The role of RET genomic variants in infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis (IHPS) is a common childhood pathology affecting approximately 1-5 children pro 1000 newborns, with a genetic background as suggested by the familial occurrence. RET is a candidate gene for IHPS due to its role in the development of the intrinsic innervation and ganglia of the smooth musculature and the association of RET variants with another motility disorder (Hirschsprung's disease). Accordingly, we investigated RET-IHPS associations through sequencing of the complete RET coding region in 32 IHPS patients. Genotype frequencies were compared between patients and 48 controls using the Cochran-Armitage trend test or Fischer's test for exact p-values. We found 19 RET variants in IHPS, including polymorphisms in the promoter region (c.-200 G>A and c.-196 C>A). There was no statistically significant difference between the frequencies of the variants in both groups. There was no deviation from the Hardy-Weinberg equilibrium, yet a significant correlation (linkage disequilibrium) for variants in the promoter region, in exons 11, 13, 14 and 19 and in the 3' UTR. We conclude that RET variants are present in IHPS patients yet show no significant statistical association with the IHPS phenotype, suggesting at best an adjuvant role for RET in IHPS.

Analysis of RET, ZEB2, EDN3 and GDNF genomic rearrangements in 80 patients with Hirschsprung disease (using multiplex ligation-dependent probe amplification).

Hirschsprung disease (HSCR) is transmitted in a complex pattern of inheritance and is mostly associated with variants in the RET proto-oncogene. However, RET mutations are only identified in 15-20% of sporadic HSCR cases and solely in 50% of the familial cases. Since genomic rearrangements in particularly sensitive areas of the RET proto-oncogene and/or associated genes may account for the HSCR phenotype in patients without other detectable RET variants, the aim of the present study was to identify rearrangements in the coding sequence of RET as well as in three HSCR-associated genes (ZEB2, EDN3 and GDNF) in HSCR patients by using Multiplex Ligation-dependent Probe Amplification (MLPA). We have screened 80 HSCR patients for genomic rearrangements in RET, ZEB2, EDN3 and GDNF and did not identify any deletion or amplification in these four genes in all patients. We conclude that genomic rearrangements in RET are rare and were not responsible for the HSCR phenotype in individuals without identifiable germline RET variants in our group of patients, yet this possibility cannot be excluded altogether because the confidence to identify variation in at least two percent of the individuals was only 95%.

Cathepsin C gene variants in aggressive periodontitis.

Cathepsin C (CTSC) mutations are known to cause Papillon-Lefèvre syndrome. The aim of this study was to examine the association of CTSC genotype with susceptibility to non-syndromic aggressive periodontitis. The CTSC gene was analyzed in 110 persons with generalized aggressive periodontitis in comparison with 78 control individuals, after identifying different variants in a cohort of 100 persons. Five out of 19 discovered variants were included in this association study, representing 5 single-nucleotide polymorphism groups in tight linkage disequilibrium. The relevance of genotypes on enzyme function was examined. The carrier frequency of the missense variant p.I453V was significantly increased in persons with disease compared with healthy control individuals (17.3% vs. 6.4%, p < 0.05). CTSC activity in leukocytes from individuals harboring this variant was significantly reduced (119.8 Delta OD/min*10(5) cells, 95% confidence interval 17.4-174.9, p = 0.018). No influence of promoter variants was found on mRNA expression. The results support the hypothesis that CTSC gene variants contribute to increased susceptibility in generalized aggressive periodontitis.

[Inherited tumors of the gastrointestinal tract. Diagnosis and therapeutic aspects]. / Erbliche Tumoren im Gastrointestinaltrakt. Diagnostik und therapeutische Konsequenzen.

Familial tumors of the gastrointestinal tract, which often appear as autosomal-dominantly inherited tumor syndromes, account for only a small proportion of all gastrointestinal tumors. With the opportunities of modern molecular diagnostics, identifying the pathogenic mutation in families is often possible, with the option of predictive molecular testing and differentiation between mutation carriers and noncarriers. Thus a good chance exists for detection of early tumor stages by individually tailored surveillance programs and for improving prognosis by early intervention and prophylactic resection. Clinical manifestation, molecular basis at the root, individual surveillance programs, and their consequences for the treatment of familial gastric cancer, familial adenomatous polyposis coli, hereditary nonpolyposis colorectal cancer, Peutz-Jeghers syndrome, juvenile polyposis, hyperplastic polyposis, and familial pancreatic cancer are presented.

Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy.

