotyping procedures, sequence data sets of the 7 housekeeping genes of 1,126 strains with different genotypes were obtained from the pubMLST database (62 from hpAsia2, 493 from hpEurope, 76 from hpNEAfrica, 50 from hpSahul, 28 from hpAfrica2, 279 from hpEastAsia, and 138 from hpAfrica1) (http://pubmlst.org/). These sequence data sets were compared to our data obtained from strains in Okinawa. Neighbor-joining trees were constructed by MEGA 4.0 with 1,000 bootstrappings and using Kimura-2 parameters (19, 38).
Population structure analysis of H. pylori strains.
We analyzed bacterial population structure using STRUCTURE (v.2.3.2) software (12). Markov Chain Monte Carlo simulations of STRUCTURE were run in the admixture model with burn-in of 20,000, followed by 30,000 iterations for each run. To run STRUCTURE, a hypothetical number of bacterial populations, K, must be input. We set K as 6 to 8 and performed five runs for each K.
Statistical analysis.
The associations between the diversity of cagA or vacA and the clinical outcome were analyzed with the chi-square test and the Fisher exact probability test. A multivariate logistic regression model was used to calculate the odds ratios (OR) of the clinical outcomes by including age, sex, and H. pylori genotypes. All determinants with P values of <0.10 were entered together in the full model of logistic regression, and the model was reduced by excluding variables with P values of >0.10. The OR and 95% confidence interval (CI) were used to estimate the risk. A P value of <0.05 was accepted as statistically significant. The SPSS statistical software package version 18.0 (SPSS, Inc., Chicago, IL) was used for all statistical analyses.
RESULTS
A total of 353 strains isolated from H. pylori-positive Japanese patients (216 males [age range, 15 to 89 years; mean age, 55.3 years] and 135 females [age range, 19 to 80 years; mean age, 54.7 years]) confirmed by culture were examined in the present study. Eleven strains with undetermined cagA or vacA genotypes were excluded from the study. Five strains isolated from subjects with diseases other than gastritis, GU, DU, and gastric cancer were also excluded (four with mucosa-associated lymphoid tissue lymphoma and one with malignant lymphoma). Overall, a total of 337 strains (98 from patients with gastritis, 101 from patients with GU, 114 from patients with DU, and 24 from patients with gastric cancer) were included in the final analysis. The average age was significantly higher in gastric cancer patients than in patients with gastritis (P = 0.002) (Table 1). The male/female patient ratio was significantly higher for the GU and gastric cancer strains than for the gastritis strains (P < 0.001 and 0.04, respectively) (Table 1).
Table 1.
Association between H. pylori virulence factors and clinical outcomesa
| Description or genotype | Total |
Gastritis |
Gastric ulcer |
Duodenal ulcer |
Gastric cancer |
|||||
|---|---|---|---|---|---|---|---|---|---|---|
| No. | % | No. | % | No. | % | No. | % | No. | % | |
| Total studied | 337 | 98 | 101 | 114 | 24 | |||||
| Mean age (yr) | 51.2 | 49.9 | 51.4 | 50.2 | 62.7* | |||||
| Male | 207 | 61.4 | 47 | 48.0 | 74 | 73.3* | 69 | 60.5 | 17 | 70.8* |
