MSH2 and CXCR4 involvement in malignant VIPoma.

BACKGROUND: Vasoactive intestinal polypeptide secreting tumors(VIPomas) are rare endocrine tumors of the pancreas with an estimated incidence of 0.1 per million per year. The molecular mechanisms that mediate development of VIPomas are poorly investigated and require definition. METHODS: A genome- and gene expression analysis of specimens of a primary pancreatic VIPoma with hepatic metastases was performed. The primary tumor, the metastases, the corresponding healthy tissue of the liver, and the pancreas were compared with each other using oligonucleotide microarrays and loss of heterozygosity (LOH). RESULTS: The results revealed multiple LOH events and several differentially expressed genes. Our finding of LOH and downregulation was conspicuous in the microarray analysis for the mismatch repair gene MSH2 in the primary pancreatic VIPoma tumor, the hepatic metastasis but not in the corresponding healthy tissue. Further a strong overexpression of the chemokine CXCR4 was detected in the hepatic metastases compared to its pancreatic primary. With a review of the literature we describe the molecular insights of metastatic development in VIPoma. CONCLUSION: In VIPoma, defects in the mismatch repair system especially in MSH2 may contribute to carcinogenesis, and increased CXCR4 may be associated with liver metastasis.

Human precision-cut liver tumor slices as a tumor patient-individual predictive test system for oncolytic measles vaccine viruses.

Availability of an individualized preselection of oncolytic viruses to be used for virotherapy of tumor patients would be of great help. Using primary liver tumor resection specimens we evaluated the precision-cut liver slice (PCLS) technology as a novel in vitro test system for characterization of paramount tumor infection parameters of individual patients. PCLS slices from resection specimens of 20 liver tumor patients were cultivated in vitro for up to 5 days and infected with 5 different oncolytic measles vaccine virus (MeV) strains. Effectiveness of tumor infection was monitored by viral nucleocapsid (N) protein detection in immunofluorescence staining or Western blot analysis or by detection of GFP marker gene expression. MeV spreading in PCLS cultures was visualized by confocal microscopy. Oncolytic MeV vaccine particles were demonstrated to efficiently infect PCLS slices originating from different primary and secondary tumors of the liver with MeV strains Moraten/Edmonston Zagreb and AIK-C showing highest infection rates (75% of all tested tumor specimens). Employing mixed liver tissue slices (exhibiting both tumorous and non-tumorous tissue areas on one and the same sample) a distinct tumor area favouring pattern of MeV infections was observed being in accordance with our finding that primary human hepatocytes are also permissive to MeV particles, albeit at a much lower rate and with a much less pronounced cytopathic effect. Furthermore, confocal microscopy demonstrated virus penetration throughout tumor tissues into deep cell layers. In conclusion, the PCLS technology is suitable to perform a tumor-patient individualized preselection of oncolytic agents prior to clinical virotherapeutic applications.

PGK1 a potential marker for peritoneal dissemination in gastric cancer.

BACKGROUND/AIMS: Peritoneal carcinomatosis, which is caused by the dissemination of cancer cells into the abdominal cavity is a frequent finding in patients with primary gastric cancer, and it is associated with a poor prognosis. The mechanisms that mediate peritoneal carcinomatosis in diffuse primary gastric tumours require definition. METHODS: We therefore compared the gene expression profile in diffuse primary gastric cancer patients with and without peritoneal carcinomatosis (n=13). Human specimens from consecutive gastric cancer patients with and without peritoneal carcinomatosis were investigated using oligonucleotide microarrays. Differentially expressed genes of interest were further evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The results reveal a significant overexpression of phosphoglycerate kinase 1 (PGK1), the chemokine CXCR4 and its ligand CXCL12 in specimens from diffuse gastric cancer patients with peritoneal carcinomatosis. Overexpression of PGK1 is known to increase the expression of CXCR4. CXCR4 on its part can increase CXCL12 expression. Elevated levels of CXCR4 and CXCL12 are associated with an increase in the metastatic rate and play an important role in the metastatic homing of malignant cells. CONCLUSION: The overexpression of PGK1 and its signalling targets may be a expression-pathway in diffuse primary gastric carcinomas promoting peritoneal dissemination and may function as prognostic markers and/or be potential therapeutic targets to prevent the migration of gastric carcinoma cells into the peritoneum.

Large proportion of low frequency microsatellite-instability and loss of heterozygosity in pheochromocytoma and endocrine tumors detected with an extended marker panel.

