S1 Genotype Research Articles

Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases.

Our aim was to detect Helicobacter pylori (H. pylori) from gastric biopsies of 248 patients using a novel, polymerase chain reaction (PCR)-based methodology, which simultaneously facilitates the determination of H. pylori vacA genotypes and cagA gene. A simple methodology for sample preparation was established and PCR was performed with primer systems for the 16S rRNA, vacA, and cagA genes, thus circumventing the need to culture H. pylori and to extract DNA from biopsy samples. Infection with H. pylori was detected in 147 (59.3%) of 248 patients. The vacA signal sequence genotype s1 was present in 104 (81.3%) of 128 H. pylori-positive patients, and 24 (18.8%) patients had the genotype s2. The vacA middle region types m1 and m2 were detected in 46 (35.9%) and 79 (61.7%) patients, respectively. The combinations s1/m2 (43%) and s1/m1 (35.9%) were found more frequently than s2/m2 (18.8%). The cagA gene was detected in 75 (72.1%) of 104 H. pylori-positive biopsies with the vacA genotype s1. All 24 biopsies with the type s2 were cagA negative. Strains of the type vacA s1 were found in 97% of H. pylori-positive patients with peptic ulcer disease and were associated with the presence of the cagA gene, whereas 96% of the strains of the type vacA s2 were detected in patients who only had nonulcer dyspepsia. Using a novel PCR-based methodology, H. pylori 16S rRNA gene, vacA genotypes, and cagA gene can now be rapidly detected directly in gastric biopsies with high accuracy. These data demonstrate that infection with H. pylori strains of the vacA s1 genotype and the cagA gene are more likely to result in peptic ulcer disease. Determination of vacA genotypes and cagA gene may contribute to the potential clinical identification of patients at different levels of risk.

Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases

OBJECTIVE: Our aim was to detect Helicobacter pylori ( H. pylori) from gastric biopsies of 248 patients using a novel, polymerase chain reaction (PCR)-based methodology, which simultaneously facilitates the determination of H. pylori vacA genotypes and cagA gene. METHODS: A simple methodology for sample preparation was established and PCR was performed with primer systems for the 16S rRNA, vacA, and cagA genes, thus circumventing the need to culture H. pylori and to extract DNA from biopsy samples. RESULTS: Infection with H. pylori was detected in 147 (59.3%) of 248 patients. The vacA signal sequence genotype s1 was present in 104 (81.3%) of 128 H. pylori-positive patients, and 24 (18.8%) patients had the genotype s2. The vacA middle region types m1 and m2 were detected in 46 (35.9%) and 79 (61.7%) patients, respectively. The combinations s1/m2 (43%) and s1/ml (35.9%) were found more frequently than s2/m2 (18.8%). The cagA gene was detected in 75 (72.1%) of 104 H. pylori-positive biopsies with the vacA genotype s1. All 24 biopsies with the type s2 were cagA negative. Strains of the type vacA s1 were found in 97% of H. pylori-positive patients with peptic ulcer disease and were associated with the presence of the cagA gene, whereas 96% of the strains of the type vacA s2 were detected in patients who only had nonulcer dyspepsia. CONCLUSIONS: Using a novel PCR-based methodology, H. pylori 16S rRNA gene, vacA genotypes, and cagA gene can now be rapidly detected directly in gastric biopsies with high accuracy. These data demonstrate that infection with H. pylori strains of the vacA s1 genotype and the cagA gene are more likely to result in peptic ulcer disease. Determination of vacA genotypes and cagA gene may contribute to the potential clinical identification of patients at different levels of risk.

Genotypes of Helicobacter pylori obtained from gastric ulcer patients taking or not taking NSAIDs