| cagA positive | 291 | 86.4 | 76 | 77.6 | 89 | 88.1* | 103 | 90.4* | 23 | 95.8* |
| East-Asian-type cagA | 237 | 70.3 | 59 | 60.2 | 84 | 83.2* | 73 | 64.0 | 21 | 87.5* |
| Western-type cagA | 54 | 16.0 | 17 | 17.3 | 5 | 5.0 | 30 | 26.3 | 2 | 8.3 |
| vacA s1 | 288 | 85.5 | 75 | 76.5 | 88 | 87.1 | 102 | 89.5* | 23 | 95.8* |
| vacA s2 | 49 | 14.5 | 23 | 23.5 | 13 | 12.9 | 12 | 10.5 | 1 | 4.2 |
| vacA m1 | 232 | 68.8 | 59 | 60.2 | 81 | 80.2* | 71 | 62.3 | 21 | 87.5* |
| vacA m2 | 105 | 31.2 | 39 | 39.8 | 20 | 19.8 | 43 | 37.7 | 3 | 12.5 |
| vacA s1m1 | 230 | 68.2 | 58 | 59.2 | 80 | 79.2* | 71 | 62.3 | 21 | 87.5* |
| vacA s1m2 | 58 | 17.2 | 17 | 17.3 | 8 | 7.9* | 31 | 27.2 | 2 | 8.3 |
| vacA s2m1 | 2 | 0.6 | 1 | 1.0 | 1 | 1.0 | 0 | 0.0 | 0 | 0.0 |
| vacA s2m2 | 47 | 13.9 | 22 | 22.4 | 12 | 11.9* | 12 | 10.5* | 1 | 4.2* |
| East-Asian cagA/vacA s1m1 | 216 | 64.1 | 55 | 56.1 | 77 | 76.2* | 63 | 55.3 | 21 | 87.5* |
| East-Asian cagA/vacA s1m2 | 18 | 5.3 | 3 | 3.1 | 6 | 5.9 | 9 | 7.9 | 0 | 0.0 |
| Western cagA/vacA s1m1 | 14 | 4.2 | 3 | 3.1 | 3 | 3.0 | 8 | 7.0 | 0 | 0.0 |
| Western cagA/vacA s1m2 | 40 | 11.9 | 14 | 14.3 | 2 | 2.0* | 22 | 19.3 | 2 | 8.3 |
| Western cagA/vacA s2m2 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 |
All values indicate the number of samples and the percentage unless noted otherwise in column 1.
, P < 0.05 compared to gastritis.
cagA and vacA status in Okinawa.
The distribution of the cagA and vacA genotypes in Okinawa is shown in Table 1. Prevalence of cagA was 86.4% (291/337) and the rest (46/337, 13.6%) was cagA negative. The major cagA genotype was East-Asian-type cagA (237/337, 70.3%), and Western-type cagA was found in 16.0% (54/337). The vacA s1 genotype was the most common (288/337, 85.5%), and the vacA s2 genotype was found in 14.5% (49/337). The prevalence of the vacA m1 genotype was 68.8% (232/337), and the vacA m2 genotype was found in 31.2% (105/337). For the combination of the vacA s region and m region, 230 strains (68.3%) were s1m1, 58 (17.2%) were s1m2, 2 (0.6%) were s2m1, and 47 (13.9%) were s2m2. Regarding the combination of the cagA and vacA genotypes, the vacA s1m1 genotype was the most prevalent in East-Asian-type cagA strains (216/237, 91.1%). In Western-type cagA strains, the vacA s1m2 genotype was the most prevalent (40/54, 74.0%), and 14 strains (25.9%) were the vacA s1m1 genotype. All cagA-negative strains possessed the vacA s2/m2 genotype.
Association between virulence factors and clinical outcomes.
The prevalence of cagA was significantly higher in strains from GU (88.1%), DU (90.4%), and gastric cancer (95.8%) than those from gastritis (77.6%) (P = 0.04, 0.01 and 0.04, respectively) (Table 1). East-Asian-type cagA genotype was significantly more prevalent in strains from GU (83.2%) and gastric cancer (87.5%) than those from gastritis (60.2%) (P < 0.001 and P = 0.01, respectively). The prevalence of East-Asian-type cagA genotype was also significantly higher in strains from GU (83.2%) and gastric cancer (87.5%) than those from DU (64.0%) (P = 0.001 and 0.02, respectively). There was no significant difference between the prevalence of East-Asian-type cagA in DU and gastritis (64.0 versus 60.2%).