PURPOSE: Pheochromocytoma (PCC) is a usually benign tumor originated in the majority of patients from the adrenal medulla. Regarding sporadic forms of PCC, mechanisms of pathogenesis are largely unknown. Recently, microsatellite-instability (MSI) was discussed as genetic factor contributing to PCC development. Since microsatellite markers used for MSI detection have only been recommended for colorectal carcinoma (CRC), we established an extended marker set for MSI detection in PCC. METHODS: Twenty-two PCC patients were analyzed applying 11 microsatellite markers. Our marker set comprised the reference panel for CRC and six additional markers, which have already been described to detect MSI in tumors other than CRC. Moreover, 23 endocrine tumors with gastrointestinal origin were examined in order to test the applicability of this marker panel. RESULTS: Microsatellite-instability was detected in 41% of PCCs. Twenty-seven percent showed loss of heterozygosity (LOH) events affecting different chromosomal regions. Among the 23 patients with endocrine tumors, only three (one pancreatic endocrine tumor, one duodenal neuro-endocrine tumor, one hepatic metastasis of a primary tumor with unknown origin) demonstrated MSI. CONCLUSIONS: The extended microsatellite panel is qualified to detect MSI in PCC. Nine percent of MSI-positive cases would have not been noticed by the use of the reference panel alone. PCCs are characterized by low frequency MSI pointing to failures in factors involved in DNA replication.

Cancer immunoediting by GITR (glucocorticoid-induced TNF-related protein) ligand in humans: NK cell/tumor cell interactions.

Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-beta by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-gamma production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRL-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-kappaB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans.

Coincidence of mutations in different connexin genes in Hungarian patients.

Mutations in the GJB2 gene are the most common cause of hereditary prelingual sensorineural hearing impairment in Europe. Several studies indicate that different members of the connexin protein family interact to form gap junctions in the inner ear. Mutations in different connexin genes may accumulate and, consequently lead to hearing impairment. Therefore, we screened 47 Hungarian GJB2- heterozygous (one mutation in coding exon of the GJB2 gene) patients with hearing impairment for DNA changes in two further connexin genes (GJB6 and GJB3) and in the 5' non-coding region of GJB2 including the splice sites. Eleven out of 47 GJB2-heterozygous patients analyzed carried the splice site mutation -3170G>A in the 5'UTR region of GJB2. One out of these 11 patients showed homozygous -3170G>A genotype in combination with p.R127H. Next to the GJB2 mutations we noted 2 cases of deletion in GJB6 [Delta(GJB6-D13S1830)] and 3 (2 new and 1 described) base substitutions in GJB3 [c.357C>T, c.798C>T and c.94C>T (p.R32W)] which are unlikely disease-causing. Our results suggest the importance of routine screening for the rather frequent -3170G>A mutation (in addition to c.35delG) in patients with hearing impairment.

Influence of gain of function epithelial chloride channel ClC-Kb mutation on hearing thresholds.

Hearing depends on functional ClC-K-type chloride channels composed of barttin with ClC-Ka or ClC-Kb. Loss-of-function mutations of the barttin gene BSND or of both, the ClC-Ka gene CLNKA and the ClC-Kb gene CLNKB lead to congenital deafness and renal salt wasting. Recently, we identified the gain-of-function mutation ClC-Kb(T481S) which is associated with increased blood pressure. To explore the impact of ClC-Kb(T481S) on hearing, healthy volunteers (n=329) and individuals suffering from tinnitus (n=246) volunteered for hearing tests (n=348) and genetic analysis (n=575). 19.1% of the individuals were heterozygote (ClC-Kb(T481S)/ClC-Kb) and 1.7% homozygote carriers. Pure tone average hearing threshold (PTAt) for air conduction was significantly (p<0.033) lower in ClC-Kb(T481S) carriers (13.2+/-1.2dB) than in wild-type individuals (17.1+/-0.9dB). The prevalence of ClC-Kb(T481S) carriers was significantly increased (29.7%) in individuals with PTAt<15dB (p<0.05) and significantly decreased (13.2%) in individuals with PTAt>30 dB (p<0.017). The difference was largely due to the female population. Bone conduction was less affected pointing to an effect of the mutation on middle ear function. Tinnitus tended to be more frequent in ClC-Kb(T481S) carriers, a difference, however, not statistically significant. In conclusion, hearing thresholds are slightly lower in carriers of ClC-Kb(T481S), i.e., the gain-of-function polymorphism ClC-Kb(T481S) exerts a subtle but significant protective effect against hearing loss.

High frequency of ENG and ALK1/ACVRL1 mutations in German HHT patients.

