Gene expression analysis in chronic postradiation proctopathy.

PURPOSE: Radiotherapy is one of the important treatment modalities for tumors of pelvic organs. The fixed location of the rectum and its anatomic relationship with other pelvic organs makes it prone to radiation injury resulting in chronic radiation proctopathy in 5% to 20% of patients. Endothelial dysfunction has been associated with a number of pathophysiological processes. Endothelial cells synthesize and release various factors that regulate angiogenesis, inflammatory responses, hemostasis, as well as vascular tone and permeability. METHODS: Rectum tissue samples from 20 patients with established chronic radiation proctopathy were analysed for the expression of genes related to oxidative stress, tissue hypoxia, angiogenesis, and inflammation [endoglin (ENG), activin receptor-like kinase 1 (ALK1), platelet endothelial cell adhesion molecule 1 (PECAM), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), hypoxia-inducible factor 1 (HIF-1), and interleukin-1 beta (IL-1ß)]. RESULTS: Overexpression of HIF-1, VEGF, FGF2, and IL-1ß was detected in affected tissue. For the first time, a significant suppression of activin receptor-like kinase 1 and ENG could be revealed. CONCLUSION: The data provided here allow further insight into the pathogenesis of radiation-induced rectum injury. Radiation-induced damage is not confined to a single event but involves complex signaling between different pathways, enhancing and maintaining the processes that lead to mucosal damage. The results indicate that postradiation tissue hypoxia is critical for fibrosis, which involves changes in the expression of profibrotic and angiogenic factors in rectal tissue.

Increased translocator protein (TSPO) mRNA levels in colon but not in rectum carcinoma.

BACKGROUND: The peripheral-type benzodiazepine receptor or translocator protein (TSPO) is an 18-kDa protein involved in cell proliferation and apoptosis. TSPO was shown to be overexpressed in malignant tumors and cancer cell lines, correlating with enhanced malignant behavior. The present study analyzed the role of TSPO in patients with colorectal carcinomas. METHODS: Tumor tissues and corresponding normal mucosa from 55 patients who underwent resection for colorectal carcinomas were analyzed for TSPO expression in correlation to GAPDH expression(glyceraldehyde-3-phosphate dehydrogenase) using a multiplex RT-PCR assay. RESULTS: TSPO was overexpressed in 67% of the tumors in comparison to corresponding normal mucosa, and positivity as well as expression levels in colon carcinomas were significantly higher than in the rectum carcinomas. In contrast, TSPO expression was not different in intermediate versus high-grade tumors or in lymph node-positive versus -negative patients. CONCLUSION: The differences in TSPO expression between colon and rectum carcinoma may imply that these tumors are of different biological behavior.

Deficient membrane integration of the novel p.N14D-GJB2 mutant associated with non-syndromic hearing impairment.

Mutations in GJB2, the gene encoding for the Gap Junction protein Connexin 26 (Cx26), have been established as the major cause of hereditary, non-syndromic hearing impairment (HI). We report here the identification of a novel point mutation in GJB2, c.40A>G [p.N14D], detected in compound heterozygosity with the c.35delG mutation in two brothers with moderate non-syndromic sensorineural HI. The mother who carried one wildtype and a p.N14D allele displayed normal hearing. The mutation leads to substitution of the neutral amino acid asparagine (N) by the negatively charged aspartic acid (D) at amino acid number 14, a position that is conserved among Cx26 of different organisms and among many other connexin isoforms. To investigate the impact of this mutation on protein function, Cx26 activity was measured by depolarization activated hemichannel conductance in non-coupled Xenopus laevis oocytes. Oocytes injected with the p.N14D mutant cRNA showed strongly reduced currents compared to wildtype. Coinjection of wildtype and mutant cRNA at equimolar levels restored the conductive properties supporting the recessive character of this mutation. Total Cx26 protein expression and cell surface abundance examined by western blotting and by quantitative immunoassays revealed that the hemichannel was properly synthesized but not integrated into the plasma membrane. In this study we have shown that the GJB2 mutation p.N14D is associated with recessively inherited HI and exhibits a defective phenotype due to diminished expression at the cell surface.