OBJECTIVE: Whether Helicobacter pylori infection and use of nonsteroidal antiinflammatory drugs (NSAIDs) are independent risk factors for ulcerogenesis remains unclear. We undertook this study to evaluate H. pylori isolates from gastric ulcer patients to determine whether the genotype of the infecting isolate could be correlated with the use or nonuse of NSAIDs. METHODS: Fifty-two patients presenting with gastric ulcer and infected with H. pylori were included; 26 patients were taking NSAIDs or aspirin (ASA) regularly at the time of ulcer diagnosis. Polymerase chain reaction (PCR) was employed to assess the presence and mosaicism of the following H. pylori genes: cagA, vacA, iceA, and picB. RESULTS: We found no statistical differences in the presence of these genes in H. pylori isolates from gastric ulcer patients taking or not taking prescription NSAIDs or ASA. A 297-bp fragment of the cagA gene was detected in 96% of the isolates from the NSAID and ASA users and 100% from the non-NSAID users ( p = 1.0). A larger and more variable region of the cagA gene was detected more frequently among the isolates from non-NSAID users than those from NSAID users ( p = 0.05). Ninety-two percent of the isolates were identified as vacA genotype s1. The dominant vacA subtype was s1b, 76.9% and 65.4% in isolates from non-NSAID–taking or NSAID–taking patients, respectively ( p = 0.4). iceA1 genotype was not correlated with gastric ulcer as this allele was only detected in 17.3% of all isolates. CONCLUSIONS: No significant differences in the presence of the candidate virulence genes vacA, cagA, picB, or iceA were detected in isolates from gastric ulcer patients taking prescription NSAIDs or ASA, compared with those not taking these drugs, indicating that single gene presence does not allow discrimination of isolates that may be important in NSAID-induced ulcerogenesis. A variable region of the cagA gene was more frequently detected in isolates from patients not taking NSAIDs or ASA, suggesting that this gene may be modified by NSAID- or ASA-related factors or that certain strains may be selected for in patients taking these medications.

Genotypes of Helicobacter pylori obtained from gastric ulcer patients taking or not taking NSAIDs.

Whether Helicobacter pylori infection and use of nonsteroidal antiinflammatory drugs (NSAIDs) are independent risk factors for ulcerogenesis remains unclear. We undertook this study to evaluate H. pylori isolates from gastric ulcer patients to determine whether the genotype of the infecting isolate could be correlated with the use or nonuse of NSAIDs. Fifty-two patients presenting with gastric ulcer and infected with H. pylori were included; 26 patients were taking NSAIDs or aspirin (ASA) regularly at the time of ulcer diagnosis. Polymerase chain reaction (PCR) was employed to assess the presence and mosaicism of the following H. pylori genes: cagA, vacA, iceA, and picB. We found no statistical differences in the presence of these genes in H. pylori isolates from gastric ulcer patients taking or not taking prescription NSAIDs or ASA. A 297-bp fragment of the cagA gene was detected in 96% of the isolates from the NSAID and ASA users and 100% from the non-NSAID users (p = 1.0). A larger and more variable region of the cagA gene was detected more frequently among the isolates from non-NSAID users than those from NSAID users (p = 0.05). Ninety-two percent of the isolates were identified as vacA genotype s1. The dominant vacA subtype was s1b, 76.9% and 65.4% in isolates from non-NSAID-taking or NSAID-taking patients, respectively (p = 0.4). iceA1 genotype was not correlated with gastric ulcer as this allele was only detected in 17.3% of all isolates. No significant differences in the presence of the candidate virulence genes vacA, cagA, picB, or iceA were detected in isolates from gastric ulcer patients taking prescription NSAIDs or ASA, compared with those not taking these drugs, indicating that single gene presence does not allow discrimination of isolates that may be important in NSAID-induced ulcerogenesis. A variable region of the cagA gene was more frequently detected in isolates from patients not taking NSAIDs or ASA, suggesting that this gene may be modified by NSAID- or ASA-related factors or that certain strains may be selected for in patients taking these medications.

VacA Genotyping Directly from Gastric Biopsy Specimens and Estimation of Mixed Helicobacter pylori Infections in Patients with Duodenal Ulcer and Gastritis

Background: The vacA genotypes and the cagA gene status were investigated in 80 Helicobacter pylori-infected patients with duodenal ulcer (DU) and 49 with gastritis only. Methods: Lysates of gastric biopsy specimens were used directly for polymerase chain reaction-based detection. Results: The m1 subtype was found in 36% and 31% and the m2 in 36% and 46% of specimens from patients with DU and gastritis, respectively (P > 0.05). In 15% of samples the midregion remained unclassified. The prevalence rate of s1 subtypes was higher in cases of DU (69%) than in gastritis (43%) (P < 0.0001); the opposite correlation was observed for s2. The cagA gene was detected in 80% of patients with DU and in 52% of those with gastritis (P < 0.0001). Infections with multiple H. pylori strains exceeded 50% in both groups. Conclusions: These results suggest that vacA s1 genotype and cagA + status are associated with higher DU prevalence and that mixed H. pylori infections are very common in our geographic region.

Relationship of vacA genotypes of Helicobacter pylori to cagA status, cytotoxin production, and clinical outcome.