Morbus Osler or HHT (hereditary hemorrhagic telangiectasia) is a disorder of the fibrovascular tissue that is inherited in an autosomal dominant way with frequency rates between 1:2,500 and 1:40,000. The disease provokes malformations of the blood vessels sometimes resulting in life-threatening complications. Presently, two genes involved in the development of HHT have been identified: ACVRL1 and ENG. Both of them encode proteins that belong to the TGF-beta receptor complex family and play an essential role in the formation of the vascular system. Recently, several mutations in ACVRL1 and ENG have been described in other European populations. However, no data concerning mutation frequencies in the German population have been reported so far. Therefore, we screened our collective of German HHT patients (28 single cases and 11 familial cases) for mutations in both genes by direct sequencing. We detected 11 mutations already described elsewhere and 19 novel ones. Furthermore, evidence for the pathogenic role of four new missense mutations was collected by screening a healthy control collective using RFLP analysis. Interestingly, the majority of ACVRL1 mutations represented missense mutations, whereas mutations in ENG mostly resulted in a shortened protein. Our results demonstrate the importance of ACVRL1 and ENG mutations in German HHT patients displaying mutation frequencies over 80%.

Active succinate dehydrogenase (SDH) and lack of SDHD mutations in sporadic paragangliomas.

BACKGROUND: Paragangliomas are benign, slow-growing tumours of the head and neck region. The candidate gene for familial and some sporadic paragangliomas, SDHD (succinate dehydrogenase, subunit D), has been mapped to the PGL1 locus in 11q23.3. MATERIALS AND METHODS: Normal and tumour DNA of 17 patients with sporadic paragangliomas were analysed by sequencing (SDHD, SDHB and SDHC genes), fluorescence in situ hybridisation (FISH). In addition, loss of heterozygosity (LOH) and succinate dehydrogenase (SDH) enzyme activity assays were performed. RESULTS AND CONCLUSION: Only two patients from our collective showed SDH gene mutations, one in SDHD and one in SDHB, respectively. Moreover, SDH activity detected in 5/8 patients confirmed the fact that SDH inactivation is not a major event in sporadic paragangliomas. LOH and FISH analysis demonstrated a frequent loss of regions within chromosome 11, indicating that additional genes in 11q may play a role in tumour genesis of sporadic paragangliomas.

GJB2 mutations in patients with non-syndromic hearing loss from Northeastern Hungary.

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, c.35delG, accounts for the majority of mutations in Caucasian patients with HI. In the present study we screened 500 healthy control individuals and a group of patients with HI from Northeastern Hungary for GJB2 mutations. The patients' group consisted of 102 familial from 28 families and 92 non-familial cases. The most common mutation in the Hungarian population is the c.35delG, followed by the c.71G>A (p.W24X) mutation. 34.3% of the patients in the familial group were homozygous, and 17.6% heterozygous for 35delG. In the non-familial group the respective values were 37% and 18% (allele frequency: 46.2%). In the general population an allele frequency of 2.4% was determined. Several patients were identified with additional, already described or new GJB2 mutations, mostly in heterozygous state. The mutation c.380G>A (p.R127H) was formerly found only in heterozygous state and its disease relation was controversial. We demonstrated the presence of this mutation in a family with three homozygous patients and 4 heterozygous unaffected family members, a clear indication of recessively inherited HI. Furthermore, we provided evidence for the pathogenic role of two new mutations, c.51C>A (p.S17Y) and c.177G>T (p.G59V), detected in the present study. In the latter case the pattern of inheritance might be dominant. Our results confirm the importance of GJB2 mutations in the Hungarian population displaying mutation frequencies that are comparable with those in the Mediterranean area.

Mutation A1555G in the 12S rRNA gene and its epidemiological importance in German, Hungarian, and Polish patients.

The A1555G mutation in the 12SrRNA gene has been associated with aminoglycoside induced and nonsyndromic sensorineural hearing impairment. In this study we analyzed Hungarian, Polish and German patients with nonsyndromic severe to profound hearing impairment of unknown origin for this mutation. The frequency of the A1555G mutation in the Hungarian hearing impaired population was below 1.8 %. Three out of 125 Polish patients carrying the A1555G mutation were identified. Among German patients one carrier was found (0.7 %) revealing a homoplastic A1555G mutation, whereas no mutation was detected in control individuals with normal hearing (frequency < 0.6%). In summary the frequencies of the A1555G mutation are low in the hearing impaired as well as in the normal population in Hungary, Poland and Germany. Since the importance of this mutation and its relationship with aminoglycoside exposure is not well understood yet, patients with nonsyndromic hearing impairment should be routinely screened for this mutation to avoid aminoglycoside induced hearing impairment due to increased sensitivity of maternal relatives.

