Dietmar R Lohmann

Publications of Dietmar R Lohmann

• EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation.

  Authors: Lisa C Neumann, Andreas Weinhäusel, Stefanie Thomas, Bernhard Horsthemke, Dietmar R Lohmann, Michael Zeschnigk

  BMC cancer. 08/2011; 11:380.

  Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantlyUveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression. EFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients. Analysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples. Biallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.

• Loss at chromosome arm 16q in retinoblastoma: confirmation of the association with diffuse vitreous seeding and refinement of the recurrently deleted region.

  Authors: Sebastian Gustmann, Ludger Klein-Hitpass, Harald Stephan, Susanne Weber, Norbert Bornfeld, Marc Kaulisch, Dietmar R Lohmann, Nicole Dünker

  Genes, chromosomes & cancer. 02/2011; 50(5):327-37.

  In addition to mutations in both alleles of the retinoblastoma gene (RB1) alleles, retinoblastomas frequently show additional alterations including loss of chromosome arm 16q. In a previous study,In addition to mutations in both alleles of the retinoblastoma gene (RB1) alleles, retinoblastomas frequently show additional alterations including loss of chromosome arm 16q. In a previous study, the presence of 16q alterations was found to be associated with diffuse vitreous seeding of this tumor. This growth pattern is clinically important as it determines therapeutic decisions. The present study was designed to test this association and to narrow down the list of candidate genes in the minimal region of genomic loss on chromosome arm 16q. Our data confirm the association of 16q loss and diffuse vitreous seeding and define a minimal region of genomic loss of 6.6 Mb on 16q containing 86 known genes. As retinoblastoma is an embryonic tumor, we assumed that any gene relevant for its progression is likely to show regulated expression during retinogenesis. Microarray expression analysis of RNA from a continuous developmental series of murine retinas identified murine orthologs with regulated expression and these data helped to narrow the number of candidate genes in minimal region to 35. Analysis of gene expression in retinoblastomas with and without the loss of heterozygosity (LOH) on chromosome 16q further reduced this number to 26 candidate genes. One of these genes is cadherin 13 (CDH13) and notably, downregulation of CHD13 has previously been associated with poorer prognosis in various other cancers.

• Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

  Authors: Johannes G Dauwerse, Jill Dixon, Saskia Seland, Claudia A L Ruivenkamp, Arie van Haeringen, Lies H Hoefsloot, Dorien J M Peters, Agnes Clement-de Boers, Cornelia Daumer-Haas, Robert Maiwald, Christiane Zweier, Bronwyn Kerr, Ana M Cobo, Joaquín F Toral, A Jeannette M Hoogeboom, Dietmar R Lohmann, Ute Hehr, Michael J Dixon, Martijn H Breuning, Dagmar Wieczorek

  Nature genetics. 01/2011; 43(1):20-2.

  We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygousWe identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both alleles of POLR1C in three individuals with TCS. These findings identify two additional genes involved in TCS, confirm the genetic heterogeneity of TCS and support the hypothesis that TCS is a ribosomopathy.

• Microcephaly, microtia, preauricular tags, choanal atresia and developmental delay in three unrelated patients: A mandibulofacial dysostosis distinct from Treacher Collins syndrome.

  Authors: Dagmar Wieczorek, Blanca Gener, Ma Jesús Martínez González, Saskia Seland, Sven Fischer, Ute Hehr, Alma Kuechler, Lies H Hoefsloot, Nicole de Leeuw, Gabriele Gillessen-Kaesbach, Dietmar R Lohmann

  American journal of medical genetics. Part A. 04/2009;

  Treacher Collins syndrome (TCS, OMIM 154500) is a well-defined mandibulofacial dysostosis characterized by symmetric facial anomalies consisting of malar hypoplasia, coloboma of the lower eyelid,Treacher Collins syndrome (TCS, OMIM 154500) is a well-defined mandibulofacial dysostosis characterized by symmetric facial anomalies consisting of malar hypoplasia, coloboma of the lower eyelid, dysplastic ears, micrognathia, cleft palate and deafness. Other mandibulofacial dysostoses (MDs) such as Toriello (OMIM 301950), Bauru (OMIM 604830), Hedera-Toriello-Petty (OMIM 608257), and Guion-Almeida (OMIM 610536) syndromes are less well characterized and much rarer. Here we describe three unrelated patients showing clinical features overlapping with TCS, but who in addition have developmental delay, microcephaly and a distinct facial gestalt. Because of the distinct ear anomalies and the hearing loss a HOXA2 mutation was taken into account. CHARGE syndrome was discussed because of ear anomalies, choanal atresia, and developmental delay in our patients. But mutational analyses including sequencing of the TCOF1, the HOXA2, and the CHD7 genes, deletion screening of the TCOF1 gene as well as genomewide array analyses revealed normal results. We suggest that these three patients have a new type of mandibulofacial dysostosis. As all three cases are sporadic and both sexes are affected the pattern of inheritance might be autosomal dominant or autosomal recessive. Identification of additional patients will allow to further delineate the phenotype, to assign the inheritance pattern and to identify the molecular basis. (c) 2009 Wiley-Liss, Inc.

