Methylation-specific Multiplex Ligation-dependent Probe Amplification Research Articles

Consistent DNA Hypermethylation Patterns in Laryngeal Papillomas

INTRODUCTION: This study examined the contribution of promoter hypermethylation to the pathogenesis of respiratory papillomatosis (RP), including recurrences (RRP) and progression to squamous cell carcinoma (SSC). MATERIALS AND METHODS: A retrospective cohort of 25 laryngeal papilloma cases included 21 RRP, two of which progressed to SCC. Aberrant methylation status was determined using the multi-gene (22 tumor suppressor genes) methylation-specific multiplex ligation-dependent probe amplification assay and confirmed using methylation specific PCR. RESULTS: Twenty genes had altered DNA methylation in 22 of 25 cases. Aberrant methylation of CDKN2B and TIMP3 was most frequent. Promoter hypermethylation of BRCA2, APC, CDKN2A and CDKN2B was detected in 2 RRP cases with subsequent progression to SCC. Of the 25 cases, 22 were positive for HPV-6, 2 for HPV-11 and 1 for HPV-16 and 33. CONCLUSIONS: Consistent aberrant methylation of multiple tumor suppressor genes contributes to the pathogenesis of laryngeal papillomas. Persistent aberrant DNA methylation events in 2 RRP cases that progressed to cancer indicate an epigenetic monoclonal progression continuum to SCC.

DNA Promoter Methylation in Breast Tumors Is Not Associated with Polymorphism in the GSTP1 Gene.

Abstract The gluthathione S-transferases (GSTs) are a family of enzymes that play an important role in detoxification of a variety of potential carcinogenic, hydrophobic and electrophilic compounds. Glutathione-S-transferase P1 (GSTP1) is involved in thiol-mediated detoxification and breakdown of reactive oxygen species created by anticancer drug exposure. It is also an inhibitor of c-Jun N-terminal kinase 1 which is involved in stress response, apoptosis and cellular proliferation. It has been postulated that a decrease in GST enzyme activity could result in higher frequency and a specific pattern of mutations in cancer. Loss of GSTP1 expression is observed in approximately two thirds of breast cancer cases, which indicates its potential role in breast carcinogenesis. The GSTP1 gene exhibits deletion polymorphisms and homozygous deletion of the gene may result in lack of enzyme activity. The polymorphism in the GSTP1 gene results from a base substitution and leads to an amino acid change at codon 105. This amino acid resides at the substrate binding site of the enzyme and may modify the catalytic activity of the enzyme several-fold.DNA methylation is an important epigenetic modification of the genome. A specific methylation pattern has been observed in various types of cancers. Hypermethylation of the GSTP1 promoter has also been associated with gene silencing in different forms of cancer. In a previous study we have found that the GSTP1 gene was the second most frequently methylated gene in breast cancer. However, a possible relationship between the methylation status of the gene promoter and GSTP1 polymorphic variants has not been investigated.In this study we investigated the role of the different allelic variants in the methylation status of the GSTP1 gene in patients with breast cancer. A total of 48 patients with breast cancer and 32 healthy women were analyzed. The GSTP1 polymorphism was determined by PCR-RFLP. Methylation of the GSTP1 gene was evaluated by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) using probes targeting the GSTP1 gene and metylation-sensitive enzyme digestion of the probe-DNA hybrids. The amplified products were analyzed by sequence-type capillary electrophoresis. Subsequent quantification of the methylation status was performed by comparing the relative signal peaks from the digested and undigested samples.The null allele was observed in % 56,2 of the patients and % 46.8 of the control group, respectively. No significant difference was observed between the distribution of the genotypes in the patients and the controls. The GSTP1 gene promoter was methylated in % 39.6 of the patients. However, the distribution of the genotypes and allele frequencies did not reveal any preferential methylation of a specific allele. The genotypes were not associated with increased likelihood of promoter methylation. Our data indicate that the deletion allele of the GSTP1 gene does affect the methylation status of the gene in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1150.