The vacA s1 genotype was more prevalent in DU (89.5%) and gastric cancer strains (95.8%) than in gastritis strains (76.5%) (P = 0.01 and 0.03, respectively). The vacA m1 genotype was more prevalent in GU (80.2%) and gastric cancer (87.5%) than gastritis (60.2%) (P = 0.002 and 0.01, respectively). Regarding the combination of the vacA s and m genotypes, the vacA s1m1 genotype was significantly higher in strains from GU (79.2%) and gastric cancer (87.5%) than those from gastritis (59.2%) (P = 0.002 and 0.006, respectively); on the other hand, there was no significant difference between DU (62.3%) and gastritis (59.2%). The vacA s1m2 genotype was significantly prevalent in strains from gastritis than those from GU (17.3 versus 7.9%, P = 0.04). The prevalence of the vacA s1m2 genotype tended to be higher in strains from patients with DU than those from patients with gastritis (27.2 versus 17.3%), although the difference did not reach statistical significance (P = 0.08). The prevalence of the vacA s2m2 genotype was significantly higher in strains from gastritis patients than in those from GU, DU, and gastric cancer patients (22.4 versus 11.9, 10.5, and 4.2%, P = 0.04, 0.01 and 0.04, respectively).
The prevalence of East-Asian-type cagA/vacA s1m1 genotype was significantly higher in strains from GU and gastric cancer patients than in those from gastritis patients (76.2, 87.5 versus 56.1%, P = 0.002, 0.003, respectively). After adjustment by age and sex in multivariate analysis, the presence of the East-Asian-type cagA/vacA s1m1 genotype was significantly associated with GU compared to gastritis (OR = 2.73, 95% CI = 1.44 to 5.16). It was also significantly associated with gastric cancer compared to gastritis after being adjusted by age and sex (OR = 6.68, 95% CI = 1.73 to 25.8). Western-type cagA/vacA s1m2 strains were significantly prevalent in strains from DU and gastritis than those from GU (19.3, 14.3 versus 2.0%; P < 0.001 and P = 0.001, respectively).
Association between the EPIYA segment types of CagA and clinical outcomes.
Among 236 East-Asian-type CagA strains, 223 (94.4%) contained ABD type. On the other hand, most Western-type-CagA contained ABC type (45/55; 81.8%) (Table 2). The prevalence of strains with multiple C segments (i.e., ABCC and ABCCC) was 0% in gastric cancer patients but 2.8% (2/72) in gastritis patients, 2.5% (2/81) in GU patients, and 1.0% (1/98) in DU patients. The ratio of (ABCC and ABCCC)/ABC was not significantly different in these three groups.
Table 2.
Association between EPIYA segment types of CagA and clinical outcomes
| Type | No. of samplesb |
||||
|---|---|---|---|---|---|
| Total | Gastritis | GU | DU | GC | |
| Western-type CagA | |||||
| ABa | 4 | 3 | 0 | 1 | 0 |
| AC | 1 | 0 | 0 | 1 | 0 |
| ABC | 45 | 12 | 3 | 28 | 2 |
| ABCC | 4 | 2 | 2 | 0 | 0 |
| ABCCC | 1 | 0 | 0 | 1 | 0 |
| Total | 55 | 17 | 5 | 31 | 2 |
| East-Asian-type CagA | |||||
| AD | 4 | 0 | 3 | 1 | 0 |
| ABD | 223 | 58 | 76 | 69 | 20 |
| ABBD | 6 | 0 | 4 | 2 | 0 |
| ABABD | 3 | 1 | 1 | 0 | 1 |
| Total | 236 | 59 | 84 | 72 | 21 |
AB type was defined as Western-CagA according to the sequence of B segment (TGQVASPEEPIYAQVAKKVKAKIDRLDQIASGLGGVGQAG for Western-type B segment and AGQVASPEEPIYAQVAKKVSAKIDQLNEATS for East-Asian-type B segment).
