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Gastroduodenal Diseases in Okinawa, Japan

Osamu Matsunari,a,bSeiji Shiota,a,cRumiko Suzuki,aMasahide Watada,a,bNagisa Kinjo,dKazunari Murakami,bToshio Fujioka,b Fukunori Kinjo,dand Yoshio Yamaokaa,e

Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu-City, Oita, Japana; Department of Gastroenterology, Oita University Faculty of Medicine, Yufu-City, Oita, Japanb; Department of General Medicine, Oita University Faculty of Medicine, Yufu-City, Oita, Japanc; Department of Endoscopy, University Hospital, University of the Ryukyus, Nishihara, Okinawa, Japand; and Department of Medicine-Gastroenterology, Baylor College of Medicine and Michael E. Debakey Veterans Affairs Medical Center, Houston, Texas, USAe

The incidence of gastric cancer in Okinawa is lowest in Japan. Some previous reports using small number of strains suggested that the high prevalence ofHelicobacter pyloriwith Western-typecagAin Okinawa compared to other areas in Japan might con-tribute to the low incidence of gastric cancer. It has still not been confirmed why the prevalence of Western-typecagAstrains is high in Okinawa. We examined the association between the virulence factors ofH. pyloriand gastroduodenal diseases in Oki-nawa. The genotypes ofcagAandvacAof 337H. pyloristrains were determined by PCR and gene sequencing. The genealogy of these Western-typecagAstrains in Okinawa was analyzed by multilocus sequence typing (MLST). Overall, 86.4% of the strains possessedcagA: 70.3% were Asian type and 16.0% were Western type. After adjustment by age and sex, the presence of East-Asian-typecagA/vacAs1m1 genotypes was significantly associated with gastric cancer compared to gastritis (odds ratio6.68, 95% confidence interval1.73 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-typecagA

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-typecagAstrains. The origin of Western-typecagAstrains in Okinawa may be different from those of Western countries.

H

elicobacter pyloriinfection is now accepted as the major cause of chronic gastritis. Several epidemiological studies have shown thatH. pyloriinfection 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 withH. pyloriinfection ever develops it. One possible reason for the various outcomes ofH. pylori infec-tion relates to differences in the virulence ofH. pyloristrains in addition to host, environmental, and dietary factors. SeveralH. pylorivirulence factors, includingcagAandvacA, have been re-ported to be associated with the severe outcomes (4, 8, 45).

cagA, which encodes a highly immunogenic protein (CagA), is the most extensively studiedH. pylorivirulence factor (8, 40).cagA is a polymorphic gene; there are different numbers of repeat se-quences located in the 3=region ofcagAof differentH. pylori strains (46, 48, 51). The repeat regions were initially classified into two types; the first repeat and the second repeat (48). The se-quence of the second repeat region was found to differ consider-ably between East-Asian strains (East-Asian-typecagA) and West-ern strains (WestWest-ern-typecagA) (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 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-typecagAstrains have an increased risk of peptic ulcer and/or gastric cancer compared to those infected with non-East-Asian-typecagAstrains (18, 42). Recent report us-ing Korean population showed that individuals infected with East-Asian-typecagAstrains have an increased risk of gastric can-cer compared to those infected with non-East-Asian-typecagA 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 be-tween East-Asian strains and Western strains although the func-tional 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 re-sponsible 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 studiedH. pylorivirulence factor. VacA can induce vacuolation and multiple cellular activities, in-cluding membrane-channel formation, cytochromecrelease from

Received26 August 2011 Returned for modification25 September 2011

Accepted11 December 2011

Published ahead of print21 December 2011

Address correspondence to Yoshio Yamaoka, yyamaoka@oita-u.ac.jp. Supplemental material for this article may be found athttp://jcm.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.05562-11

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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 differ-entH. pyloristrains (10, 21), primarily due to differences invacA structure in the signal (s) regions (s1 and s2) and the middle (m) regions (m1 and m2) (4). The status ofvacAcan 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, fol-lowed by s1m2 strains, whereas s2m2 strains have no cytotoxic activity, and s2m1 strains are rare (4). In agreement within vitro data, many studies showed that individuals infected withvacAs1 or m1 H. pyloristrains 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 ofH. pyloriin Okinawa is not significantly different from other parts of Japan (17, 25), the inci-dence of gastric cancer (6.3 deaths/100,000 population) in Oki-nawa 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 es-timated 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 rea-son 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-typecagAstrains in Okinawa compared to other areas in Japan (7, 30, 52). However, the num-ber of subjects in the previous studies was too small to confirm the association between differentcagAtypes and clinical outcomes in Okinawa (e.g., only four strains from gastric cancer). These re-ports also did not examine the association betweenvacA, another important virulence factor ofH. pylori, and gastric cancer in Oki-nawa (7, 30, 52). In the present study, we therefore aimed to ex-amine the association betweenH. pylori cagAandvacAgenotypes and gastroduodenal diseases in Okinawa.