Frequencies of GJB2 mutations in German control individuals and patients showing sporadic non-syndromic hearing impairment.

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, 35delG (30delG), accounts for up to 70% of all analyzed European patients with autosomal recessive inherited HI and 10% of patients with HI of unknown origin, respectively. We screened 188 control individuals and 342 German patients with non-syndromic sporadic HI for the 35delG, compound heterozygosity and other GJB2 mutations by PCR, restriction enzyme based screening, SSCP and sequencing. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. This study revealed one novel silent mutation (438C/T), three novel gene variants resulting in amino acid substitutions (K112E, T123S, K223R) and two novel HI-related mutations (I82M, 313del14).

Chromosome 11 monosomy in conjunction with a mutated SDHD initiation codon in nonfamilial paraganglioma cases.

Paragangliomas of the head and neck region are a group of rare, usually benign, slow-growing tumors developing from paraganglionic chemoreceptors in most patients. Mutations in a subunit of the mitochondrial enzyme II complex (succinate dehydrogenase [SDHD]) were shown to be responsible for the formation of paragangliomas. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), was observed recently. We analyzed DNA derived from tumor sections of three unrelated paraganglioma patients (one case with multiple paragangliomas, two cases with single tumors; all of them sporadic cases) for mutations in the SDHD gene by direct sequencing. Microsatellite-based LOH was performed, and events of chromosomal loss were validated by fluorescence in situ hybridization (FISH) on paraffin-embedded tumor and normal tissue by using centromeric satellite DNA. Sequence analysis revealed mutations in SDHD exon 1 in all patients, affecting the initiation codon (M1V). Another alteration was detected in exon 2 but was lacking in tumor DNA and therefore classified as polymorphism (H50R). LOH and FISH analyses demonstrated partial/total monosomy for chromosome 11 in the tumor samples tested. A common genetic mechanism appears to be the pathophysiologic basis for sporadic tumor development because the proposed two-hit model comprising both LOH and point mutation is manifest in our patients. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors in one of the patients.

Analysis of candidate genes for genotypic diagnosis in the long QT syndrome.

Patients with the long QT syndrome (LQTS) suffer from cardiac arrhythmias that can lead to abrupt loss of consciousness and sudden death, already in young individuals. Thus, an early diagnosis of LQTS is essential for patients and their family members. So far, six genes (KCNQ1, HERG, SCN5A, ANK2, KCNE1, KCNE2) have been demonstrated to be involved in the development of LQTS. Since this syndrome is genetically heterogeneous and large-sized families are often not available for linkage analysis, alternative tools are required for a genetic diagnosis. To investigate genes with numerous exons, like KCNQ1, HERG, SCN5A and ANK2, segregation analysis of a Polish Romano-Ward family with eight members was performed as a reliable method faster than linkage analysis or direct sequencing. To test these four LQT loci, an appropriate selection of microsatellite markers covering different chromosomal regions was applied. Furthermore, two small genes KCNE1 and KCNE2 (at the LQT5 and LQT6 loci), and the SGK1 gene (encoding a kinase regulating KCNE1 and SCN5A channels) were sequenced. All six LQT loci and the SGK1 gene were excluded by these analyses, thus a different pathogenic mechanism of LQT syndromes can be presumed.

[Frequency of the Connexin26/35delG mutation and its characteristic phenotype in patients with hearing impairment and controls in Northeastern Hungary]. / Connexin 26/35delG mutáció gyakorisága és jellemzó fenotípusa Magyarország északkeleti régiójában vizsgált hallássérült és kontroll esetekben.

INTRODUCTION: Hereditary hearing impairment is a heterogeneous disorder showing different pattern of inheritance and involving a multitude of different genes. Mutations in the GJB2 gene, especially the 35delG mutation, have been established as a major cause of inherited and sporadic non-syndromic deafness in different populations. Mutations in GJB2 gene, encoding gap junction protein (Connexin 26), may be responsible for up to 50% of cases of autosomal recessive non-syndromic hearing impairment and in 15-30% of sporadic cases. STUDY DESIGN: The authors analyzed 15 north east Hungarian families and 30 sporadic cases with nonsyndromic hearing impairment for the 35delG mutation. METHODS: DNA were tested for the common 35delG mutation by a polymerase chain reaction based restriction enzyme assay (BsiYl). RESULTS: Fifty two patients showing a homozygous 35delG mutation were audiological examined. Ordinarily these patients showed a prelingual, sensorineural, bilateral, symmetric hearing loss without progression. The audiograms were characterized by sloping or flat patterns. The carrier frequency of the 35delG mutation among control group was 5.1%. CONCLUSION: The phenotypic manifestation varied in 30% of all analyzed patients, making genetic counseling extremely difficult. Due to this knowledge mutation analysis of GJB2 cannot distinctly predict the degree of hearing impairment.