[Mitochondrial A1555G mutation. Molecular genetic diagnosis in sporadic cases of non-syndromic hearing impairment]. / Mitochondriale A1555G-Mutation. Molekulargenetische Diagnostik bei sporadischen Fällen nichtsyndromaler Schwerhörigkeit.

BACKGROUND: The A1555G mutation in mitochondrial DNA is the cause of hearing impairment in about 50% of all carriers. The severity and onset of this impairment is predominantly affected by the use of aminoglycosides. PATIENTS AND METHODS: A total of 391 patients displaying sporadic, non-syndromic, mild to severe hearing impairment were analyzed for the A1555G mutation using molecular genetic methods. RESULTS: We analysed additional family members of the two patients (0.5% of the total) who had the mutation. All maternal relatives carried the mutation, but only three individuals from the two families displayed a variable sensorineural hearing loss. CONCLUSION: The A1555G mutation is infrequently involved as a genetic cause of sporadic, non-syndromic hearing impairment. Nevertheless, based on the variable clinical outcome of hearing impairment and the possibility of preventive steps, a genetic test in this patient subgroup is indicated.

[Mitochondrial hearing impairment. Background, genetic predisposition and possibilities for diagnosis]. / Mitochondrial bedingte SchwerhörigkeitenHintergründe, genetische Prädisposition und Möglichkeiten der Diagnostik.

Hearing impairment (HI) is one of the most common neurosensory disorders, with sensorineural hereditary HI being the most common form. Mitochondrial maternally inherited HI appears to be increasing in frequency. The incidence of mitochondrial defects causing HI is estimated to be between 6 and 33% of all hearing deficiencies, with an even higher percentage for some syndromic cases. This review summarises the syndromic and non-syndromic characteristics of sensorineural HI based on mutations in mitochondrially encoded genes, the relationship to aminoglycoside-induced HI and related diagnostic tools.

Refinement of the DFNA4 locus to a 1.44 Mb region in 19q13.33.

Many forms of autosomal dominant non-syndromic hearing impairment are known. While the underlying gene defects and causative mutations have been discovered for some forms, the gene responsible for DFNA4 has remained elusive to date. Examination of a German four-generation kindred led to the identification of a 1.44 Mb map segment in contig NT_011109 as being the most likely DFNA4 candidate region in 19q13.33. The recombination breakpoints in this family and the intervals of two previously reported DFNA4 families allowed us to delineate a minimum consensus region between the markers D19S879 and D19S246. In our family, a maximum two-point LOD score of 4.5 was obtained at theta = 0 for the marker D19S867. Within the refined DFNA4 interval the public databases list more than 50 genes, from which several appear to be promising DFNA4 candidates due to similarities with animal models and with other causative genes involved in hearing disability.

[Phenotypic characterization of a DFNA6 family with low-frequency hearing loss]. / Phänotypische Charakterisierung einer DFNA6-Familie mit Tieftonschwerhörigkeit.

BACKGROUND: Hereditary hearing impairment is a heterogeneous sensory defect with approximately two-thirds of all cases being nonsyndromic. Only two loci (DFNA1 and DFNA6/14/38) are associated with low frequency sensorineural nonsyndromic hearing impairment. DFNA6 was mapped to chromosome 4p16. Recessive mutations in the WFS1 gene are responsible for Wolfram syndrome; missense mutations inherited as an autosomal dominant result in low frequency sensorineural hearing impairment (LFSNHI). PATIENTS AND METHODS: In this study we analyzed the phenotype of a large Hungarian family with LFSNHI and linkage to DFNA6. The family contains 14 affected persons. RESULTS AND CONCLUSION: In general, these patients show a postlingual, sensorineural, bilateral, symmetric, nonsyndromic low frequency hearing impairment with a slow progression. This impairment is accompanied by normal vision and normal vestibular responses.