A Methylation Profile of Tumor Suppressor Genes Predicts a Poorer Survival in Patients with Refractory Anemia with Ringed Sideroblasts.

Abstract 2780Poster Board II-756Patients with refractory anemia with ring sideroblasts (RARS) are considered to have good prognosis and anemia is usually managed with best supportive care and erythroid stimulating agents. Nowadays, no specific molecular marker related to outcome and disease progression has been identified. Several genes involved in cell cycle and apoptosis that may become inactivated by aberrant hypermethylation have been identified in patients with myelodysplastic syndromes (MDS) but the significance of a methylation profile (studying several genes at the same time) in RARS is unknown, mainly because the number of patients with RARS in published reports is rather low. To assess the implication of aberrant methylation in RARS, we studied the methylation status of 25 sequences of a set of tumor suppressor genes in 40 patients (median age 70 yr, 25 male gender) with RARS according to FAB criteria [WHO classification, RARS in 22 patients (55%); refractory citopenia with multilineage dysplasia and ring sideroblasts, 18 (45%)] by means of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. The MS-MLPA procedure (SALSA MLPA kit ME001, MRC-Holland, Amsterdam, The Netherlands) has been developed for detecting in a semi-quantitative manner changes in the methylation status of 25 tumor suppressor genes in a single experiment. Briefly, approximately 50 ng of DNA were denatured and hybridized with MLPA probes. Subsequently, the probes were ligated in half of every sample, whereas for the rest of the sample, ligation was combined with an endonuclease HhaI digestion resulting in ligation of the methylated sequences only. PCR was performed as described by the manufacturer, and then separated by capillary gel electrophoresis and quantified using the Genemapper software (ABI 310, Applied Biosystems, Foster City, CA). Quantification of the methylation status was done by dividing the peak area with the combined areas of the control probes lacking the target sequence of the HhaI. Finally, the relative peak area of each target probe from the digested sample was compared with those obtained from the undigested sample. Aberrant methylation was scored when the calculated methylation percentage was >10%. To validate the MS-MLPA method the methylation status of CDKN2B was also analyzed by methylation-specific PCR (MSP). Actuarial curves of OS were built by Kaplan-Meier method and differences between curves compared with log-rank tests. Small numbers precluded the use of multivariate methodology. The MS-MLPA analysis detected methylation of at least one gene in 17 patients (42.5%). The genes aberrantly methylated were CDKN2B (n = 8, 20%), RASSF1 (n = 7, 17%), RARB (n = 3, 7.5%), CDH13 (n = 3, 7.5%) and FHIT (n = 2; 5%). Of the 17 patients, five (12%) presented methylation in two genes (RASSF1-FHIT in 2, RASSF1-RARB in 1, RASSF1-CDH13 in 1, and CDKN2B-CDH13 in 1, who was the only patient who progressed to AML). The presence of aberrant gene methylation was not significantly associated with any clinical or biological characteristic or WHO morphological subtype. Patients with aberrant gene methylation had a significantly shorter overall survival (OS) than patients without methylated genes (median OS, 72 mo vs 114 mo, respectively; P = 0.03). Patients with hemoglobin level below 10 g/dL and platelet count below 150 ×109/L also had a significantly poorer OS (P= 0.01 and P= 0.02, respectively). As the majority of probes used in the MS-MPLA method detect methylation in only one CpG pair, the results of CDKN2B methylation were validated by MSP, which yielded the same methylation results than with MS-MPLA methodology. The 8 patients with methylated CDKN2B show a trend for a shorter survival than the remaining 32 with unmethylated CDKN2B (median 72 mo vs 114 mo, P = 0.08). The results of this study indicate that aberrant methylation of several tumor suppressor genes is observed in a substantial proportion of patients with RARS. As occurs in patients with high-risk MDS, our results suggest that aberrant gene methylation confers a poor prognosis in RARS, but these data and their potential independent value require confirmation in larger series that employ multivariate methods. Finally, these findings provide a strong rationale for the use of hypomethylating agents (e.g., azacitidine or decitabine) in patients with RARS.This work has been partially supported by ISCIII grants RD06/0020/0031 and CA08/00141. Disclosures:No relevant conflicts of interest to declare.