A 4-bp insertion in the eya-homologous region (eyaHR) of EYA4 causes hearing impairment in a Hungarian family linked to DFNA10.

BACKGROUND: Hereditary hearing impairment (HHI) is a heterogeneous class of disorders that shows various patterns of inheritance and involves a multitude of different genes. Mutations in the EYA4 gene are responsible for postlingual, progressive, autosomal dominant hearing loss at the DFNA10 locus. EYA4 is orthologous to the Drosophila gene eya ("eyes absent"), a key regulator of eye formation. EYA4 plays an important role in several developmental processes. MATERIAL AND METHODS: Here we report a Hungarian family displaying sensorineural, progressive hearing impairment. The family comprising four generations with 11 affected and 8 unaffected members was subjected to genome-wide linkage analysis and candidate gene sequencing. RESULTS: By linkage analysis, the chromosomal region 6q22.3 was shown to segregate with the disease. Mutation analysis of the EYA4 gene, which maps to 6q22.3, revealed an insertion of 4 bp (1558insTTTG) in all affected family members. This insertion creates a frameshift and results in a stop codon at position 379. Hence, nearly the complete "eya homologous region" (eyaHR), which is essential for the protein function, would be deleted in the mutant EYA4 protein if the transcription were found to be stable. CONCLUSIONS: This family is the third one linked to DFNA10 and revealing a mutation in the EYA4 gene. In all three families, the mutations are localized in different regions of the eyaHR, suggesting that this protein contains several functional subregions with different tissue-specific importance.

A genotype-phenotype correlation with gender-effect for hearing impairment caused by TECTA mutations.

BACKGROUND: Alpha-tectorin is a noncollagenous component of the tectorial membrane which plays an essential role in auditory transduction. In several DFNA12 families mutations in TECTA, the gene encoding alpha-tectorin, were shown to cause hearing impairment (HI) with different phenotypes depending on the location of the mutation. METHODS/RESULTS: Here we report a Turkish family displaying autosomal dominant inherited HI. Linkage analysis revealed significant cosegregation (LOD score: 4.6) of the disease to markers on chromosome 11q23.3- q24. This region contains the TECTA gene which was subsequently sequenced. A nucleotide change in exon 13, 4526T>G, was detected leading to a substitution from cysteine to glycine at codon 1509 of the TECTA protein. This cysteine is located in vWFD4 domain, a protein domain which is supposed to be involved in disulfide bonds and protein-protein interactions. CONCLUSIONS: It is conspicuous that the phenotype in this family correlates with other families, also displaying mutations involving conserved cysteines. In all three families these mutations result in progressive HI involving high frequencies. In contrast, mutations which are not affecting the vWFD domains seem to provoke mid-frequency sensorineural HI. Furthermore, evaluation of clinical data in our family revealed a gender effect for the severity of hearing impairment. Males were significantly more affected than females. The identification of the third family displaying a missense mutation in the vWFD domain of alpha-tectorin underlines the phenotype-genotype correlation based on different mutations in TECTA.

Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations.

Hereditary hearing impairment is an extremely heterogeneous trait, with more than 70 identified loci. Only two of these loci are associated with an auditory phenotype that predominantly affects the low frequencies (DFNA1 and DFNA6/14). In this study, we have completed mutation screening of the WFS1 gene in eight autosomal dominant families and twelve sporadic cases in which affected persons have low-frequency sensorineural hearing impairment (LFSNHI). Mutations in this gene are known to be responsible for Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), which is an autosomal recessive trait. We have identified seven missense mutations and a single amino acid deletion affecting conserved amino acids in six families and one sporadic case, indicating that mutations in WFS1 are a major cause of inherited but not sporadic low-frequency hearing impairment. Among the ten WFS1 mutations reported in LFSNHI, none is expected to lead to premature protein truncation, and nine cluster in the C-terminal protein domain. In contrast, 64% of the Wolfram syndrome mutations are inactivating. Our results indicate that only non-inactivating mutations in WFS1 are responsible for non-syndromic low-frequency hearing impairment.