Recently, genomic diversity withinH. pyloripopulations was examined by the multilocus sequence typing (MLST) method us-ing seven housekeepus-ing genes (atpA,efp,mutY,ppa,trpC,ureI, andyphC) (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. pyloristrains with Western-typecagAare mainly isolated from European countries and belong to the bacterial pop-ulation hpEurope, and strains from African countries belong to hpAfrica1 and hpNEAfrica. On the other hand, mostH. pylori isolates with East-Asian-typecagAare isolated from East-Asian counties and belong to hpEastAsia (13, 22, 23). Although there is a report that the structure of Western-typecagAin Okinawa is rather different from that of native Western countries (39), there is no report that analyzed the population type ofH. pyloriisolate from Okinawa. In particular, the population type of Western-type cagAstrains isolated from Okinawa has not been elucidated. We

hypothesized that Western-typecagAstrains 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 ofH. pyloriisolated from Okinawa by MLST to investigate the genealogy of Western-typecagAstrains.

MATERIALS AND METHODS

Patients andH. pylori.H. pyloristrains were obtained from the gastric mucosa ofH. 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, embed-ded in paraffin, and cut into sequential 4-␮m sections. DU, GU, and gastric cancer were identified by endoscopy, and gastric cancer was further confirmed by histopathology (29). Gastritis was defined asH. pylori gas-tritis in the absence of peptic ulcer or gastric malignancy. Patients with a history of partial gastric resection were excluded. Written informed con-sent was obtained from the all participants, and the protocol was ap-proved by the Ethics Committee of University of the Ryukyus.

Isolation and genotyping ofH. pylori.Antral biopsy specimens were obtained for the isolation ofH. pyloriusing standard culture methods as previously described (49).H. pyloriDNA was extracted from confluent plate cultures expanded from a single colony using a commercially avail-able kit (Qiagen, Inc., Santa Clarita, CA). The status ofcagAwas deter-mined by PCR for conserved region ofcagAand 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 ofcagA, as described previously (51). To confirm the presence ofcagA, we also constructed a new primer pair for conserved region ofcagA: cagOMF (5=-AGC AAA AAG CGA CCT TGA AA-3=) and cagOMR (5=-AGT GGC TCA AGC TCG TGA AT-3=). The PCR condi-tions 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 BlendTaqDNA polymerase (Toyobo, Japan). The absence ofcagAwas confirmed by the presence ofcagAempty site, as previously described (2). PCR products were purified using a QIAquick purification kit (Qiagen) according to the manufacturer’s instructions, and thecagAgenotype was confirmed by sequencing of PCR products (East-Asian type and Western type). DNA direct sequencing was per-formed 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 seg-ment pattern as described previously (44). These EPIYA segseg-ment types of CagA were compared to previous data from strains in Okinawa obtained from GenBank. The CRPIA motifs from strains in Okinawa were com-pared to those of Western-CagA strains from GenBank by using the pro-gram WebLogo (version 3) (http://weblogo.threeplusone.com/) (11, 31). ThevacAgenotyping (s1, s2, m1, and m2) were performed as de-scribed 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 ofH. pyloristrains.MLST of the seven house-keeping genes (atpA,efp,mutY,ppa,trpC,ureI, andyphC) were deter-mined by PCR-based sequencing as described previously (1). For con-struction of phylogenetic tree based on MLST genotyping procedures, sequence data sets of the 7 housekeeping genes of 1,126 strains with dif-ferent 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

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obtained from strains in Okinawa. Neighbor-joining trees were con-structed by MEGA 4.0 with 1,000 bootstrappings and using Kimura-2 parameters (19, 38).