DNA Methylation Changes Following 5-Azacitidine Treatment in Patients with Myelodysplastic Syndrome.

Abstract 2791Poster Board II-767Gene silencing by promoter methylation is as potent as functional inactivating of tumor suppressor genes by mutations. DNA methyltransferase inhibitor, 5-azacytidine (AC) and 5-aza-2 -deoxycitidine (DAC), which is proved to be effective in myelodysplastic syndromes (MDS) can induce re-expression in cancer; however their mechanism remains controversial. 25 tumor suppressor genes by MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification) were analyzed in 44 MDS patients treated Vidaza® (5-azacitidine, AC). Hypermethylation of at least one gene was detected in 9/44 patients (20.5%), including four genes CDKN2B, FHIT, ESR1 and IGSF4. Interestingly, of 9 hypermethylated patients, 8 patients showed demethylation in concordance with their clinical responses after three to five cycles AC treatment. Lack or decrease methylation was observed in four patients with hematological improvements. Persistence methylation was observed in four others who became AML transformation or no response after treatment, especially reinforcing methylated gene in a case progressed to leukemia later. Our study also founds out IGSF4 gene hypermethylation in MDS as a first report. Additionally, mRNA expression of CDKN2B, IGSF4, and ESR1 in MDS were significantly lower than those in the control group (p < 0.05).Our results suggest that the methylation changes of specific genes contributes to disease pathogenesis and might present a molecular marker that can be used to monitor the efficacy of AC treatment in MDS. Disclosures:No relevant conflicts of interest to declare.

Hypermethylation of CD44 Is Characteristic of Specific Lymphoma Subtypes.

Abstract 3469Poster Board III-357Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a common hallmark of cancer. It is generally agreed, that CpG island hypermethylation profiles are specific for different tumor types [Costello et al., 2000, Nat Genet 25:132-138]. Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification), methylation of 24 different TSG was analyzed in 40 lymphoma cell lines representing Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). On average 8 ± 2.8 TSG out of the 24 analyzed were methylated per lymphoma cell line, whereas 0/24 TSG were methylated in healthy donor tonsils. Methylation frequencies decreased from HL, ALCL, BL, FL, DLBCL to MCL cell lines. The TSG methylation status of the most relevant genes was verified by methylation-specific PCR. Moreover, TSG hypermethylation correlated with transcriptional silencing as assessed by quantitative real time PCR. While our studies on the methylation status of TSG in lymphoma cell lines support previous methylation analyses performed on primary lymphoma patient material for many of the genes analyzed here, MS-MLPA screening also revealed a new interesting candidate: CD44. Hypermethylation of CD44 was characteristic of ALCL, BL, FL and DLBCL cell lines and allowed their discrimination from MCL and HL cell lines. In CD44 hypermethylated cell lines expression of CD44 was re-inducible at mRNA and protein levels by treatment with the demethylating agent 5-Aza-2'-deoxycytidine, confirming its epigenetic regulation. Furthermore, CD44 ligation with an anti-CD44 antibody induced apoptosis in CD44+ (CD44 unmethylated) lymphoma cell lines whereas CD44 hypermethylated cell lines showed no response. Thus, CD44 might be an interesting new epigenetic marker and a potential molecular target for the diagnosis and treatment of specific lymphoma subtypes. DisclosuresNo relevant conflicts of interest to declare.

Hypomethylation of Tumor Suppressor Genes in Acute Myeloid Leukemia: Characteristic of Cell Lines with MLL Abnormalities?.

Abstract 365▪▪This icon denotes an abstract that is clinically relevant. Background:Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines. Results:Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion:These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins. Disclosures:No relevant conflicts of interest to declare.

Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in Acute Myeloid Leukemia

BackgroundTranslocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines.ResultsUsing MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLLmu AML cells, while 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu AML cell lines. MLLwt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene.ConclusionThese results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

Clinical and histomolecular endometrial tumor characterization of patients at-risk for Lynch syndrome in South of Brazil

Lynch syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in one of the mismatch repair (MMR) genes: MLH1, MSH2, MSH6 and PMS2. Clinically, Lynch syndrome is characterized by early onset (45 years) of colorectal cancer (CRC), as well as extra-colonic cancer. Male and female carriers of Lynch syndrome-associated mutations have different lifetime risks for CRC and in women endometrial cancer (EC) may be the most common tumor. Whenever Amsterdam criteria are not fulfilled, the currently recommended laboratory screening strategies involve microsatellite instability testing and immunohistochemistry staining of the tumor for the major MMR proteins. The aim of this study was to estimate the frequency of MMR deficiencies in women diagnosed with EC who are at-risk for Lynch syndrome. Thirty women diagnosed with EC under the age of 50 years and/or women with EC and a first degree relative diagnosed with a Lynch syndrome-associated tumor were included. To assess MMR deficiencies four methods were used: multiplex PCR, Single Strand Conformation Polymorphism, Immunohistochemistry and Methylation Specific-Multiplex Ligation-dependent Probe Amplification. Twelve (40%) patients with EC fulfilling one of the inclusion criteria had results indicative of MMR deficiency. The identification of 5 women with clear evidence of MMR deficiency and absence of either Amsterdam or Bethesda criteria among 10 diagnosed with EC under the age of 50 years reinforces previous suggestions by some authors that these women should be considered at risk and always screened for Lynch syndrome after informed consent.

Reply to Camprubí et al

Reply to Camprubí et al

Analysis of microsatellite instability in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. The presence of microsatellite instability (MSI) in brain tumors, particularly medulloblastomas, has not been properly addressed. The aim of the present study was to evaluate the role of MSI in medulloblastoma carcinogenesis. MSI status was determined in 36 patients using a pentaplex PCR of quasimonomorphic markers (NR27, NR21, NR24, BAT25, and BAT26). Methylation status of mismatch repair (MMR) genes was achieved by methylation-specific multiplex ligation-dependent probe amplification (MLPA). In addition, MutS homolog 6 (MSH6) expression was determined by immunohistochemistry. Mutations of 10 MSI target genes (TCF4, XRCC2, MBD4, MRE11, ATR, MSH3, TGFBR2, RAD50, MSH6, and BAX) were studied by pentaplex PCR followed by analysis with GeneScan 3.7 software. Mutation analysis of hotspot regions of beta-catenin (CTNNB1) and BRAF (v-raf murine sarcoma viral oncogene homolog B1) oncogenes was performed by PCR single-strand conformation polymorphism analysis followed by direct sequencing. Among the 36 tumors, we found four (11%) cases with instability, one with high MSI and three with low MSI. Methylation analysis of MMR genes in cases presenting shifts on the MSI markers revealed mild hypermethylation of MSH6 in 75% of cases, yet MSH6 was expressed in all the tumors. The MSI target genes MBD4 (methyl-CpG binding domain protein 4) and MRE11 (meiotic recombination 11 homolog A) were mutated in two different tumors. No CTNNB1 or BRAF mutations were found. This study is the most comprehensive analysis of MSI in medulloblastomas to date. We observed the presence of MSI together with mutations of MSI target genes in a small fraction of cases, suggesting a new genetic pathway for a role in medulloblastoma development.

Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness and poor survival of renal-cell carcinoma

Background:The von Hippel–Lindau gene (VHL) alteration, a common event in sporadic clear-cell renal-cell carcinoma (CCRCC), leads to highly vascularised tumours. Vascular endothelial growth factor (VEGF) is the major factor involved in angiogenesis, but the prognostic significance of both VHL inactivation and VEGF expression remain controversial. The aims of this study were to analyse the relationship between VHL genetic and epigenetic alterations, VHL expression and VEGF tumour or plasma expression, and to analyse their respective prognostic value in patients with CCRCC.Methods:A total of 102 patients with CCRCC were prospectively analysed. Alterations in VHL were determined by sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA. Expression of pVHL and VEGF was determined by immunohistochemistry. Plasma VEGF was measured by enzyme-linked immunosorbent assay (ELISA).Results:VHL mutation, deletion and promoter methylation were identified in 70, 76 and 14 cases, respectively. Overall, at least one VHL-gene alteration occurred in 91 cases (89.2%). Both VEGF tumour and plasma expression appeared to be decreased in case of VHL alteration. Median progression-free survival and CCRCC-specific survival were significantly reduced in patients with wild-type VHL or altered VHL and high VEGF expression, which, therefore, represent two markers of tumour aggressiveness in CCRCC.Conclusion:Stratifying CCRCCs according to VHL and VEGF status may help tailor therapeutic strategy.

Detection and Discrimination between Deletional and Non-Deletional Prader-Willi and Angelman Syndromes by Methylation-Specific PCR and Quantitative Melting Curve Analysis

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are clinically distinct neurological disorders caused by a lack of expression of oppositely imprinted genes in chromosomal region 15q11-13. The loss of expression can be due to parent-specific segmental deletions or can arise from non-deletional mechanisms, such as uniparental disomy of chromosome 15 or defects in imprinting. Most current diagnostic methods to distinguish PWS from AS require separate amplification and detection steps, and some methods cannot differentiate between deletional and non-deletional forms of these syndromes. We have developed a single-step, methylation-specific PCR, and quantitative melting curve analysis assay to identify methylation differences and copy number changes in PWS and AS. In this strategy, duplex amplification followed by melting curve analysis was performed to detect the maternally and paternally imprinted SNRPN alleles and LIS1 reference gene. To discriminate between deletional and non-deletional PWS and AS, relative peak height ratios of maternal or paternal SNRPN:LIS1 were determined, respectively. To validate the diagnostic accuracy of the analysis, methylation-specific multiplex ligation-dependent probe amplification was performed on all PWS and AS samples. Complete concordance between the melting curve analysis and methylation-specific multiplex ligation-dependent probe amplification results was observed for all PWS and AS samples. Methylation-specific PCR and quantitative melting curve analysis represents a simple, rapid, and robust alternative to methylation-specific multiplex ligation-dependent probe amplification for the detection of and discrimination between deletional and non-deletional forms of PWS and AS.

G.P.7.01 Analysis of the nebulin gene by multiplex-ligation dependent probe amplification (MLPA) and methylation-specific MLPA

Nebulin (NEB) is the main causative gene for autosomal recessive nemaline myopathy (NM). There are no clear mutational hotspots in the 183 NEB exons, and mutations are rarely recurrent among the usually compound heterozygous patients. The development of practically applicable mutation analysis methods and the efficient identification of new NEB mutations is important for better understanding of any genotype–phenotype correlations. Denaturing high-performance liquid chromatography (dHPLC) has been efficient in identifying small, heterozygous mutations but in many cases analysed by this method, the second mutation remains unidentified.