• Patterns of missplicing caused by RB1 gene mutations in patients with retinoblastoma and association with phenotypic expression.

  Authors: Katherine Zhang, Inga Nowak, Diane Rushlow, Brenda L Gallie, Dietmar R Lohmann

  Human mutation. 05/2008; 29(4):475-84.

  We have analyzed RNA from retinoblastoma patients and unaffected carriers with various RB1 gene mutations to determine the patterns of missplicing and associations with phenotypic expression. MostWe have analyzed RNA from retinoblastoma patients and unaffected carriers with various RB1 gene mutations to determine the patterns of missplicing and associations with phenotypic expression. Most sequence alterations in or in the neighborhood of conserved splice signals that we tested resulted in simple exon skipping (15 mutations) or intron inclusion (new acceptor AG-sites, four mutations) as expected. Two mutations resulted in skipping of a neighboring exon (exon 11), a complex pattern indicating competition for correct lariat formation. We observed no activation of a cryptic splice site but found that a recurrent missense mutation in exon 7 creates a new splice site (two families). RT-PCR analysis enabled us to confirm the presence and to characterize the transcriptional consequences of gross insertions and deletions in the RB1 gene in six patients, including two patients with mutational mosaicism. We also used RT-PCR analysis to search for unknown mutations in 15 patients and identified three oncogenic point mutations deep in introns. Two of these mutations are recurrent thus indicating that, despite the vast extent of the introns of the RB1 gene, few bases are effective targets for oncogenic mutations. When analyzing associations between phenotypic expression (16 families) and mutational consequences we observed no link to the presence or absence of a premature termination codon in the mutant transcript. However, the location of a mutation relative to the splice sequence has a strong and consistent influence on phenotypic expression.

• Neurotrophin receptor expression in human primary retinoblastomas and retinoblastoma cell lines.

  Authors: Harald Stephan, Johannes L Zakrzewski, Réka Bölöni, Corinna Grasemann, Dietmar R Lohmann, Angelika Eggert

  Pediatric blood & cancer. 03/2008; 50(2):218-22.

  BACKGROUND: Neurotrophin receptor signaling regulates proliferation, differentiation and death of neuronal cells. Expression of Trk receptors has been implicated in the pathogenesis and prognosis ofBACKGROUND: Neurotrophin receptor signaling regulates proliferation, differentiation and death of neuronal cells. Expression of Trk receptors has been implicated in the pathogenesis and prognosis of embryonal tumors, including neuroblastoma, nephroblastoma, and medulloblastoma. PROCEDURE: We analyzed TrkA, TrkB, TrkC, and p75 expression using semi-quantitative RT-PCR in 23 retinoblastomas and 8 retinoblastoma cell lines. Comparison of mRNA expression with clinical variables as well as the proliferation (PI) and apoptotic index (AI) of the tumor, was performed by Pearson correlation analysis and two-sample t-test. RESULTS: Almost all tumor samples and cell lines demonstrated high expression of all Trk receptors. Expression of TrkB and its ligand, BDNF, was most pronounced, suggesting TrkB to be the major Trk receptor involved in retinoblastoma biology. In contrast, p75 expression was substantially reduced in a subset of tumors and cell lines, in particular compared to its expression in normal retina. Tumors with infiltrative growth demonstrated significantly lower relative levels of TrkC expression than localized tumors (P = 0.004). High expression of TrkA was associated with a higher AI (P = 0.04), and high expression of TrkC was associated with a younger age of the patients (P = 0.03). Inhibition of Trk signaling by K252a resulted in marked growth inhibition of retinoblastoma cells in vitro. CONCLUSIONS: Our findings suggest a role for neurotrophin signaling in the biology of retinoblastoma. General Trk inhibitors are effective in decreasing growth rates of retinoblastoma cells in vitro, and should be evaluated in in vivo studies.