Population structure analysis ofH. pyloristrains.We analyzed bac-terial 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 pop-ulations, 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 ofcagAor

vacAand 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, andH. pylorigenotypes. All determinants withPvalues of⬍0.10 were entered together in the full model of logistic regression, and the model was reduced by excluding variables withPvalues of⬎0.10. The OR and 95% confidence interval (CI) were used to estimate the risk. AP

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 fromH. 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 undeterminedcagAorvacAgenotypes 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 lym-phoma and one with malignant lymlym-phoma). 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).

cagAandvacAstatus in Okinawa.The distribution of thecagA andvacAgenotypes in Okinawa is shown in Table 1. Prevalence of cagAwas 86.4% (291/337) and the rest (46/337, 13.6%) wascagA negative. The major cagA genotype was East-Asian-type cagA (237/337, 70.3%), and Western-typecagAwas found in 16.0% (54/337). ThevacAs1 genotype was the most common (288/337, 85.5%), and the vacA s2 genotype was found in 14.5% (49/ 337). The prevalence of thevacAm1 genotype was 68.8% (232/ 337), and thevacAm2 genotype was found in 31.2% (105/337). For the combination of thevacAs 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 cagAandvacAgenotypes, thevacAs1m1 genotype was the most prevalent in East-Asian-typecagAstrains (216/237, 91.1%). In Western-typecagAstrains, thevacAs1m2 genotype was the most prevalent (40/54, 74.0%), and 14 strains (25.9%) were thevacA s1m1 genotype. All cagA-negative strains possessed the vacA s2/m2 genotype.

Association between virulence factors and clinical outcomes. The prevalence ofcagAwas 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-typecagAgenotype was significantly more prevalent in strains from GU (83.2%) and gastric cancer (87.5%) than those from gastritis (60.2%) (P⬍0.001 andP⫽0.01, respectively). The prevalence of East-Asian-typecagAgenotype 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-typecagAin DU and gastritis (64.0 versus 60.2%).

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ThevacAs1 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). ThevacAm1 genotype was more TABLE 1Association betweenH. pylorivirulence 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*

cagApositive 291 86.4 76 77.6 89 88.1* 103 90.4* 23 95.8* East-Asian-typecagA 237 70.3 59 60.2 84 83.2* 73 64.0 21 87.5* Western-typecagA 54 16.0 17 17.3 5 5.0 30 26.3 2 8.3

vacAs1 288 85.5 75 76.5 88 87.1 102 89.5* 23 95.8*

vacAs2 49 14.5 23 23.5 13 12.9 12 10.5 1 4.2

vacAm1 232 68.8 59 60.2 81 80.2* 71 62.3 21 87.5*

vacAm2 105 31.2 39 39.8 20 19.8 43 37.7 3 12.5

vacAs1m1 230 68.2 58 59.2 80 79.2* 71 62.3 21 87.5*

vacAs1m2 58 17.2 17 17.3 8 7.9* 31 27.2 2 8.3

vacAs2m1 2 0.6 1 1.0 1 1.0 0 0.0 0 0.0

vacAs2m2 47 13.9 22 22.4 12 11.9* 12 10.5* 1 4.2* East-AsiancagA/vacAs1m1 216 64.1 55 56.1 77 76.2* 63 55.3 21 87.5* East-AsiancagA/vacAs1m2 18 5.3 3 3.1 6 5.9 9 7.9 0 0.0 WesterncagA/vacAs1m1 14 4.2 3 3.1 3 3.0 8 7.0 0 0.0 WesterncagA/vacAs1m2 40 11.9 14 14.3 2 2.0* 22 19.3 2 8.3 WesterncagA/vacAs2m2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0

aAll values indicate the number of samples and the percentage unless noted otherwise in column 1. *,P0.05 compared to gastritis.

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prevalent in GU (80.2%) and gastric cancer (87.5%) than gastritis (60.2%) (P⫽0.002 and 0.01, respectively). Regarding the combi-nation of thevacAs and m genotypes, thevacAs1m1 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 differ-ence between DU (62.3%) and gastritis (59.2%). ThevacAs1m2 genotype was significantly prevalent in strains from gastritis than those from GU (17.3 versus 7.9%,P⫽0.04). The prevalence of the vacAs1m2 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 signifi-cance (P⫽0.08). The prevalence of thevacAs2m2 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-typecagA/vacAs1m1 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-typecagA/vacAs1m1 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-typecagA/vacAs1m2 strains were signifi-cantly prevalent in strains from DU and gastritis than those from GU (19.3, 14.3 versus 2.0%;P⬍0.001 andP⫽0.001, respec-tively).

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 sig-nificantly different in these three groups.

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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 TABLE 2Association 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

aAB type was defined as Western-CagA according to the sequence of B segment (TGQ

VASPEEPIYAQVAKKVKAKIDRLDQIASGLGGVGQAG for Western-type B segment and AGQVASPEEPIYAQVAKKVSAKIDQLNEATS for East-Asian-type B segment).

bGU, gastric ulcer; DU, duodenal ulcer; GC, gastric cancer.