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A(*)0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

CARD15 gene variants in aggressive periodontitis.

OBJECTIVE: The CARD15 gene encodes a protein that acts as an intracellular receptor of bacterial products, thus playing an important role in the innate immune response. Recently, CARD15 gene variants have been identified as a cause of increased susceptibility to Crohn's disease. The present study aimed to examine a potential association of CARD15 gene variants with aggressive periodontitis susceptibility. MATERIAL AND METHODS: The three main known CARD15 gene variants (p.R702W, p.G908R, and p.L1007fsX1008) were analysed by direct sequencing of exon 4, 8, and 11 of the gene in a total of 86 generalized aggressive periodontitis patients in comparison with 67 healthy controls. RESULTS: The mutant allele frequencies of the CARD15 variants were low in the generalized aggressive periodontitis group as well as in the control group and not significantly different (R702W: 3.5% versus 5.2%; G908R: 1.7% versus 1.5%; L1007fsX1008: 5.2% versus 4.5%). Two rare variants (A755V and R791Q), previously described only in patients with other inflammatory diseases, were observed in three patients having aggressive periodontitis but not in controls. CONCLUSIONS: Unlike in Crohn's disease, our results did not show an association between the three main CARD15 mutations and aggressive periodontitis. The role of rare variants remains unclear.

The p53 codon 72 variation is associated with the age of onset of hereditary non-polyposis colorectal cancer (HNPCC).

The polymorphic variants at codon 72 of the p53 gene were shown to be functionally distinct in vitro, whereby the arginine (arg) variant induces apoptosis more efficiently than the proline (pro) variant. From the evidence that the DNA mismatch repair system and p53 interact to maintain genomic integrity, we hypothesized that the codon 72 variation may influence the age of onset of disease in HNPCC patients. We tested 538 patients for p53 codon 72 variants, including 167 unrelated patients with pathogenic germline mutations in MSH2 or MLH1 and colorectal carcinoma as first tumour, 126 patients with sporadic microsatellite stable colorectal cancers, and 245 healthy controls. The median age of onset was 41, 36, and 32 years for MSH2 or MLH1 mutation carriers with arg/arg, arg/pro, and pro/pro genotypes, respectively. The log rank test revealed significant differences in the age of onset between arg/arg and pro/pro individuals (p = 0.0002) and in arg/pro versus arg/arg and pro/pro individuals (p = 0.0026 and p = 0.0217, respectively). A Cox regression model indicated an additive mode of inheritance. No significant differences in age of onset were observed among different genotype carriers with microsatellite stable tumours. Our results suggest that p53 codon 72 genotypes are associated with the age of onset of colorectal carcinoma in a mismatch repair deficient background in a dose dependent manner. These findings may be relevant for preventive strategies in HNPCC.

Inhibition of malignant glioma cell growth by a survivin mutant retrovirus.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor that is resistant to conventional radiotherapy and chemotherapy. The median survival time of patients with GBM has remained less than 2 years despite concerted efforts to improve therapy. As a new approach to treat GBM we generated retroviral particles encoding mutant survivin for transduction of glioma cells. We demonstrate here that retroviral overexpression of a nonphosphorylatable Thr-34 --> Ala mutant of survivin (survivinT34A), in the glioma cell lines U373 and H4 resulted in a marked increase in the percentage of cells bearing multiple nuclei, which was accompanied by significantly decreased cell proliferation, and in greater numbers of cells with hypodiploid DNA content. Administration of the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl-ketone did not reduce the cell death rate. Yet increased nuclear translocation of apoptosis-inducing factor (AIF) was observed in cells transduced with survivinT34A, indicating caspase-independent cell death. Transduction of retroviral vectors encoding wild-type survivin also led to the appearance of multinuclear cells. In contrast to mutant survivin, overexpressed wild-type survivin did not increase the cell death rate and no enhanced nuclear AIF translocation was observed. We suggest that retroviral vectors delivering mutant survivinT34A might be employed for the treatment of glioblastoma.

Low-level microsatellite instability phenotype in sporadic glioblastoma multiforme.