• Allelic loss in a minimal region on chromosome 16q24 is associated with vitreous seeding of retinoblastoma.

  Authors: Sandrine Gratias, Harald Rieder, Reinhard Ullmann, Ludger Klein-Hitpass, Stephanie Schneider, Réka Bölöni, Martin Kappler, Dietmar R Lohmann

  Cancer research. 02/2007; 67(1):408-16.

  In addition to RB1 gene mutations, retinoblastomas frequently show gains of 1q and 6p and losses of 16q. To identify suppressor genes on 16q, we analyzed 22 short tandem repeat loci in 58 patientsIn addition to RB1 gene mutations, retinoblastomas frequently show gains of 1q and 6p and losses of 16q. To identify suppressor genes on 16q, we analyzed 22 short tandem repeat loci in 58 patients with known RB1 mutations. A subset of tumors was also investigated by conventional and matrix comparative genomic hybridization. In 40 of 58 (69%) tumors, we found no loss of heterozygosity (LOH) at any 16q marker. LOH was detected in 18 of 58 (31%) tumors, including five with allelic imbalance at some markers. In one tumor LOH was only observed at 16q24. As the parental origin of allele loss was unbiased, an imprinted locus is unlikely to be involved. Analysis of gene expression by microarray hybridization and quantitative RT real-time PCR did not identify a candidate suppressor in 16q24. Cadherin 13 (CDH13), CBFA2T3, and WFDC1, which are candidate suppressors in other tumor entities with 16q24 loss, did not show loss of expression. In addition, mutation and methylation analysis showed no somatic alteration of CDH13. Results in all tumors with chromosome 16 alterations define a single minimal deleted region of 5.7 Mb in the telomeric part of 16q24 with the centromeric boundary defined by retention of heterozygosity for a single nucleotide variant in exon 10 of CDH13 (Mb 82.7). Interestingly, clinical presentation of tumors with and without 16q alterations was distinct. Specifically, almost all retinoblastomas with 16q24 loss showed diffuse intraocular seeding. This suggests that genetic alterations in the minimal deleted region are associated with impaired cell-to-cell adhesion.

• Establishment and characterization of two uveal melanoma cell lines derived from tumors with loss of one chromosome 3.

  Authors: Gordon Nareyeck, Michael Zeschnigk, Gabriele Prescher, Dietmar R Lohmann, Gerasimos Anastassiou

  Experimental eye research. 11/2006; 83(4):858-64.

  Uveal melanoma (UM) is the most common intraocular malignancy. Approximately 50% of UM patients die of metastases, which mainly arise from primary tumors with loss of an entire chromosome 3 (monosomyUveal melanoma (UM) is the most common intraocular malignancy. Approximately 50% of UM patients die of metastases, which mainly arise from primary tumors with loss of an entire chromosome 3 (monosomy 3). To identify cell lines with monosomy 3 that may serve as a model system for UM with high metastatic potential, we determined the chromosome 3 status of previously established and frequently used UM cell lines by microsatellite analysis (Mel202, Mel285, Mel290, 92-1, OMM-1, OCM-1, OCM-3, OCM-8) and cytogenetic analysis (Mel202, Mel285, OCM-8). We found that none of these cell lines has monosomy 3. Therefore we established and characterized two novel cell lines, UPMM-1 and UPMM-2 that are both developed from primary uveal melanoma tissue samples with monosomy 3. The cell line UPMM-1 has retained the chromosome 3 status of the primary tumor. In UPMM-2 chromosome 3 has undergone duplication (isodisomy) and is present on the background of a hypotetraploid karyotype. Our data suggest that, UPMM-1 may serve as a model system to study the mechanisms underlying the metastatic potential of uveal melanomas with monosomy 3.

• Minimal region of deletion on chromosomal arm 3p25.1-p25.2 in uveal melanoma.

  Authors: Dietmar R Lohmann, Thomas Häusler, Bernhard Horsthemke, Michael Zeschnigk

  Cancer research. 12/2005; 65(22):10634; author reply 10634.

• Loss of heterozygosity of 1p in uveal melanomas with monosomy 3.