ATM gene analysis in neuroblastoma: A report from the COG

10058 Background: Neuroblastoma (NB) is the most common malignant disease of infancy and accounts for approximately 8% of all childhood cancers. The clinical hallmark of this tumor is the marked variability in prognosis depending of the age, stage, and biological characteristics. There is evidence to suggest that the long arm of chromosome 11 (11q) plays a role in NB biology. The ATM gene is located at 11q22–23 and hereditary mutations of this gene cause ataxia-telangiectasia, a recessive disorder associated with a high incidence of neoplasia. The aim of this project was to determine the prevalence of ATM gene mutation and ATM methylation status in 50 NB samples. Methods: Following DNA extraction, PCR products of the 65 exons of the ATM gene and its promoter were screened by DHPLC. This screening was also performed on DNA from 60 blood donors. Alterations detected were analyzed by direct sequencing. Direct and indirect criteria were used to classify the observed nucleotide alterations as mutation (if pathogenic), rare variant (if the allelic frequency in controls was &lt; or = 1%), variant (1.1–2.4%) or polymorphism (&gt; or = 2.5%). The ATM methylation status was analyzed by MS-MLPA (Methylation-Specific Multiplex Ligation-dependent Probe Amplification). Results: Except polymorphisms, 17 different sequence alterations were found in 17 NB samples (34%). Ten of these 17 alterations, found in 11 NB (22%), were rare variants (RV). In 5 NB (10%), RV were found homozygous. At the same time, we found 20 different sequence alterations in 19 controls (32%). Sixteen of these 20 alterations were RV and one was a heterozygous pathogenic mutation. These 17 alterations concern 15 controls (25%). No homozygous RV was found in controls. We found no evidence of ATM promoter hypermethylation in the 48 NB samples analyzed. Conclusions: We found no difference in ATMvariant and RV frequency between NB and control samples. However, as ATM deletion is a frequent event in NB, we found a high frequency of homozygous RV (10%). At present, we are completing this study by screening ATMlarge genomic deletion/duplication using MLPA. Finally, our observations indicate that epigenetic ATM silencing by methylation is uncommon in neuroblastoma. No significant financial relationships to disclose.

DNA Hypermethylation Profiles in Squamous Cell Carcinoma of the Vulva

Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: tumor protein p73 (TP73), fragile histidine triad (FHIT), von Hippel-Lindau (VHL), adenomatosis polyposis coli (APC), estrogen receptor 1 (ESR1), cyclin-dependent kinase inhibitor 2B (CDKN2B), death-associated protein kinase 1 (DAPK1), glutathione S-transferase pi (GSTP1), and immunoglobin superfamily, member 4 (IGSF4). The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1, and FHIT in 3 of 13 cell lines. Methylation-specific polymerase chain reaction was performed for TP73 and FHIT to confirm aberrant methylation by methylation-specific multiplex ligation-dependent probe amplification. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by reverse transcription polymerase chain reaction confirmed aberrant methylation. Frequent genetic alterations of loss and gain of gene copy number included gain of GSTP1 and multiple endocrine neoplasia type 1 (MEN1), and loss of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) and IGSF4 in over 50% of the squamous cell carcinoma of the vulva cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

Methylation Profiles in Breast Cancer

In this study, we sought to assess the aberrant methylation of multiple tumor-suppressor genes in a single reaction by using methylation-specific multiplex ligation-dependent probe amplification. Breast tumors and corresponding normal tissues of 77 patients were analyzed. In this study, 17 of 24 genes displayed promoter methylation in one or more of the tumor samples. The most frequently methylated genes were RASSF1 and GSTP1, followed by DAPK1 and CDKN2B. Our data indicate that the methylation of specific genes is a frequent event in breast cancer and show that MS-MLPA is a powerful tool to analyze epigenetic alterations for diagnostic, as well as therapeutic, purposes.

Methylation-specific multiplex ligation-dependent probe amplification in diagnosis of Prader-Willi syndrome and Angelman syndrome

To verify the sensitivity and reliability of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and to develop a simple, accurate, reliability method of genetic diagnosis for AS and PWS. Peripheral blood samples were collected from 4 suspected AS patients, 2 suspected PWS patients, 2 normal persons, and 2 molecular biologically proven positive controls (1 AS patient and 1 PWS patient). DNA was extracted and purified. MS-MLPA was used to detect the methylation of the CpG dinucleotide and the copy number in the 15q-q13 region. The results of MS-MLPA were confirmed by MSP. Three cases with maternal deletion on 15q11-q13 region and one case with paternal uniparental disomy (UPD) or imprinting center defect in 15q11-q13 region were found in the 4 suspected AS patients. One PWS case was found to be with paternal deletion in 15q11-q13 region and the other with paternal deletion in 15q11-q13 region or UPD or imprinting center defect in 15q11-q13 region. MS-MLPA is a simple, rapid, accurate, and reliable method of genetic test.