PURPOSE: Genetic instability is a hallmark of glioblastoma multiforme (GBM). Microsatellite instability (MSI) is a significant event in the tumorigenesis of many sporadic malignancies. The aim of our investigation was to study microsatellite instability in newly diagnosed glioblastomas. METHODS: MSI was investigated in 109 GBMs with 15 microsatellite markers. Immunohistochemistry was performed for the mismatch repair (MMR) proteins hMLH1, hMSH2, hPMS2, and hMSH6 in cases showing MSI. Sequence and promoter methylation status of hMLH1 were analyzed in the case of a decreased hMLH1 protein expression as well. To further investigate MSI(+) GBMs we carried out studies of LOH at selected chromosome regions, EGFR amplification, and sequence of p53 and PTEN. RESULTS: MSI was observed in six GBMs (5.5%) and it was more frequent in GBMs with a previous lower grade astrocytoma (18.8% vs. 3.2%). MMR protein staining was positive in all MSI(+) GBMs except in one case, which showed an aberrant expression of hMLH1 and hPMS2 without hMLH1 inactivation. Among MSI(+) GBMs, one tumor corresponded to the GBM molecular type 1 (p53 mutation, no EGFR amplification), another tumor to type 2 (wild-type p53, EGFR amplification), and four tumors to neither type (wild-type p53, no EGFR amplification). None of the six tumors carried a PTEN mutation. CONCLUSIONS: MSI in GBM might be caused by inactivation of minor MMR genes rather than by a deficiency of hMLH1 or hMSH2 and it appears not to play a decisive role in the pathogenesis of these tumors. MSI(+) GBMs predominantly showed a profile which included wild-type of p53 and PTEN and absence of EGFR amplification but MSI occurred in all GBM molecular subtypes.

[Management of multiple endocrine neoplasia syndrome type 2 families in association with rare germline mutations of the RET proto-oncogene]. / Management von Familien mit einem multiplen endokrinen Neoplasie-Syndrom Typ 2 in Assoziation mit seltenen Keimbahnmutationen des RET-Protoonkogens.

INTRODUCTION: Heredity of MEN2 syndromes is caused by a heterozygous germline mutation in the RET proto-oncogene. This study describes families with rare noncysteine codon 790/791 mutations and discusses the genotype-phenotype correlation plus the therapeutic options. PATIENTS AND METHODS: Forty-five patients with a putative sporadic MTC were screened for RET germline mutations by direct DNA sequencing. Family members of identified index cases underwent genetic analysis. Gene carriers were examined clinically and biochemicaly and underwent prophylactic thyroidectomy. RESULTS: Five index patients were identified. In the kindreds three L790F and one Y791F carriers were detected. The thyroid gland histology of L790F carriers revealed MTC in 2 patients and C-cell hyperplasia in 2 additional patients. The Y791F carrier had a normal histology. CONCLUSIONS: Codon 790/791 mutations had diverse penetrance: prophylactic thyreoidectomy in children is a justifiable approach for codon 790 mutation carriers, but should depend on the clinical course of codon 791 carriers.

Novel mutations in the cathepsin C gene in patients with pre-pubertal aggressive periodontitis and Papillon-Lefèvre syndrome.

Aggressive periodontitis (AP) in pre-pubertal children is often associated with genetic disorders like Papillon-Lefèvre syndrome (PLS). PLS is caused by mutations in the cathepsin C (CTSC) gene. We report a novel CTSC mutation (c.566-572del) in an otherwise healthy AP child and two novel compound heterozygous mutations (c.947T>G, c.1268G>C) in a PLS patient. We conclude that at least a subset of pre-pubertal AP is due to CTSC mutations and therefore may be an allelic variant of PLS.

ADAM9 expression in pancreatic cancer is associated with tumour type and is a prognostic factor in ductal adenocarcinoma.

Gene expression profiling revealed ADAM9 to be distinctly overexpressed in pancreatic ductal adenocarcinoma (PDAC). We examined the relevance of ADAM9 expression in PDAC diagnosis and prognosis. A total of 59 infiltrating PDACs, 32 specimens from patients with chronic pancreatitis, 11 endocrine tumours and 24 acinar cell carcinomas were immunohistochemically analysed for ADAM9 expression. Staining for ADAM9 was detected in 58 out of 59 (98.3%) PDACs and in two out of 24 (8.3%) acinar cell carcinomas, but not in endocrine tumours. In the non-neoplastic pancreas, whether normal or chronically inflamed, ADAM9 was expressed in centroacinar and intralobular duct cells, but not in interlobular duct cells and their hyperplastic lesions. Pancreatic ductal adenocarcinomas showing cytoplasmic ADAM9 expression correlated with poor tumour differentiation and also with shorter overall survival than in cases showing only an apical membranous staining pattern (P=0.001). Multivariate analysis identified cytoplasmic ADAM9 expression as an independent marker of shortened survival in a set of 42 curatively (R0) resected PDAC (P<0.05, hazard ratio 2.85, 95% confidence interval: 1.21-6.71). The results show that ADAM9 expression distinguishes PDACs from other solid pancreatic tumours. In addition, cytoplasmic ADAM9 overexpression is associated with poor differentiation and shortened survival. Therefore, ADAM9 overexpression might contribute to the aggressiveness of PDACs.