  Authors: Thomas Häusler, Andreas Stang, Gerasimos Anastassiou, Karl-Heinz Jöckel, Stefanie Mrzyk, Bernhard Horsthemke, Dietmar R Lohmann, Michael Zeschnigk

  International journal of cancer. Journal international du cancer. 11/2005; 116(6):909-13.

  Gains and losses of chromosomes 1, 3, 6 and 8 are nonrandom chromosomal aberrations in uveal melanoma. Monosomy 3 is the most frequent abnormality and is associated with poor prognosis. To identifyGains and losses of chromosomes 1, 3, 6 and 8 are nonrandom chromosomal aberrations in uveal melanoma. Monosomy 3 is the most frequent abnormality and is associated with poor prognosis. To identify regions of allelic loss on the short arm of chromosome 1 and to investigate if these alterations contribute to uveal melanoma progression, we performed microsatellite analysis of 10 loci in 70 uveal melanomas. A total of 51 tumors were obtained from patients with clinical follow-up data, 19 tumors were from recent patients without follow-up. Loss of heterozygosity (LOH) of at least 1 marker was more frequent in tumors with monosomy 3 (40%) than in tumors with disomy 3 (10%). In particular, loss of the entire short arm of chromosome 1 was only observed in tumors with monosomy 3 (p = 0.0001). By comparing the extent of 1p LOH in all tumors with monosomy 3, we were able to define a smallest region of overlap (SRO) of approximately 55 Mb, which is flanked by markers D1S507 and D1S198. On the basis of our data and published cytogenetic data, we propose that 1p31 harbors genes involved in the progression of uveal melanoma with monosomy 3.

• Gains and overexpression identify DEK and E2F3 as targets of chromosome 6p gains in retinoblastoma.

  Authors: Corinna Grasemann, Sandrine Gratias, Harald Stephan, Andreas Schüler, Alexander Schramm, Ludger Klein-Hitpass, Harald Rieder, Stephanie Schneider, Ferdinand Kappes, Angelika Eggert, Dietmar R Lohmann

  Oncogene. 10/2005; 24(42):6441-9.

  The paediatric eye tumour retinoblastoma is initiated by inactivation of RB1, a tumour suppressor on chromosome 13q. In addition to RB1 loss, many retinoblastomas show other genetic alterationsThe paediatric eye tumour retinoblastoma is initiated by inactivation of RB1, a tumour suppressor on chromosome 13q. In addition to RB1 loss, many retinoblastomas show other genetic alterations including gains on chromosomes 6p21-pter and 1q31-q32. Recently, the minimal region of gains on chromosome 6 was narrowed to band p22. We examined genomic gains and expression changes in primary retinoblastomas to identify potential target genes in 6p22. Quantitative multiplex PCR detected copy numbers > or = 3 in 25 (33%) tumours and no gains in 31 of 76 (40%) tumours. The remaining 20 (26%) samples showed gains only at some loci, most often including E2F3 and DEK in 6p22.3. Analysis of RNA from 21 primary retinoblastomas showed that expression levels of these and some other genes in 6p22 correspond to DNA gains. However, KIF 13A, a reported candidate oncogene on 6p, was expressed at low levels or absent. Clinical manifestation of tumours with gains at all 6p22 loci was distinct in that distribution of age at diagnosis was markedly shifted to older age compared to tumours with no or partial gains. In summary, our results suggest that DEK and E2F3 are potential targets of 6p gains in retinoblastoma.

• Genomic gains on chromosome 1q in retinoblastoma: consequences on gene expression and association with clinical manifestation.

  Authors: Sandrine Gratias, Andreas Schüler, Ludger Klein-Hitpass, Harald Stephan, Harald Rieder, Stephanie Schneider, Bernhard Horsthemke, Dietmar R Lohmann

  International journal of cancer. Journal international du cancer. 10/2005; 116(4):555-63.

  Many retinoblastomas (Rbs) show genomic alterations in addition to mutational loss of both normal RB1 alleles. The most frequent of these changes are gains on chromosomes 1q and 6p and losses on 16q.Many retinoblastomas (Rbs) show genomic alterations in addition to mutational loss of both normal RB1 alleles. The most frequent of these changes are gains on chromosomes 1q and 6p and losses on 16q. To identify the genes targeted by gains on chromosome 1q, we used quantitative-multiplex PCR to determine DNA copy number changes in 76 primary tumors and 6 Rb cell lines. In addition, in 21 of these tumors, gene expression was analyzed by cDNA microarray hybridization. Increased copy numbers of loci on chromosome 1q were present in 34 (45%) primary tumors and in all 6 cell lines. Two regions of gain emerged, one in 1q32 and another in 1q21. Tumors with 1q gains showed higher RNA expression of several genes in these 2 regions. The clinical manifestation of tumors with and without gains was similar with regard to many aspects, including size, necrosis and calcification. However, the distribution of age at diagnosis was remarkably distinct, with earlier diagnosis in tumors without gains. This suggests that these tumors either are initiated earlier or grow faster than tumors with gains. This association with clinical manifestation indicates that gains on 1q are significant for the biology of Rb. The genes on 1q with copy number gains and overexpression are candidates that need to be tested for their individual contribution to the progression of Rb.

• Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization.

  Authors: Boris Zielinski, Sandrine Gratias, Grischa Toedt, Frank Mendrzyk, Daniel E Stange, Bernhard Radlwimmer, Dietmar R Lohmann, Peter Lichter

  Genes, chromosomes & cancer. 08/2005; 43(3):294-301.

  The genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on aThe genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on a genomewide level, we studied a series of 17 primary retinoblastomas by matrix-based comparative genomic hybridization (matrix-CGH). The matrix-CGH chip contained 6,000 immobilized genomic DNA fragments covering the human genome, with an average resolution of about 500 kb. The most frequent imbalances detected were gains on chromosome arms 1q (12 of 17), 6p (10 of 17), 2p (5 of 17), and 19q (4 of 17) and loss on 16q (7 of 17). Candidate regions could be narrowed to small intervals by the identified minimally overlapping regions on 1q22, 1q32.1q32.2, 2p24.1, and 6p21.33-p21.31. Furthermore, two as-yet-unknown high-level amplifications were detected, each in a single patient, on chromosome bands 1p34.2 and 1p33. Thus, this study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma.

• Genotyping in 46 patients with tentative diagnosis of Treacher Collins syndrome revealed unexpected phenotypic variation.

  Authors: Ozge Altug-Teber, Gabriele Gillessen-Kaesbach, Sven Fischer, Stefan Böhringer, Beate Albrecht, Angelika Albert, Mine Arslan-Kirchner, Eric Haan, Monika Hagedorn-Greiwe, Christof Hammans [......] Luitgard M Neumann, Eva-Christina Prott, Anita Rauch, Hans-Dieter Rott, Heide Seidel, Stephanie Spranger, Martin Sprengel, Barbara Zoll, Dietmar R Lohmann, Dagmar Wieczorek

  European journal of human genetics : EJHG. 12/2004; 12(11):879-90.

  To define the range of phenotypic expression in Treacher Collins syndrome (TCS; Franceschetti-Klein syndrome), we performed mutation analysis in the TCOF1 gene in 46 patients with tentative diagnosisTo define the range of phenotypic expression in Treacher Collins syndrome (TCS; Franceschetti-Klein syndrome), we performed mutation analysis in the TCOF1 gene in 46 patients with tentative diagnosis of TCS and evaluated the clinical data, including a scoring system. A total of 27 coding exons of TCOF1 and adjacent splice junctions were analysed by direct sequencing. In 36 patients with a clinically unequivocal diagnosis of TCS, we detected 28 pathogenic mutations, including 25 novel alterations. No mutation was identified in the remaining eight patients with unequivocal diagnosis of TCS and 10 further patients, in whom the referring diagnosis of TCS was clinically doubtful. There is no overt genotype-phenotype correlation except that conductive deafness is significantly less frequent in patients with mutations in the 3' part of the open reading frame. Inter- and intrafamilial variation is wide. Some mutation carriers, parents of typically affected patients, are so mildly affected that the diagnosis might be overlooked clinically. This suggests that modifying factors are important for phenotypic expression. Based on these findings, minimal diagnostic criteria were defined: downward slanting palpebral fissures and hypoplasia of the zygomatic arch. The difficulties in genetic counselling, especially diagnosis of family members with a mild phenotype, are described.

• Retinoblastoma: revisiting the model prototype of inherited cancer.

  Authors: Dietmar R Lohmann, Brenda L Gallie

  American journal of medical genetics. Part C, Seminars in medical genetics. 09/2004; 129C(1):23-8.