Mosaicism in vacA alleles with three distinct families of vacA signal sequences (s1a, s1b and s2) and two distinct families of middle region alleles (m1 and m2) has been reported. It was suggested that the vacA s1a genotype was closely associated with duodenal ulcer disease and with high cytotoxin production. The aim of this study was to evaluate the role of vacA genotyping with respect to gastric inflammation and injury, cytotoxin activity, and clinical presentation. H. pylori from patients with gastritis, peptic ulcer disease, or gastric cancer were characterized by vacA typing by polymerase chain reaction (PCR) and DNA sequencing. In vitro cytotoxin activity was assessed by vacuolation assay using Vero cells as well as with Hela cells. Four hundred ninety-one strains were tested. vacA genotype s1a/m1 was present in more than 95% of strains independent of presentation with gastritis, peptic ulcer, or gastric cancer. No vacA genotype was associated with high average cytotoxin activity. The s2/m2 isolates had low or absent cytotoxin activity. All cagA negative strains (n = 18) were s1a strains and both s2/m2 strains were cagA positive. One strain that was a recombinant of m1 and m2 strains was identified and had low cytotoxin activity. The nucleotide and amino acid sequences between original m1 strains and Japanese m1 strains (new m1 strains) were about 85% and 81%, respectively. Strains with the new m1 genotype had nucleotide and amino acid sequences similarity of more than 96%. There was no difference in cytotoxin activity between strains with the Western type m1 and the new type m1 genotype. In this as in other reported studies ( approximately 1500 patients overall) vacA genotype was strongly but not exclusively associated with the presence of cagA. Overall, the studies did not support a role for vacA genotyping in relation to cytotoxin activity, virulence, histologic finding, or risk of a particular H. pylori disease. vacA genotype s1 is likely to be a surrogate marker for the presence of the cag pathogenicity island.

Helicobacter pylori cagA status, vacA genotypes and ulcer disease.

Helicobacter pylori is the causative organism of peptic ulcer disease and has two putative virulence determinants: the cagA gene which encodes a protein of unknown function in 60% of strains, and the vacA gene, which is present in all strains, although active cytotoxin is produced in only about 50% of these. The relationship between genotypes of both cagA and vacA and resultant gastroduodenal pathology is unclear. The objective of this study was to correlate vacA genotype and cagA status with gastroduodenal pathology. One hundred and six dyspeptic patients were studied (average age 56 years, range 19-86 years, 56 men) referred for routine endoscopy. Macroscopic evidence of gastroduodenal disease was noted and antral biopsies taken for culture and genotyping of H. pylori. The polymerase chain reaction (PCR) was used to detect the cagA and vacA genes of H. pylori using specific primers. Seventy eight of the 106 (73.6%) patients were cagA positive. Of those who had peptic ulcer disease 29/32 (90.6%) were cagA positive. The presence of the cagA gene was significantly associated with peptic ulcer disease (P = 0.006). The presence of the vacA s1 genotype was also significantly associated with peptic ulcer disease (P = 0.01). The presence of the cagA gene was significantly associated with the vacA s1 genotype (P < 0.001). There was no significant difference in the distribution of the s1/m1 and s1/m2 strains between ulcer and non-ulcer patients. There is a significant association of the cagA gene and vacA s1 signal sequence with gastroduodenal ulcer disease. The relationship of the various other vacA genotypes to gastroduodenal ulcer disease is less clear.

Identification and analysis of a new vacA genotype variant of Helicobacter pylori in different patient groups in Germany.

The vacuolating cytotoxin of Helicobacter pylori (VacA) is known to cause cell damage to mammalian cells and is suspected to give rise to gastric epithelial lesions that might lead to peptic ulcer disease. As shown recently, the gene encoding VacA exhibits genetic variation, with three different families of signal sequences (s1a, s1b, and s2) and two families of midregion sequences (m1 and m2). In order to investigate the relationship between the presence of specific vacA genotypes and peptic ulceration, the vacA genotypes of 158 clinical isolates of H. pylori were determined. The study group consisted of 106 patients with duodenal ulceration; 52 patients with nonulcer dyspepsia (NUD) were used as controls. H. pylori of genotype s1 was isolated from 96% of the patients with ulcerations, whereas genotype s2 was only present in 4%, indicating a strong correlation between the vacA genotype and peptic ulceration (P < 0.001). In contrast, 31% of the patients from the NUD control group were infected with strains of vacA genotype s2. Particular midregion genotypes (m1 and m2) were not associated with clinical manifestations. The midregions from 18% of the isolates could not be classified by the proposed scheme. DNA sequencing revealed high homology between the untypeable midregions and that of genotype m1, with multiple base pair exchanges, some affecting the primer annealing site. Compared to those of m1 and m2 alleles, the divergent midregions from untypeable strains showed clustering, indicating the presence of a further subfamily of sequences in the midregion of vacA in German isolates, for which we propose the term "m1a." A new specific primer that we designed for typing m1a isolates might be useful in other studies.