TABLE 3Frequencies 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

aThe use of boldfacing indicates EPIYA sequences and data.

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EPIYA (793/877⫽90.4%), EPIYT (6.4%), and ESIYA (1.5%), in agreement with our previous study that examined 560 CagAs de-posited 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 pat-tern was different from typical Wespat-tern-typecagAfrom Western counties.

The CagA CRPIA motifs of Western-type CagA strains in Oki-nawa, in particular, the first motifs, were also different from those of Western-type CagA strains obtained from GenBank (FSLK ver-sus 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.

Population type ofH. pylori in Okinawa. The population types of 54 Western-typecagAand 46cagA-negative strains were analyzed by MLST. Seventeen East-Asian-typecagAstrains were also randomly selected and included as a reference. Intriguingly, MLST analysis revealed that the majority of Western-typecagA strains (38/54, 70.3%) belonged to the sub-branch between hpAsia2 and hspAmerind but not in hpEurope (Fig. 2). Moreover, 11 Western-typecagAstrains (20.3%) were located in hspEAsia. Only four Western-typecagAstrains (4/54, 7.4%) were classified as hpEurope, and one strain was classified as hpAsia2. MostcagA -negative strains (38/46, 82.6%) formed a cluster in hpEastAsia, adjacent to hspMaori. All East-Asian-typecagAstrains were clas-sified as hpEastAsia.

Based on the MLST phylogenetic analysis, we categorized Oki-nawan 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 sub-jected 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 pop-ulation (K) was set to 7. The major poppop-ulation 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 popula-tion 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 (tur-quoise).

Regarding Western-typecagAstrains 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, mostcagA-negative strains (38/46) belonged to group B. FIG 1CRPIA 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.

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Nucleotide sequence accession numbers. Nucleotide se-quence data reported are available under the DDBJ accession numbersAB664064toAB665054.

DISCUSSION

Gastric cancer is more prevalent in East Asian countries than in Western countries (14). Many studies have shown that infection with East-Asian-typecagAstrains contributes to the high preva-lence of gastric cancer in East-Asian countries. However, many articles led to this conclusion based on the evidence that the prev-alence of East-Asian-typecagAstrains 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-typecagAin 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 cagAstrains in Thailand (42). However, we could not exclude the possibility that the different ethnicity might contribute to the re-sults 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 betweenH. pylorigenotypes 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 cagAstrains was significantly higher in strains from gastric cancer patients than in gastritis patients in same location. Although Ya-mazaki et al. previously showed that the East-Asian-typecagA 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-typecagAas the significant factor for the risk of GU and gastric cancer. In addition, our study revealed that an ex-tremely high prevalence (29.3%) of non-East-Asian-type cagA strains was detected in Okinawa. In particular, 13.6% strains were cagAnegative. In contrast, we previously reported that non-East-Asian-typecagAstrains could not be found in strains isolated in other parts of Japan (48, 50). For example, we previously exam-ined 155cagA-positive strains isolated in Kyoto, located in middle of Japan, and found that all strains possessed East-Asian-typecagA (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-typecagAand that the remaining four strains (1.9%) werecagAnegative (50). We also examined FIG 2Phylogenetic tree based on the seven housekeeping genes ofH. pylori.

Sequence data sets of the seven housekeeping genes of 1,126 strains with dif-ferent 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 in-cluded. Neighbor-joining trees were constructed in MEGA 4.0 using boot-strapping at 1,000 bootstrap trials and through Kimura-2 parameters. The scale bar indicates the number of amino acid substitutions per site.

FIG 3Population 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 com-ponents, 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, includ-ing strains other than those from Okinawa, is available in Fig. S1 in the sup-plemental material.

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strains isolated in other East-Asian countries (i.e., South Korea, Taiwan, Hong Kong, and Vietnam); however, none had Western-typecagA; 259 of 264 (98.1%) were East-Asian-typecagA, and 5 (1.9%) werecagAnegative (50). This difference inH. 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.