Loss of BRCA2 correlates with reduced long-term survival of sporadic breast cancer patients.

BACKGROUND: The present study was undertaken to analyze the prognostic value of loss of heterozygosity (LOH) at 13q12-13, 17q21 and 17p13, harboring BRCA2, BRCA1 and p53 to predict the clinical course of sporadic breast cancer patients. MATERIALS AND METHODS: LOH analysis was performed by PCR amplification of genomic DNA using nine microsatellite markers. Fifty-three sporadic breast cancer patients were followed clinically for a median of 55 months. Disease-free and overall survival was documented as the endpoint for statistical evaluation. RESULTS: Patients presenting with LOH in their tumor samples at at least one of the loci examined were found to have a reduced overall survival time compared to those retaining heterozygosity (61% versus 48%). Focusing on the three target regions, patients with LOH at the BRCA2 locus died earlier compared to patients retaining heterozygosity (69% versus 50%) and, in addition, BRCA2 LOH-positive patients showed a shorter metastasis-free interval (30 versus 37 months). In a multivariate analysis, LOH at the 13q12-13 locus was found to be a significant predictor for reduced long-term survival (risk ratio 2.33, 95% C.I., 1.0-5.3; p<0.05) and earlier metastases manifestation (risk ratio 2.32, 95% C.I., 1.0-5.3, p<0.05). CONCLUSION: Allelic loss at the BRCA2 locus may be of use as a negative predictor for metastases-free and overall survival.

Within-gene interaction between c.135 G/A genotypes and RET proto-oncogene germline mutations in HSCR families.

Hirschsprung disease (HSCR) is considered a model for a complex inheritance disorder. Several genes, including the major HSCR-susceptibility RET proto-oncogene, play an aetiological role in the development of HSCR. Genetic linkage analysis in familial HSCR with both long- and short-segment phenotypes has demonstrated a tight linkage to the RET locus, while the phenotype within a HSCR family is characterised by an incomplete penetrance or a variable extension of the aganglionosis. Therefore, additional genetic alterations of RET are postulated in the aetiology or modification of the HSCR phenotype. In this study, the coding region of all 21 exons of the RET proto-oncogene, including the flanking intronic sequences, were investigated by direct DNA sequencing in a HSCR population. We genotyped the c.135 G/A polymorphism and resolved haplotypes comprising the mutation locus and the c.135 G/A polymorphism. Twenty different mutations were detected in 18 of 76 HSCR patients. In ten families the mutations were inherited from the parents, while only four patients had a positive family history for the disease. Moreover, in all ten families an incomplete penetrance of the HSCR phenotype was observed. We have investigated the effect of the non-mutated wild-type allele as well as the c.135 G/A polymorphism on the phenotype within the HSCR families. Our findings support the notion that both RET alleles are involved in the pathogenesis of a subgroup of HSCR patients in a dose-dependent fashion. Additionally, we have shown a modifying effect of the c.135 G/A polymorphism on the HSCR phenotype within HSCR families.

Genomic rearrangements of hMSH6 contribute to the genetic predisposition in suspected hereditary non-polyposis colorectal cancer syndrome.

BACKGROUND: Germline mutations in mismatch repair genes, mainly in hMLH1, hMSH2, and hMSH6, predispose to the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. A substantial fraction of these mutations exists in genomic rearrangements of hMSH2 and hMLH1. In contrast, genomic rearrangements have not been reported in hMSH6. METHODS: Out of 15 HNPCC or HNPCC-like patients who developed tumours with loss of hMSH6 protein expression, we selected three patients who still had no germline mutations after gene sequencing. Genomic DNA of these patients was analysed using PCR based relative quantification of hMSH6 fragments. Indicated exon deletions and amplifications were characterised by long range PCR and sequencing. RESULTS: Genomic rearrangements were identified in two of the three patients. Breakpoint analyses showed an Alu repeat mediated deletion of 13.0 kb affecting the promoter region, exon 1, and exon 2 in one patient, and a duplication of 4.9 kb containing 1.6 kb of the 3' end of exon 4 and exon 5, integrated into intron 5, in the other patient. CONCLUSIONS: Although genomic rearrangements of hMSH6 only play a small role in the spectrum of all mutations predisposing to HNPCC, our results suggest that up to 10-20% of patients with hMSH6 negative tumours harbour germline rearrangements in this gene.