GU, gastric ulcer; DU, duodenal ulcer; GC, gastric cancer.
The EPIYA motifs in these strains were also evaluated (Table 3). We obtained seven types of EPIYA or EPIYA-like sequences. In total, 877 EPIYA motifs were obtained from the 291 CagAs. On average, each CagA sequence contained approximately three EPIYA motifs. The three most common EPIYA motifs were EPIYA (793/877 = 90.4%), EPIYT (6.4%), and ESIYA (1.5%), in agreement with our previous study that examined 560 CagAs deposited in GenBank (44). Segment B displayed the biggest change in the five amino acids, and EPIYT was the more predominant than EPIYA in Western-type CagA (85% versus 3.7%). This pattern was different from typical Western-type cagA from Western counties.
Table 3.
Frequencies of the seven types of EPIYA motifsa
| Type | All motifs |
A motif |
B motif |
C or D motif |
||||
|---|---|---|---|---|---|---|---|---|
| Motif | No. | Motif | No. | Motif | No. | Motif | No. | |
| All CagA type | EPIYA | 793 | EPIYA | 289 | EPIYA | 211 | EPIYA | 293 |
| EPIYT | 56 | QPIYA | 2 | EPIYT | 56 | EPVYA | 1 | |
| ESIYA | 13 | ESIYA | 13 | |||||
| ESIYT | 10 | ESIYT | 10 | |||||
| ELIYA | 2 | ELIYA | 2 | |||||
| QPIYA | 2 | |||||||
| EPVYA | 1 | |||||||
| Total | 877 | 291 | 292 | 294 | ||||
| Western-type CagA | EPIYA | 114 | EPIYA | 55 | EPIYT | 46 | EPIYA | 57 |
| EPIYT | 46 | ESIYT | 6 | EPVYA | 1 | |||
| ESIYT | 6 | EPIYA | 2 | |||||
| EPVYA | 1 | |||||||
| Total | 167 | 55 | 54 | 58 | ||||
| East-Asian-type CagA | EPIYA | 679 | EPIYA | 234 | EPIYA | 209 | EPIYA | 236 |
| ESIYA | 13 | QPIYA | 2 | ESIYA | 13 | |||
| EPIYT | 10 | EPIYT | 10 | |||||
| ESIYT | 4 | ESIYT | 4 | |||||
| ELIYA | 2 | ELIYA | 2 | |||||
| QPIYA | 2 | |||||||
| Total | 710 | 236 | 238 | 236 | ||||
The use of boldfacing indicates EPIYA sequences and data.
The CagA CRPIA motifs of Western-type CagA strains in Okinawa, in particular, the first motifs, were also different from those of Western-type CagA strains obtained from GenBank (FSLK versus FPLK) (Fig. 1). Interestingly, the first CRPIA motifs of Western-type CagA strains in Okinawa were different from those of the second motifs in Okinawa.
Fig 1.
CRPIA motifs of Western-type CagA strains from Okinawa and Western-type CagA strains from GenBank. The CRPIA motifs were obtained from 45 Western-type CagA (ABC type) strains and 159 Western-type CagA strains from GenBank. Sequence logos were determined by using the program in WebLogo, v3.
Population type of H. pylori in Okinawa.
The population types of 54 Western-type cagA and 46 cagA-negative strains were analyzed by MLST. Seventeen East-Asian-type cagA strains were also randomly selected and included as a reference. Intriguingly, MLST analysis revealed that the majority of Western-type cagA strains (38/54, 70.3%) belonged to the sub-branch between hpAsia2 and hspAmerind but not in hpEurope (Fig. 2). Moreover, 11 Western-type cagA strains (20.3%) were located in hspEAsia. Only four Western-type cagA strains (4/54, 7.4%) were classified as hpEurope, and one strain was classified as hpAsia2. Most cagA-negative strains (38/46, 82.6%) formed a cluster in hpEastAsia, adjacent to hspMaori. All East-Asian-type cagA strains were classified as hpEastAsia.