The Helicobacter pylori vaca S1 genotype and the cagE gene are associated with gastroduodenal disease

The Helicobacter pylori vaca S1 genotype and the cagE gene are associated with gastroduodenal disease

Diversity of Helicobacter pylori vacA and cagA genes and relationship to VacA and CagA protein expression, cytotoxin production, and associated diseases.

The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by vacA and cagA, respectively, are important virulence determinants of Helicobacter pylori. Sixty-five H. pylori strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the vacA and cagA genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the vacA signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the vacA middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) (P = 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis (P = 0.02). The vacA genotype s1 was associated with the presence of cagA (P < 0.0001), VacA expression (P < 0.0001), and cytotoxin activity (P = 0.003). The cagA gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis (P = 0.17). The vacA genotypes of German H. pylori isolates are identical to those previously reported. H. pylori strains of vacA type s1 are associated with the occurrence of peptic ulceration and the presence of cagA, cytotoxin activity, and VacA expression.

Freezing tolerance, cold acclimation and oxidative stress in potato. Paraquat tolerance is related to acclimation but is a poor indicator of freezing tolerance

Potato is a species commonly cultivated in temperate areas where the growing season may be interrupted by frosts, resulting in loss of yield. Cultivated potato, Solanum tuberosum, is freezing sensitive, but it has several freezing‐tolerant wild potato relatives, one of which is S. commersonii. Our study was aimed to resolve the relationship between enhanced freezing tolerance, acclimation capacity and capacity to tolerate active oxygen species. To be able to characterize freezing tolerant ideotypes, a potato population (S1), which segregates in freezing tolerance, acclimation capacity and capacity to tolerate superoxide radicals, was produced by selfing a somatic hybrid between a freezing‐tolerant Solanum commersonii (LT50=‐4.6°C) and ‐sensitive S. tuberosum (LT50=‐3.0°C). The distribution of non‐acclimated freezing tolerance (NA‐freezing tolerance) of the S1 population varied between the parental lines and we were able to identify genotypes, having significantly high or low NA‐freezing tolerance. When a population of 25 genotypes was tested both for NA‐freezing and paraquat (PQ) tolerance, no correlation was found between these two traits (R = 0.02). However, the most NA‐freezing tolerant genotypes were also among the most PQ tolerant plants. Simultaneously, one of the NA‐freezing sensitive genotypes (2022) (LT50=‐3.0°C) was observed to be PQ tolerant. These conflicting results may reflect a significant, but not obligatory, role of superoxide scavenging mechanisms in the NA‐freezing tolerance of S. commersonii. The freezing tolerance after cold acclimation (CA‐freezing tolerance) and the acclimation capacity (AC) was measured after acclimation for 7 days at 4/2°C. Lack of correlation between NA‐freezing tolerance and AC (R =‐0.05) in the S1 population points to independent genetic control of NA‐freezing tolerance and AC in Solanum sp. Increased freezing tolerance after cold acclimation was clearly related to PQ tolerance of all S1 genotypes, especially those having good acclimation capacity. The rapid loss of improved PQ tolerance under deacclimation conditions confirmed the close relationship between the process of cold acclimation and enhanced PQ tolerance. Here, we report an increased PQ tolerance in cold‐acclimated plants compared to non‐acclimated controls. However, we concluded that high PQ tolerance is not a good indicator of actual freezing tolerance and should not be used as a selectable marker for the identification of a freezing‐tolerant genotype.