  Hereditary retinoblastoma is an autosomal dominant disorder caused by mutations in the RB1 gene. Analysis of this rare condition has helped to elucidate the mechanisms underlying hereditary cancerHereditary retinoblastoma is an autosomal dominant disorder caused by mutations in the RB1 gene. Analysis of this rare condition has helped to elucidate the mechanisms underlying hereditary cancer predisposition in general. As identification of RB1 gene mutations has become a part of clinical management of patients with retinoblastoma, there is now a wealth of data. In this article, we summarize the current knowledge on the relations between the genotype and phenotypic expression. Moreover, detailed analysis of genotype-phenotype relations shows that hereditary retinoblastoma has features of a complex trait.

• A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.

  Authors: Michael Zeschnigk, Stefan Böhringer, Elizabeth Ann Price, Zerrin Onadim, Lars Masshöfer, Dietmar R Lohmann

  Nucleic acids research. 02/2004; 32(16):e125.

  Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore,Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.

• Methylation analysis of several tumour suppressor genes shows a low frequency of methylation of CDKN2A and RARB in uveal melanomas.

  Authors: Michael Zeschnigk, Frank Tschentscher, Christina Lich, Birgit Brandt, Bernhard Horsthemke, Dietmar R Lohmann

  Comparative and functional genomics. 01/2003; 4(3):329-36.

  We have investigated the frequency of methylation of several tumour suppressor genes in uveal melanoma. As the loss of one copy of chromosome 3 (monosomy 3), which is found in about half of theseWe have investigated the frequency of methylation of several tumour suppressor genes in uveal melanoma. As the loss of one copy of chromosome 3 (monosomy 3), which is found in about half of these tumours, is tightly associated with metastatic disease, a special emphasis was laid on genes located on this chromosome, including the fragile histidine triad (FHIT), von Hippel-Lindau (VHL), beta-catenin (CTNNB1), activated leukocyte cell adhesion molecule (ALCAM) and retinoic acid receptor-beta2 (RARB) genes. In addition, the methylation patterns of the CpG-rich regions 5' of the E-cadherin (CDH1), p16/cyclin-dependent kinase inhibitor 2 A (CDKN2A) and retinoblastoma (RB1) genes were analysed by bisulphite genomic sequencing or methylation-specific PCR (MSP). Furthermore, the SNRPN and D15S63 loci, which are located in the imprinted region of chromosome 15, were included in the study. Aberrant methylation was detected in nine of 40 tumours analysed: The imprinted SNRPN and D15S63 loci were hypermethylated in three tumours, all of which retained both copies of chromosome 3. Methylated RARB alleles were detected in three tumours, whereas in three other tumours CDKN2A was found to be methylated. As we did not find RARB and CDKN2A preferentially methylated in tumours with monosomy 3, which is a significant predictor of metastatic disease, we suggest that these genes may play a causative role in the formation of uveal melanoma but not in the development of metastases.

• A parent-of-origin effect in two families with retinoblastoma is associated with a distinct splice mutation in the RB1 gene.

  Authors: Martina Klutz, Dieter Brockmann, Dietmar R Lohmann

  American journal of human genetics. 07/2002; 71(1):174-9.

  We have identified a splice-site mutation (IVS6+1G-->T) in the RB1 gene, in two unrelated families with incomplete-penetrance retinoblastoma. Analysis of RNA from white blood cells showed that thisWe have identified a splice-site mutation (IVS6+1G-->T) in the RB1 gene, in two unrelated families with incomplete-penetrance retinoblastoma. Analysis of RNA from white blood cells showed that this mutation causes skipping of exon 6. Although this deletion results in a frameshift, most carriers of the mutation did not develop retinoblastoma. Interestingly, the relative abundance of the resultant nonsense messenger RNA varies between members of the same family and is either similar to or considerably lower than the transcript level of the normal allele. Moreover, variation of relative transcript levels is associated with both the sex of the parent that transmitted the mutant allele and phenotypic expression: All eight carriers with similar abundance of nonsense and normal transcript have received the mutant allele from their mother, and only one of them has developed retinoblastoma; by contrast, all eight carriers with reduced abundance of the nonsense transcript have received the mutant allele from their father, and all but two them have retinoblastoma. After treatment with cycloheximide, the relative abundance of transcripts from paternally inherited mutant alleles was partly restored, thus indicating that posttranscriptional mechanisms, rather than transcriptional silencing, are responsible for low levels of mutant messenger RNA. Our data suggest that a specific RB1 mutation can be associated with differential penetrance, on the basis of the sex of the transmitting parent.

• Retinoblastoma

  Authors: Dietmar R Lohmann, Brenda L Gallie