Prevention of brain metastasis formation by local expression of interleukin-4 or hemagglutinin antigen.

INTRODUCTION: Expression of hemagglutinin antigen of influenza virus (HA) by the murine colon carcinoma cell line (CT-26) produces systemic immunization against tumor challenges in the cecum, liver and lungs but not in the brain of BALB/c-mice. Immunization with IL-4 expressing CT-26 cells inhibits lung metastases formation. The purpose of our study was to examine the effects of HA or IL-4 expression on brain metastases formation. METHODS: Using selective internal carotid artery injections, brain metastases formation of HA or IL-4 expressing CT-26 cells with and without subcutaneous pre-immunization was evaluated in Balb/c mice. RESULTS: Systemic pre-immunization with HA or IL-4 expressing tumor cells cannot protect against brain metastases, while the local, intracerebral expression of HA or IL-4 inhibits the growth of hematogenous brain metastases. CONCLUSION: Pre-immunization with HA or IL-4 expressing tumor cells did produce systemic immunity against liver and lung metastases but not against brain metastases. Local, intracerebral expression of HA or IL-4 prevents from cerebral metastases formation in an animal model.

Infrequent genetic alterations of the tumor suppressor gene PTEN/MMAC1 in squamous cell carcinoma of the oral cavity.

BACKGROUND: Fifty tumor specimens from primarily untreated patients were analyzed to elucidate the involvement of the tumor suppressor gene PTEN/MMAC1 in the development of oral squamous cell cancer. METHODS: Eight microsatellite markers, spanning 10 cM of genomic DNA located centromeric, telomeric or intragenic of PTEN/MMAC1 were used for loss of heterozygosity (LOH) and breakpoint analysis. The microsatellite panel within or in close proximity (1 cM) to the 10q23.3 locus showed a LOH rate of 12%. Complete sequence analysis of the genes coding region was performed in all 10 cases that exhibited LOH in one of the eight microsatellite markers within or around the PTEN/MMAC1 gene. Comparative multiplex PCR reactions served to screen for homozygous deletions. RESULTS: There was no association between allelic loss of the gene, overall patient survival and recurrence-free survival. Sequencing did not reveal any mutation in the coding region of PTEN/MMAC1. Differential PCR analysis failed to detect any homozygous deletion. CONCLUSIONS: We conclude that PTEN/MMAC1 gene alterations do not play a key role in tumorigenesis of oral squamous cell cancers.

Lack of association between endoglin intron 7 insertion polymorphism and intracranial aneurysms in a white population: evidence of racial/ethnic differences.

BACKGROUND AND PURPOSE: Endoglin is a component of the transforming growth factor-beta receptor complex and is predominantly expressed on cell surfaces of endothelial cells. A polymorphism of the endoglin gene has previously been found to be associated with the occurrence of intracranial aneurysms in a Japanese population. In our study, we investigated whether this polymorphism is associated with the development of cerebral aneurysm in a white population. METHODS: The study population consisted of 121 white patients who had been treated for intracranial aneurysms, 124 healthy white blood donors, and 15 Japanese volunteers. Exon 7 of the endoglin gene and adjacent intronic sequences were amplified by polymerase chain reaction and analyzed by using an automated laser fluorescence detection system. RESULTS: A well-known insertion polymorphism (5'-TCCCCC-3', starting 23 bp distal from the 3' end of exon 7) was identified. The allele frequencies of the polymorphism were 35 (14.5%) of 242 alleles in the aneurysm group and 35 (14.1%) of 248 alleles in the white control group, which does not represent a statistically significant difference (P>0.85). The sequence of the polymorphism is complementary to that reported in the previously mentioned Japanese study. However, the 2 polymorphisms are identical. Under this assumption, the allele frequencies differ significantly among the Japanese controls in that particular study and the white controls in our study (27.8% versus 14.1%, respectively; P=0.0003). CONCLUSIONS: The genetic polymorphism in the vicinity of 3' end of exon 7 in the endoglin gene was not significantly associated with the occurrence of intracranial aneurysms in the white population. There are ethnic-related differences of allele frequencies between our white controls and the previously reported Japanese controls.