[Paraganglioma in the area of the head and neck. A review of molecular genetic research]. / Paragangliome der Kopf-Hals-Region. Ein Uberblick über die molekulargenetische Forschung.

Paragangliomas of the head and neck region are usually benign tumors that develop from chemoreceptors of paraganglionic origin in the majority of patients. These receptors play an important role in sensing and regulation of the blood CO(2) level. Genetic alterations in the mitochondrial enzyme complex II (SDH), which is involved in respiratory chain and citric acid cycle reactions, have been shown to lead to sporadic as well as familial cases of these tumors. The gene encoding the subunit SDHD shows mutations in up to 50% of these cases. In addition, loss of heterozygosity (LOH) was demonstrated in these tumor samples and has been shown to be connected with oncogenesis of paragangliomas. Thus, SDHD is the first known tumor suppressor gene encoding a mitochondrial protein. In this article we summarize the current state of knowledge concerning the development of paragangliomas.

[Clinical and molecular genetic analysis of monozygotic twins displaying stapes gusher syndrome (DFN3)]. / Klinische und molekulargenetische Analyse monozygoter Zwillinge mit Stapes-Gusher-Syndrom (DFN3).

BACKGROUND: DFN3 ( "stapes gusher") is the most frequent form of X-linked hearing impairment. It accounts for up to 0.5% of all cases of severe childhood hearing disorders. PATIENTS AND METHODS: Monozygotic twins with suspected stapes gusher syndrome, their mother, and control individuals were analyzed clinically and genetically. RESULTS: The clinical investigations confirmed a DFN3 phenotype in both brothers who displayed all typical symptoms. A molecular genetic investigation of the POU3F4 gene, which plays an essential role in the development of DFN3, was also performed. No chromosomal aberrations within the coding region of POU3F4were detected. Since several authors have described mutations in the 5' untranslated region of the gene also resulting in a DFN3 phenotype, we screened this area by microsatellite analysis and detected a double deletion localized in the critical interval. This is the first description of a double deletion in the non-coding region of POU3F4 leading to DFN3 phenotype. CONCLUSION: Interestingly, in spite of having an identical genotype, the twins displayed significant phenotypic differences. This underlines the importance of exogenous factors in the development of inherited pathological processes.

[Phenotype of patients showing hearing impairment based on the 35delG mutation in the connexin 26 gene]. / Phänotypische Charakterisierung schwerhöriger Patienten mit homozygoter 35delG-Mutation im Connexin-26-Gen.

BACKGROUND: Hereditary hearing impairment constitutes a heterogeneous class of disorders showing different patterns of inheritance and involving multiple genes. Mutations in the GJB2 gene, especially the 35delG mutation, have been established as a major cause of inherited and sporadic nonsyndromic hearing impairment in different populations. METHODS: We analyzed 14 northeast Hungarian families and 69 sporadic cases with nonsyndromic hearing impairment for the 35delG mutation. Sixty-five patients showing a homozygous 35delG mutation were examined regarding their audiologic phenotype. RESULTS: In general, these patients (70%) showed a prelingual, sensorineural, bilateral, symmetric hearing impairment without progression. The audiograms demonstrated sloping as well as flat patterns. CONCLUSIONS: The severity of hearing impairment varied in 30% of all analyzed patients, making genetic counseling difficult.

Substitutions in the conserved C2C domain of otoferlin cause DFNB9, a form of nonsyndromic autosomal recessive deafness.