Fig 2.
Phylogenetic tree based on the seven housekeeping genes of H. pylori. Sequence data sets of the seven housekeeping genes of 1,126 strains with different genotypes were obtained from the the pubMLST database (62 from hpAsia2, 493 from hpEurope, 76 from hpNEAfrica, 50 from hpSahul, 28 from hpAfrica2, 279 from hpEastAsia, and 138 from hpAfrica1). The 1,126 reference strains from the GenBank database and 117 strains from Okinawa were included. Neighbor-joining trees were constructed in MEGA 4.0 using bootstrapping at 1,000 bootstrap trials and through Kimura-2 parameters. The scale bar indicates the number of amino acid substitutions per site.
Based on the MLST phylogenetic analysis, we categorized Okinawan strains into three groups: strains that were located among hspEAsia strains (Group A), strains that were clustered between hspEAsia and hspMaori (Group B), and strains that were clustered between hspAmerind and hpAsia2 (Group C) (Fig. 2).
To investigate the population structure of Okinawan strains, we performed population analysis using STRUCTURE software (12). For this analysis, we used Okinawan strains that were subjected for MLST analysis and strains of typical hpEurope, hpSahul, hpAsia2, hspMaori, hspAmerind, and hspEAsia, deposited in the pubMLST database (20 for each group). Figure 3 and Fig. S1 in the supplemental material show the results when the number of population (K) was set to 7. The major population component (the component that showed the highest probability for an individual) of all strains that belonged to group A was same as hspEAsia (olive green). Population components shown in light green and pink were specific to Okinawan strains. All of the strains in group B showed light green as a major population component, and 82.5% (33/40) of the strains in group C showed pink as a major population component. The rest of the strains in group C (17.5%) showed as a dark yellow (common with hspEAsia) as a major component. In the MLST phylogenetic tree, 12 Okinawan strains were included among hpEurope strains. Of these, 83.3% (10/12) has the same major population component to hpEurope (turquoise).
Fig 3.
Population structure of Okinawa strains. Population components of 117 Okinawa strains were predicted by using STRUCTURE software. Each horizontal bar represents one sample. Colors represent the population components, and the lengths of the colors are proportional to the probability that the sample belongs to the population of the color. The tentative number of population is set to 7. The leftmost codes (A, B, C, hpAsia2, and hpEurope) indicate the location in the MLST phylogenetic tree (Fig. 2). The result, including strains other than those from Okinawa, is available in Fig. S1 in the supplemental material.
Regarding Western-type cagA strains in Okinawa, the majority of them (38/53) belonged to group C, which showed pink as major population component. Four strains had the same component as hpEurope, which suggests that these strains are typical European strains probably infected from people of European origin. Eleven strains had the same major component as hspEAsia. In contrast, most cagA-negative strains (38/46) belonged to group B.
Nucleotide sequence accession numbers.
Nucleotide sequence data reported are available under the DDBJ accession numbers AB664064 to AB665054.
DISCUSSION
Gastric cancer is more prevalent in East Asian countries than in Western countries (14). Many studies have shown that infection with East-Asian-type cagA strains contributes to the high prevalence of gastric cancer in East-Asian countries. However, many articles led to this conclusion based on the evidence that the prevalence of East-Asian-type cagA strains was higher in East-Asian countries than in countries other than East Asia, and there are few reports that show the significance of East-Asian-type cagA in the same location (7, 18). In our previous study, the prevalence of gastric cancer was significantly higher in patients infected with East-Asian-type cagA strains than in those with Western-type cagA strains in Thailand (42). However, we could not exclude the possibility that the different ethnicity might contribute to the results because most of the patients with gastric cancer were ethnic Chinese. It is possible that the diets and habits of the different ethnic groups vary in important and unrecognized ways, such that any linkages between H. pylori genotypes and ethnic group may be spurious and be actually related to independent environmental factors.