Failure to Confirm Association of vac A Gene Mosaicism with Duodenal Ulcer Disease

Background: Mosaicism of the Helicobacter pylori vac A gene comprises two families of allelic variations of the signal sequence region (s1, s2) and of the mid-region (m1, m2). Initial studies suggested that peptic ulcer disease correlated with the s1 subtype of vac A. We compared the prevalence of various vac A genotypes of H. pylori isolates obtained from duodenal ulcer (DU) patients and subjects with simple gastritis. Those isolates with s1 type were further examined to determine whether the specific vac A s1 (s1a versus s1b) genotype enabled prediction of gastroduodenal disease. Methods: H. pylori isolates were obtained from 38 patients with endoscopically documented DU and 39 individuals with asymptomatic H. pylori gastritis from Houston, Texas. The vac A genotype of each isolate was determined by polymerase chain reaction (PCR) amplification of genomic DNA for specific regions of the vac A gene. Those isolates with s1 vac A subtype were further examined to determine whether they had s1a or s1b mosaicism. Results: There was no difference in frequency of the s1 genotype of isolates obtained from patients with duodenal ulcer or asymptomatic H. pylori gastritis in this sample (84% versus 79%, respectively; P = 0.77). The s1/m1 vac A genotype was detected in isolates from 16 duodenal ulcer patients versus 15 with H. pylori gastritis (P = 0.82). Detailed analysis of the s1 region failed to show a correlation of either s1a or s1b with duodenal ulcer. Both s1a and s1b genotypes were detected in 24 strains, and both m1 and m2 mid-gene PCR amplicons were seen in 16 strains. Conclusions: We were unable to use H. pylori vac A genotyping to predict type of gastroduodenal disease in our patient sample. This failure to confirm an association of vac A genotype and duodenal ulcer disease differs from samples from other regions. This most likely represents an example of differences in H. pylori strains infecting host populations in different geographic regions. This study confirms the importance of establishing statistical associations with isolates from widely separate geographic regions before concluding that disease-related associations exist.

Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis.

AIMS: To determine the relation among the cytotoxin associated gene (cagA) and vacuolating cytotoxin gene (vacA) status of Helicobacter pylori isolates, the associated clinical diseases, and the severity and pattern...

Identification and expression of water stress- and abscisic acid-regulated genes in a drought-tolerant sunflower genotype.

We have studied two lines of sunflower (Helianthus annuus L.) selected in the field as drought-tolerant (R1 genotype) or drought-sensitive (S1 genotype). When subjected to drought conditions, the R1 line was able to maintain high leaf water potential longer and wilted later than the S1 line. Therefore, this indicates that R1 tolerance includes a leaf-adaptive response. By subtractive hybridization, we have isolated six different cDNAs (designated sdi for sunflower drought-induced) corresponding to transcripts accumulated in R1 and S1 leaves during adaptive response. Analysis of transcript accumulation in response to drought in both genotypes suggests a preferential expression of three sdi genes in the tolerant line. Abscisic acid-mediated induction, analysed in R1 leaves, was observed for only four sdi genes. Sequence analysis of six sdi clones revealed that five clones were related to known proteins including non specific lipid transfer proteins (nsLTP), early light-induced proteins (ELIP), l-aminocyclopropane-l-carboxylate oxidase (ACC oxidase) or dehydrins, predicted to be involved in a wide range of physiological processes.

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea.

In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S2 and S6. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S6 genotype and 350 kb in the S2 genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.

Genetic Relationship between Pollen Fertility Restoring Genes and Cytoplasmic Factors in the Male Sterile Mutants of Sugar Beets

A specific relationship between the nuclear genes and the genetic factors of cytoplasms as a consequence of cytoplasmic mutation was confirmed from the genetic interaction between the pollen fertility restoring genes and the sterile cytoplasms which were induced by gamma irradiation from the normal (N) cytoplasm, The two complementary genes Rf4 and Rf5 responsible for S1_-2 cytoplasm were different from those for S cytoplasm of spontaneous origin, and F3 lines having a genotype S1_-2 Rf4Rf4Rf5Rf5 Were bred true to the pollen fertility restored types, N or S.S.a. In the pollen fertility restoration for S1_-3 cytoplasm, an epistatic relation of two alleles (Rf6 and Rf7) which are different from S and S1_-2 showed various segregation ratios depend-ing on parental genotypes. Although the segregation mode of two additive genes was similar to those in S cytoplasm, the new genes Rf8 and Rfg were responsible fcr S1_-4 cytoplasm. Thus there is the specific relation between the pollen fertility restoring genes and the genetic factors of the male sterile cytoplasms. It is probable that the cytoplasmic mutations occurred in the strain H-2002 which contains normal (N) cytoplasm and diverse genotypes on pollen restoration and then the combination between the altered cytoplasm and the recessive type of the specific pollen fertility restoring genes indicated male sterility. If the maintainer and fcrtility restorer lines for the induced cytoplasms were selected based on the inheritance mode of pollen fertility restoration, they are also available for the use of the male sterility in hybrid seed production.