Antitumor Activity of Cda-2, An Extract from Unine, in a High-Risk Myelodysplastic Syndrome Cells in Vitro

DNA methyltransferase inhibitors (DNMTI), including 5-azacytidine and 5-aza-2′- deoxycytidine, are a new class of epigenetic drug, which exhibit higher response rates in myelodysplastic syndrome (MDS) patients. Cell differentiation agent (CDA-2) is a kind of urine extracts, which contains several DNMTIs. A phase IV clinical trials for MDS showed total response rate is 69.22%. In the present study, we investigated the mechanism of CDA-2 on MDS using high-risk MDS cell line namely MuTz-1. MTT assay results showed that CDA-2 significantly inhibit the cell growth at a dose and time-dependent manner. Flow cytometer anlyasis showed that this growth inhibition was remarkblely associated with cycle arrest in G1-phase, but not associated with apoptosis. In addition, CDA-2 increased the expression of CD11b/CD14, a pair markers representing cell differentiation. we found the spectrum of hypermethylated tumor suppressor genes (TIMP3, CDKN2B, CHFR, CD44, RASSF1, TP73, IGSF4, CDH13 and DAPK) in MuTz-1 cells by Methylation-Specific Multiplex ligation-dependent Probe amplification (MS-MLPA), but the hypermethylation of these genes were remarkable decreased, as well as the expressions of DNA methyltransferase genes DNMT1 and DNMT3B at mRNA and protein level were downregulated in the treatment for 3 days with CDA-2. Also, we used CDA-2 for treatment of three MDS patients, whose several tumor suppressor genes are hypermethylated. After tour weeks of treatment, all the hypermethylation genes were undetected, part of this result was verified by methylation specific PCR (MSP) and bisulphite sequencing. In conclusion, our results demonstrated that CDA-2 may be an effective agent targeting hepermethylated tumor suppressor genes on MDS.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification Enables a Rapid and Reliable Distinction between Male FMR1 Premutation and Full-Mutation Alleles

Fragile X syndrome is the most common cause of inherited mental retardation and the second most common cause of mental impairment after trisomy 21. It occurs because of a failure to express the fragile X mental retardation protein. The most common molecular basis for the disease is the abnormal expansion of the number of CGG repeats in the fragile X mental retardation 1 gene (FMR1). Based on the number of repeats, it is possible to distinguish four types of alleles: normal (5 to 44 repeats), intermediate (45 to 54), premutation (55 to 200), and full mutation (>200). Today, the diagnosis of fragile X syndrome is performed through a combination of PCR to identify fewer than 100 repeats and of Southern blot analysis to identify longer alleles and the methylation status of the FMR1 promoter. We have developed a methylation-specific multiplex ligation-dependent probe amplification assay to analyze male fragile X syndrome cases with long repeat tracts that are not amplifiable by PCR. This inexpensive, rapid and robust technique provides not only a clear distinction between male pre- and full-mutation FMR1 alleles, but also permits the identification of genomic deletions, a less frequent cause of fragile X syndrome.

Methylation-specific multiplex ligation-dependent probe amplification in meningiomas

Genomic loss and promotor methylation contribute to inactivation of tumor suppressor genes (TSGs). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a relatively new method for simultaneous detection of both these alterations. Here, we apply MS-MLPA to a series of 15 meningiomas of different WHO grades. The two MS-MLPA probe sets used detect copy number changes in 55 unselected TSGs and promotor methylation in a subset of 36 of these genes. Our findings concerning genomic deletions are concordant with previously published studies using alternative techniques. The number of aberrations identified per tumor increased with histopathologically determined grading. The most frequent single event was deletion of the von Hippel-Lindau (VHL) gene in 12 of the 15 tumors. Moreover, VHL deletion status was associated with presence/absence of peritumoral edema. Methylation was rare, being observed in only four tumors and in each case restricted to a single gene. We conclude that a meningioma-specific MS-MLPA probe set would be a valuable tool for both research and diagnostic approaches in these tumors.