Our study also showed that the prevalence of East-Asian-type cagA strains was significantly higher in strains from gastric cancer patients than in gastritis patients in same location. Although Yamazaki et al. previously showed that the East-Asian-type cagA strains were more prevalent in patients with gastric cancer than those with gastritis or DU in Okinawa (52), the number of patients was too small to conclude the causality (only four strains from gastric cancer). Our large-scale study confirmed the importance of East-Asian-type cagA as the significant factor for the risk of GU and gastric cancer. In addition, our study revealed that an extremely high prevalence (29.3%) of non-East-Asian-type cagA strains was detected in Okinawa. In particular, 13.6% strains were cagA negative. In contrast, we previously reported that non-East-Asian-type cagA strains could not be found in strains isolated in other parts of Japan (48, 50). For example, we previously examined 155 cagA-positive strains isolated in Kyoto, located in middle of Japan, and found that all strains possessed East-Asian-type cagA (48). In our subsequent study, we examined 210 Japanese strains isolated in Kyoto and Sapporo, located on the main island and the north island of Japan, respectively, and found that 206 (98.1%) strains possessed East-Asian-type cagA and that the remaining four strains (1.9%) were cagA negative (50). We also examined strains isolated in other East-Asian countries (i.e., South Korea, Taiwan, Hong Kong, and Vietnam); however, none had Western-type cagA; 259 of 264 (98.1%) were East-Asian-type cagA, and 5 (1.9%) were cagA negative (50). This difference in H. pylori cagA also seems to play an important role in the low incidence of gastric cancer in Okinawa and may help to explain the Asian enigmas.
The vacA s1, m1, and s1m1 genotypes were significantly associated with GU and gastric cancer, in agreement with previous reports (4). Although the vacA s1m1 genotype was the most prevalent in Okinawa, a high prevalence of other genotypes of vacA was also found compared to other parts of Japan, where most strains possess the vacA s1m1 genotypes (26). We recently reported that, in Vietnam, the vacA m2 genotype was more prevalent in Ho Chi Minh City, where the incidence of gastric cancer is lower than in Hanoi (24). The high prevalence of the vacA m2 genotype may also contribute to the low incidence of gastric cancer in Okinawa. Yamazaki et al. reported that the vacA m2 genotype was associated with peptic ulcer (52); however, these authors did not examine the association with gastric cancer due to the small number of strains from gastric cancer. Therefore, this is the first study to show the significance of vacA m1 for gastric cancer in Okinawa. In addition, East-Asian-type cagA, especially the East-Asian-type cagA/vacA s1m1 genotype, was significantly associated with GU and gastric cancer. The cagA-negative/vacA s2m2 genotypes were significantly associated with gastritis compared to other diseases, an observation that agreed with other studies from Western countries (41, 47). However, it is also true that cagA-negative/vacA s2m2 strains were evident in two gastric cancer patients. The strains were both oipA “off” and babA-negative strains (unpublished observations), and we suggest that bacterial factors would not solely determine the outcomes of gastroduodenal diseases. The number of EPIYA-C repeats was not associated with clinical outcomes, in contrast to previous reports (3, 6, 44, 46, 48). This may be due to the low number of strains with multiple EPIYA-C (n = 5) in the present study. Further study will be necessary to clarify the association between the number of EPIYA-C and clinical outcomes in Okinawa.