DFNB, the nonsyndromic hearing loss with an autosomal recessive mode of inheritance constitutes the majority of severe to profound prelingual forms of hearing impairment, usually leading to inability of speech acquisition. We analyzed a consanguineous family with autosomal recessive deafness which has been shown to segregate within chromosomal region 2p23.1 (DFNB9; MIM 601071). By SSCP analysis and DNA sequencing of the 48 exons of the DFNB9 gene, coding for otoferlin, previously reported mutations in OTOF were excluded. Next to a frequent T > C single nucleotide polymorphism in exon 8, two novel mutations linked in exon 15 of the OTOF long splice form were identified comprising substitutions at positions 490 (Pro > Gln) and 515 (Ile > Thr), both located in the conserved Ca(2+) binding C2C domain of this peptide. Comparisons of homology using human and mice otoferlins and closely related peptides and computer simulation analyses suggest that changes in the mutated segment's secondary structure affect the Ca(2+) binding capacity of the C2C domain in otoferlin.

Comparative genomic hybridization analysis of chromosomal alterations in patients with long-standing ulcerative colitis.

Patients with ulcerative colitis (UC) are prone to develop colorectal cancer which is related to the duration and extent of the disease. One of the earliest events in tumor progression is the development of aneuploidy. Aneuploidy is correlated with the grade of dysplasia which serves as a common but not always reproducible marker for the prediction of UC associated formation of cancer. We analyzed 48 biopsy samples from 5 patients with long-standing ulcerative colitis by comparative genomic hybridization (CGH). The majority of these samples represented premalignant stages which are not well characterized at the molecular level as yet. We compared biopsy samples from different colon locations in regard to chromosomal alterations, dysplasia status and DNA index. Besides chromosomal changes occurring only in certain patients in restricted areas of the colon we also detected amplifications and deletions which were common in all persons throughout the colon. The stage of dysplasia seems to have no influence on the number and appearance of chromosomal changes. Amplifications in 2, 3, 6, 9, 11, 12 and 15 were found in almost all cases. In dysplastic samples chromosomal regions 3, 6 and 11 revealed gains of DNA. Deletions were detected within 8q, 15, 18q, 20p and 22q. The affected chromosomal regions may contain yet unknown oncogenes or tumor suppressor genes participating in UC associated carcinogenesis. The conspicuous regions found in the CGH experiments allow the selective and detailed characterization at a molecular level.

Frequency of the recessive 30delG mutation in the GJB2 gene in Northeast-Hungarian individuals and patients with hearing impairment.

Mutations in the GJB2 gene, which encodes a gap junction protein (connexin 26) account for up to 50% of cases of congenital autosomal recessive non-syndromic hearing impairment. A single mutation, 30delG, is responsible for 70% of this autosomal recessive hearing loss in Europe. This study describes the 30delG mutation analysis of 23 Hungarian families (64 individuals) with at least two subjects with congenital non-syndromic hearing defect and of 52 unrelated individuals from the Northeastern population of Hungary. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. DNA was tested by PCR based restriction enzyme assay (BSiYI). Sixty-four percent of the patients displayed this one base deletion in GJB2. Out of these, 65.9% were homozygous for this mutation and 34.1% were heterozygotes. The latter showed compound heterozygosity since in these 14 patients, eight previously reported different nucleotide changes were observed on the second allele. The carrier frequency of the 30delG mutation among control group was one in 10.4 (9.6%). This high frequency of 30delG corresponds more to frequencies reported in Southern than in North Europeans.

[An attempt to identify the most frequent genomic mutations responsible for isolated deafness in patients after cochlear implantation]. / Próba identyfikacji najczestszych mutacji genowych odpowiedzialnych za gluchote izolowana u pacjentów nieslyszacych zaopatrzonych w implant slimakowy.

The aim of this study was to identify subjects with 35delG mutation of GJB2 gene as the most frequent genetic cause of deafness. Deaf patients receiving cochlear implantation at the ENT Clinic at University of Medical Sciences in Poznan and their family members were recruited to the study. Peripheral blood lymphocytes DNA was amplified in allele-specific PCR and analysed for single strand conformation polymorphism (SSCP) to detect mutation at DFNB1 locus. 35delG mutation at both alleles was found at 42.9% of deaf patients and 29.4% of health relatives were found to be carrier of the mutation at one allele. The study is thought to be a first step in analysis of typical mutations in Polish deaf population.

