The mosaic structure of the cagA gene has been suggested to affect Helicobacter pylori CagA-associated pathogenesis. An improved polymerase chain reaction assay allowed for a rapid and detailed molecular analysis of the cagA gene 3-region in a single amplification step, followed by amplicon sequencing using universal M13 and T7 sequencing primers.

UNLABELLED: ABSTRACT:

BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.

RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.

CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.

The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.

Background

The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of cagA EPIYA motifs.

Findings

MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7- sequence-tagged primers for amplification of the cagA EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the cagA EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) cagA EPIYA motif, respectively. In two cases, double cagA EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two H. pylori strains in the same biopsy.

Conclusion

Automated capillary electrophoresis and amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of cagA EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed H. pylori strains present in the same biopsy specimen.

Cell proliferation is a process that is strictly regulated by a large number of proteins. An alteration in one of the encoding genes inserts an error into the regulative protein, which may result in uncontrolled cell growth and eventually tumor formation. Lymphoma is a cancer type originating in the lymphocytes, which are part of the body’s immune defence. In the present thesis, Znfn1a1, Notch1 and Bcl11b were studied; all involved in the differentiation of T lymphocytes. The three genes are located in chromosomal regions that have previously shown frequent loss of heterozygosity in tumor DNA.

Ikaros is a protein involved in the early differentiation of T lymphocytes. In this thesis, mutation analysis of the Znfn1a1 gene in chemically induced murine lymphomas revealed point mutations and homozygous deletions in 13 % of the tumors. All of the detected deletions lead to amino acid substitutions or abrogation of the functional domains in the Ikaros protein. Our results support the role of Ikaros as a potential tumor suppressor in a subset of tumors.

Notch1 is a protein involved in many differentiation processes in the body. In lymphocytes, Notch1 drives the differentiation towards a T-cell fate and activating alterations in the Notch1 gene have been suggested to be involved in T-cell lymphoma. We identified activating mutations in Notch1 in 39 % of the chemically induced murine lymphomas, supporting the involvement of activating Notch1 mutations in the development of T-cell lymphoma.

Bcl11b has been suggested to be involved in the early T-cell specification, and mutations in the Bcl11b gene has been identified in T-cell lymphoma. In this thesis, point mutations and deletions were detected in the DNA-binding zinc finger regions of Bcl11b in 15 % of the chemically induced lymphomas in C57Bl/6×C3H/HeJ F₁ mice. A mutational hotspot was identified, where four of the tumors carried the same mutation. Three of the identified alterations, including the hotspot mutation in Bcl11b, increased cell proliferation when introduced in a cell without endogenous Bcl11b, whereas cell proliferation was suppressed by wild-type Bcl11b in the same cell line. Mutations in Bcl11b may therefore be an important contributing factor to lymphomagenesis in a subset of tumors.

A germ line point mutation was identified in BCL11B in one of 33 human B-cell lymphoma patients. Expression of BCL11B in infiltrating T cells was significantly lower in aggressive compared to indolent lymphomas, suggesting that the infiltrating T cells may affect the B-cell lymphomas.

Activating Notch1 mutations have been reported in human T-lineage acute lymphoblastic leukemia (T-ALL) and lymphomas from genetically modified mice. We report that Notch1 is a prevalent and major mutational target in chemically induced mouse lymphoma. The regions of the gene that are frequently mutated are the hterodimerization domain and the N-terminal ligand-binding region, important for protein stability, and (he polypeptide rich in proline, glutamate, serine and threonine WEST) domains, which is critical for protein degradation. Another gene, CDC4, is also involved in Notch1 degradation and shows frequent mutations. Mutations in the heterodimerization and the ligand-binding regions may cause ligand-independent signaling. whereas mutations preventing protein degradation result in accumulation of intracellular Notch1. We analyzed 103 chemical-induced mouse lymphomas for mutations in the Notch1 gene using single strand conformation analysis (SSCA) and DNA sequencing. Genetic alterations resulting in premature truncation of Notch I were identified in 28 tumors, whereas 8 revealed alterations in the heterodimerization and 16 harbored deletions in the ligand-binding region. Dideoxycytidine-induced lymphomas displayed the highest frequency of Notch1 mutations (49%). whereas in butadiene- and phenolphthalein-indced tumors showed lower frequencies (26 10%, respectively). In total, 26 novel and 3 previously reported mutations were detected. This report shows that Notch1 is a prevalent and major mtational target for 2,3-dideoxycytidine and butadiene-induced lymphoma..

Background: The telomeric region of mouse chromosome 12 has previously shown frequent allelic loss in murine lymphoma. The Bcl11b gene has been identified and suggested as a candidate tumor suppressor gene within this region. In this study, we aimed to elucidate whether Bcl11b is mutated in lymphomas with allelic loss, and whether the mutations we detected conferred any effect on cell proliferation and apoptosis.

Methods: Mouse lymphomas induced by 1,3-butadiene or 2',3'-dideoxycytidine were analysed for mutations in the Bcl11b gene using single strand conformation analysis and direct DNA sequencing. Effects on cell proliferation by the detected mutations were studied by expressing wild-type and mutant Bcl11b in the cytokine-dependent hematopoietic progenitor cell line FDC-P1, lacking endogenous Bcl11b expression.

Results: Missense and frameshift (FS) mutations were identified in 7 of 47 tumors (15%). Interestingly, all mutations were found between amino acids 778–844 which encode the three C-terminal DNA-binding zinc fingers. In FDC-P1 cells, wild-type Bcl11b suppressed cell proliferation, whereas the mutated versions (S778N, K828T, Y844C and FS823) enhanced proliferation several-fold.

Conclusion: The genetic alterations detected in this study suggest that the three C-terminal zinc fingers of Bcl11b are important for the DNA-binding. Cell proliferation was suppressed by overexpression of wild-type Bcl11b but enhanced by mutant Bcl11b, indicating that these mutations may be an important contributing factor to lymphomagenesis in a subset of tumors.

The Znfn1a1 gene encodes a zinc finger protein called Ikaros, which is criticalfor T-cell development and differentiation. The execution of normalfunction of Ikaros requires sequence-specific DNA binding, transactivation, and dimerization domains. In this study, exons 3–5 and exon 7 of the Znfn1a1 gene that encode the functional domains of Ikaros were analyzed for point mutations and deletions in murine lymphomas induced by 1,3-butadiene, 2',3'-dideoxycytidine, or phenolphthalein. Missense and frameshift mutations were identified in 11% (11 of 104) of the tumors. Interestingly, 8 of the mutations were identified in the NH₂-terminal zinc finger motifs, which are crucial for the DNA-binding function of Ikaros. The other 3 samples carried frameshift mutations in exon 7 that resulted in truncations and abrogation of both transactivation and dimerization domains. One tumor with a missense mutation in the DNA-binding domain also displayed a 45-bp deletion in the dimerization domain. Southern analysis disclosed interstitial homozygous deletions in the functional domains of Ikaros in 4% (3 of 68) of the lymphomas examined. Allelic losses on markers surrounding the Znfn1a1 gene were detected in 27% (12 of 45) of the tumors analyzed. However, only 2 tumors with allelic losses also showed mutations in the Znfn1a1 gene, indicating that other tumor suppressor genes located on this region might be involved as well. Our results suggest inactivation of Ikaros in a subset of chemically induced lymphomas and additionally support the contention of tumor-suppressor activity for Ikaros.