Recent studies have shown that Western-type cagA in Japan (J-Western type) was different from that of the typical Western-type cagA found in Western countries (39). We also reported that J-Western cagA possesses a 12-bp insertion in the cagA sequence compared to typical Western cagA strains (32). These findings showed that the Western-type cagA in Okinawa is different from the typical Western-type cagA found in Western countries. Therefore, it is probable that these strains were not derived from the U.S. populations after WWII, although it will be important to examine the genotypes of H. pylori from U.S. residents in Okinawa to further clarify these findings. Interestingly, several strains showed the unique EPIYA-like motifs (ESIYA and ESIYT), which were specific for strains isolated from Okinawa in our previous report (44). Furthermore, the CagA CRPIA motifs of Western-type cagA strains in Okinawa were also different from those of typical Western-type cagA strains. Interestingly, a recent report showed that ESIYT sequences were also common in Amerindal strains (36). In addition, a recent report showed that the CagA CRPIA motifs from Amerindal strains were different from those of typical Western-type cagA strains (36). Some CagA CRPIA motifs from Okinawan strains were started from “FSLK,” which is consistent with that from Amerindal strains. These findings suggest that these strains may derive from the same evolution. Our previous data showed that four strains isolated from the Ainu ethnic group, living in Hokkaido, the north island of Japan, were hspAmerind strains (15). There is a hypothesis that the Ainu and Okinawan people originally derive from the aboriginal Japanese who came to Japan in the Jomon period (a hunter-gatherer economy) that corresponds to the Neolithic period in Europe starting approximately 12,000 years ago. Therefore, our result may reflect the ancient population structure of Japan.
Intriguingly, MLST analysis showed that Okinawan strains could be largely divided into four groups: strains among hspEAsia that have East-Asian-type cagA or Western-type cagA, strains between hspAmerind and hpAsia2 that have Western-type cagA, and strains between hspEAsia and hspMaori that are cagA negative. Interestingly, only four Western-type cagA strains in Okinawa showed the same major population component to hpEurope. These four strains may be transferred from the United States population after WWII; however, the rest of the Western-type cagA strains should have a different origin. hspEAsia with Western-type cagA may result from recombination in the process of evolution. Furthermore, cagA-negative strains in Okinawa formed one group, which suggests that these cagA-negative strains may share a common ancestor. These groups can be derived from different ethnics. Ancient Okinawa populations were called as Ryukyuan (native Okinawan)-Japanese, which is different from the population on the main island of Japan (Hondo-Japanese). Ryukyuan-Japanese are relatively pure descendants of Jomonese (ancient Japanese, 12,000 to 2,300 years before the present), whereas Hondo-Japanese have received strong genetic infusions from migrant populations who came to western Japan (i.e., the modern Japanese, 300 BC to 700 AD) (27). It is believed that the majority of the current Okinawan populations consist of Ryukyuan (native Okinawan)-Japanese, but there are some Hondo-Japanese, and there is also a population with a mixture of the two groups to various degrees. However, the origin of the Ryukyuan-Japanese is still in question. Through population analysis, we found that Okinawan strains consisted of at least three subpopulations, and two of them were specific to Okinawa. Further study of Okinawan subpopulations would help to elucidate the human migrations that brought about this diversity. In the present study, we could not obtain enough information about the ethnics, lifestyle, and diet. These differences can influence the clinical outcomes, although it is difficult to get the information about the ethnics from the point of ethics. Further study is also necessary to elucidate the role of H. pylori virulence factors.
In conclusion, we confirmed the prevalence of the East-Asian-type cagA/vacA s1m1 genotypes for gastric cancer and GU in Okinawa, Japan. Diverse cagA and vacA genotypes contribute to the clinical outcomes in Okinawa and low incidence of gastric cancer in Okinawa. CagA sequence and MLST revealed that the origin of Western-type cagA strains is different from those of Western countries.
Supplementary Material
ACKNOWLEDGMENTS
This report is based on work supported in part by grants from the National Institutes of Health (DK62813); grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (22390085 and 22659087); Special Coordination Funds for Promoting Science and Technology from MEXT of Japan; and the Research Fund at the Discretion of the President, Oita University.
The funders had no role in study design data collection and analysis, decision to publish, or preparation of the manuscript.
We thank Y. Kudo, M. Matsuda, and A. Takahashi for their excellent technical assistance.
Footnotes
Published ahead of print 21 December 2011
Supplemental material for this article may be found at http://jcm.asm.org/.
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