ThevacAs1, m1, and s1m1 genotypes were significantly asso-ciated with GU and gastric cancer, in agreement with previous reports (4). Although thevacAs1m1 genotype was the most prev-alent in Okinawa, a high prevalence of other genotypes ofvacA was also found compared to other parts of Japan, where most strains possess thevacAs1m1 genotypes (26). We recently re-ported that, in Vietnam, thevacAm2 genotype was more preva-lent in Ho Chi Minh City, where the incidence of gastric cancer is lower than in Hanoi (24). The high prevalence of thevacAm2 genotype may also contribute to the low incidence of gastric can-cer in Okinawa. Yamazaki et al. reported that thevacAm2 geno-type 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 ofvacAm1 for gastric cancer in Okinawa. In addition, East-Asian-typecagA, especially the East-Asian-typecagA/vacAs1m1 genotype, was significantly associated with GU and gastric cancer. ThecagA-negative/vacAs2m2 geno-types 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 thatcagA -negative/vacAs2m2 strains were evident in two gastric cancer pa-tients. The strains were bothoipA“off” andbabA-negative strains (unpublished observations), and we suggest that bacterial factors would not solely determine the outcomes of gastroduodenal dis-eases. 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 nec-essary to clarify the association between the number of EPIYA-C and clinical outcomes in Okinawa.

Recent studies have shown that Western-typecagAin Japan (J-Western type) was different from that of the typical Western-typecagAfound in Western countries (39). We also reported that J-WesterncagApossesses a 12-bp insertion in thecagAsequence compared to typical WesterncagAstrains (32). These findings showed that the Western-typecagAin Okinawa is different from the typical Western-typecagAfound in Western countries. There-fore, 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 ofH. pylorifrom U.S. residents in Okinawa to fur-ther clarify these findings. Interestingly, several strains showed the unique EPIYA-like motifs (ESIYA and ESIYT), which were spe-cific 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-typecagAstrains. 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-typecagAstrains (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 cor-responds 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-typecagAor Western-typecagA, strains be-tween hspAmerind and hpAsia2 that have Western-typecagA, and strains between hspEAsia and hspMaori that arecagAnegative. Interestingly, only four Western-type cagAstrains in Okinawa showed the same major population component to hpEurope. These four strains may be transferred from the United States pop-ulation after WWII; however, the rest of the Western-typecagA strains should have a different origin. hspEAsia with Western-type cagAmay result from recombination in the process of evolution. Furthermore, cagA-negative strains in Okinawa formed one group, which suggests that thesecagA-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 popula-tion 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 mi-grant 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 Oki-nawan 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, al-though it is difficult to get the information about the ethnics from the point of ethics. Further study is also necessary to elucidate the role ofH. pylorivirulence factors.

In conclusion, we confirmed the prevalence of the East-Asian-typecagA/vacAs1m1 genotypes for gastric cancer and GU in Oki-nawa, Japan. DiversecagAandvacAgenotypes 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.

ACKNOWLEDGMENTS

This report is based on work supported in part by grants from the Na-tional Institutes of Health (DK62813); grants-in-aid for Scientific Re-search from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (22390085 and 22659087); Special Coordi-nation Funds for Promoting Science and Technology from MEXT of Ja-pan; and the Research Fund at the Discretion of the President, Oita Uni-versity.

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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.

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Association between

Helicobacter pylori

Virulence Factors and

Gastroduodenal Diseases in Okinawa, Japan

Osamu Matsunari, Seiji Shiota, Rumiko Suzuki, Masahide Watada, Nagisa Kinjo, Kazunari Murakami, Toshio Fujioka, Fukunori Kinjo, and Yoshio Yamaoka

Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu-City, Oita, Japan; Department of Gastroenterology, Oita University Faculty of Medicine, Yufu-City, Oita, Japan; Department of General Medicine, Oita University Faculty of Medicine, Yufu-City, Oita, Japan; Department of Endoscopy, University Hospital, University of the Ryukyus, Nishihara, Okinawa, Japan; and Department of Medicine-Gastroenterology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, USA

Volume 50, no. 3, p. 876 – 883, 2012. Page 877, Materials and Methods section, line 26: “cagOMR (5=-AGT GGC TCA AGC TCG TGA AT-3=)” should read “cagOMR (5=-ATT CAC GAG CTT GAG CCA CT-3=).”

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Figure

TABLE 1 Association between H. pylori virulence factors and clinical outcomesa
TABLE 2 Association between EPIYA segment types of CagA andclinical outcomes
FIG 1 CRPIA motifs of Western-type CagA strains from Okinawa and Western-type CagA strains from GenBank
FIG 2 Phylogenetic tree based on the seven housekeeping genes ofSequence data sets of the seven housekeeping genes of 1,126 strains with dif-ferent genotypes were obtained from the the pubMLST database (62 fromhpAsia2, 493 from hpEurope, 76 from hpNEAfrica

References

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