[Mutational analysis of the connexin26 gene in sporadic cases of moderate to profound deafness]. / Mutationsanalyse des Connexin26-Gens bei sporadischen Fällen mittel-bis hochgradiger Schwerhörigkeit.

Non-syndromic neurosensory recessive deafness (NSRD) is one of the most common human sensory disorders. Mutations in the connexin 26 gene have been established as a major cause of inherited and sporadic non-syndromic deafness in different populations. The CX26 gene encodes the gap junction protein connexin 26 (beta-2, GJB2), whose expression was shown in several tissues and in the cochlea. The 30delG mutation is the most frequent mutation in the CX26 gene. It represents a deletion of guanosine (G) in a sequence of six Gs extending from position 30 to 35 of the CX26 cDNA. The deletion creates a frameshift resulting in a premature stop codon and a non-functional intracellular domain in the protein. The 30delG mutation can be detected at the molecular level using PCR followed by BsiYI digestion. We screened 164 mainly German patients with non-syndromic sporadic deafness for this mutation to determine its distribution in the German population. The frequency of the mutation in our analyzed patients was lower than in other studies and therefore indicates its dependency on geographically distinct populations.

Assessing genetic heterogeneity of renal cell tumors.

Renal cell tumors display a highly variable morphology which is also reflected at the genomic level. Such heterogeneity was at first monitored by cytogenetic means (numerical and structural chromosomal aberrations); in the meantime, more refined molecular techniques allow the assessment of DNA losses or gains in metaphase chromosomes or tissue sections. Moreover, genomic instability can be monitored using microsatellite probes. All these methods document specific characteristics of certain renal cell tumor types, e.g. telomeric associations in chromophobe carcinomas or oncocytomas, typical losses in 3p in clear cell carcinomas or trisomy 7 in renal cell adenomas and carcinomas. Next to examples demonstrating these alterations the Heidelberg classification of renal cell tumors that is based on genomic observations is discussed.

[The possibilities of optimal therapy of corpus uteri cancer (author's transl)]. / Uber die Möglichkeiten einer optimalen Therapie des Carcinoma corporis uteri.

During the years 1960-1974 out of 1002 patients with endometrial carcinoma 256 underwent primary surgery and 846 primary irradiation. The evaluation of the results of 4 different surgical methods showed despite a different distribution of the stages of the cancer that Werthein's operation and abdominal hysterectomy and adnexectomy with simultaneous lymphnodectomy had statistically significant better results than abdominal hysterectomy without lymphonodectomy. By surgery a 5 years survival time was achieved in 83.6% (214 cases) with a primary mortality rate of 0.39% and an incidence of histologically proved lymphnode-metastases of 6.78%. Out of the 846 patients with generally or locally inoperable endometrial carcinomas, who underwent primary intracavitary irradiation 411 (48.6%) achieved a 5 years survival time. After irradiation in 9.9% reversible reactions (severe proctitis and cystitis) and in 2% irreversible complications (fistulas) were observed. After an observation period of 5 years 625 of the total of 1002 treated patients (62.4%) were alive and free of symptoms.

[Stage Ic cancer of the cervix. A clinical and immunological analysis (author's transl)]. / Das Zervixkarzinom im Stadium Ic. Eine klinische und immunologische Analyse.

Marked differences in cases of stage I cervical carcinoma, dependent on the extent of tissue involvement, are demonstrated by retrospective, prognostic as well als by prospective, immunological investigations. It seems recommendable to separate cases of a newly-defried stage Ib from stage Ic. The prognosis of these stage Ic cases is even worse than of stage IIa cases. As this subgrouping can be carried out only post-operatively, the preoperative assignment of cases to different stages should not be used for assessing the operative results.