Multiplex Ligation-dependent Probe Amplification Approach Research Articles

Promoter hypermethylation contributes to TIMP3 down‐regulation in high stage endometrioid endometrial carcinomas

Expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) has been found to be decreased in several types of cancer by promoter gene hypermethylation. However, little is known regarding the silencing effect of TIMP3 promoter hypermethylation on gene and protein expression in endometrial carcinomas and its prognostic significance. TIMP3 promoter hypermethylation and gene copy number variations were evaluated using a methylation-specific multiplex ligation-dependent probe amplification approach in 60 cases of endometrioid endometrial carcinomas. TIMP3 expression was also evaluated at the transcript and protein levels. Loss of TIMP-3 protein expression was found in 44 (73%) of 60 carcinomas. Promoter hypermethylation was identified in 25% (15 of 60); was more frequent in stages II-IV (55%, six of 11) than in stage I (18%, nine of 49; P=0.021); and was found more commonly in tumours with deep myometrial invasion. MLH1 and TIMP3 promoters were hypermethylated simultaneously in the same group of tumours (P<0.001). A correlation between TIMP3 methylation and microsatellite instability (MSI) was found (P=0.005). TIMP3 copy number changes were frequently a loss (35%), whereas a gain was detected in only 5%. TIMP3 promoter hypermethylation was associated with high stage endometrioid endometrial tumours with extrauterine spread. Nevertheless, promoter hypermethylation and loss of heterozygosity are not the only mechanisms for TIMP3 inactivation.

Custom-Designed MLPA Using Multiple Short Synthetic Probes: Application to Methylation Analysis of Five Promoter CpG Islands in Tumor and Urine Specimens from Patients with Bladder Cancer

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.

Apoptosis Gene Expression Profiling in Chronic Lymphocytic Leukemia Using Multiplex Ligation-Dependent Probe Amplification.

Abstract 3457Poster Board III-345One of the hallmarks of the clinically heterogeneous disease chronic lymphocytic leukemia (CLL) is defective apoptosis, which is considered to contribute not only to cell accumulation but also to disease progression and resistance to therapy. In an effort to further investigate the ‘apoptotic profile' in CLL, we have utilized the multiplex ligation-dependent probe amplification (MLPA) method to analyze the expression profile of 35 apoptosis-related genes in 67 CLL cases including 49 peripheral blood, 9 lymph node, 7 bone marrow and 2 spleen samples. CLL cells derived from peripheral blood displayed a different overall expression profile when compared to peripheral blood mononuclear cells (PBMC) from five healthy controls, with several genes including the anti-apoptotic genes BCL2A1 and MCL1 and the pro-apoptotic genes BAX and BMF all being significantly differently expressed (>2 fold difference in median expression). Few differences were detected when comparing peripheral blood CLL samples (25/49 IGHV unmutated/IGHV3-21) to lymph node samples (6/9 unmutated/IGHV3-21) or to bone marrow samples (5/7 unmutated/IGHV3-21), with lower relative expression of BIK and BAX in peripheral blood samples. Furthermore, we correlated the expression profiles of all genes included in the assay with IGHV mutation status, IGHV3-21 gene usage and genomic aberrations, but overall few differences were evident between prognostically different subgroups. An exception to this was the BIK gene which showed significantly higher expression in unmutated/IGHV3-21 compared to mutated CLL. Additionally, MCL1 and BNIP3L displayed higher expression in 17p-deleted cases compared to 13q deletion/no aberration cases. High expression of HRK and BAX indicated a correlation with poor overall survival and shorter time to treatment. However, when performing real-time quantitative PCR on an extended patient cohort (n=112) only prediction of time to treatment could be confirmed for HRK and BAX (p=0.0035 and p=0.024, respectively). Finally, in order to validate our MLPA approach, we compared the gene expression levels of four genes in 19 CLL samples using both MLPA and real-time quantitative PCR, which revealed a significant correlation for all genes tested. In summary, MLPA is a quick and cost-effective method for pathway specific gene expression analysis and the present analysis of 67 CLL cases highlights the rather homogenous apoptosis gene expression profiles among different clinical subsets of the disease. DisclosuresNo relevant conflicts of interest to declare.

Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach

BackgroundMapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.ResultsWe have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 × 104 cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.ConclusionThis new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 105 cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

Molecular screening test in familial forms of cerebral cavernous malformation: the impact of the Multiplex Ligation-dependent Probe Amplification approach

Object The purpose of this study was to underline the effectiveness of molecular analysis in cerebral cavernous angioma, with special attention to the familial forms. Methods Multiplex Ligation-dependent Probe Amplification analysis integrates the consecutive sequence analysis of the 3 genes (Krit1/CCM1, MGC4607/CCM2, and PDCD10/CCM3) known to be responsible for cerebral cavernous malformation lesions. Results The Multiplex Ligation-dependent Probe Amplification analysis revealed a new mutation, a heterozygous exon 9/10 deletion of Krit1, in the proband and in all affected family members. Conclusions The identification of the molecular defect allows physicians to screen family members at risk and to identify affected individuals before the onset of clinical symptoms caused by the presence of lesions.

NF2 Mutation Screening by Denaturing High-Performance Liquid Chromatography and High-Resolution Melting Analysis

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder caused by mutations in the NF2 gene and predisposing to the development of nervous system. Identification of germline mutations is essential to provide appropriate genetic counseling in NF2 patients, but it represents an extremely challenging task because the vast majority of mutations are unique and spread over the entire coding sequence. Moreover, about 30% of de novo patients are indeed mosaic, and direct sequencing can undetect mutated alleles present in a minority of cells. As most screening techniques do not meet the requirements for efficient NF2 testing, we have developed a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a multiplex ligation-dependent probe amplification approach to screen for gene rearrangements. In addition, we have evaluated high-resolution melting analysis (HRMA) as an exon scanning procedure to identify point mutations in the NF2 gene. The results obtained in 92 NF2 patients expand the NF2 mutational spectrum and indicate DHPLC and HRMA as good systems to screen for point mutations in diseases with a heterogeneous spectrum of alterations.

High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability

Mutation screening of the BRCA1 and BRCA2 genes in probands with familial breast/ovarian cancer has been greatly improved by the multiplex ligation-dependent probe amplification (MLPA) assay able to evidence gene rearrangements not detectable by standard screening methods. However, no criteria for selection of cases to be submitted to the MLPA test have been reported yet. We used the BRCAPro software for the selection of familial breast/ovarian cancer probands investigated with the MLPA approach after negative BRCA1/2 conventional mutation screening. One hundred and seventy-seven probands were investigated for germline BRCA1/2 mutations after assessment of genetic risk using BRCAPro. Probands were classified as BRCAPro positive (n = 67) when the carrier probability (CP) was >10% and as BRCAPro negative (n = 110), when the CP was <10%. Conventional mutational analyses of the BRCA1/2 genes and, in one case, of p53 identified 22 pathogenetic germline mutations, 12 in BRCA1, 9 in BRCA2 and 1 in p53, in 22/177 (12.4%) probands. All the mutations except one were detected in BRCAPro-positive patients. In the 46 BRCAPro-positive cases that resulted negative by BRCA1/2 mutation, screening analysis of rearrangements within BRCA1/2 by MLPA was carried out. Three patients with a very high CP showed BRCA1 deletions, consisting of deletions of exons 1–2 in two probands and of exon 24 in the third proband. In one case, the exons 1–2 deletion was shown to cosegregate with disease in the family. No BRCA2 rearrangements were detected, but one patient showed the 1100delC of the CHEK2 gene, whose probe is present in the BRCA2 kit. In our series, the highest carrier detection rate of mutation screening plus MLPA analysis (52.3%) was in patients with a BRCAPro CP >50%.

Strategies for the rapid prenatal diagnosis of chromosome aneuploidy.

Rapid diagnosis of common chromosome aneuploidies in raised risk pregnancies, usually prior to full karyotype analysis, is now carried out in a number of European genetic centres; several techniques for detecting genomic copy number changes have been described. Prenatal diagnosis of genetic disease requires accurate and robust assays; the invasive procedures are associated with a risk of pregnancy loss and an abnormal result may lead to termination of the pregnancy. The testing of prenatal material (amniotic fluid, chorionic villi or, more rarely, fetal blood) is associated with specific problems, including the quality and quantity of the tissue and difficulties of interpretation due to phenomena such as maternal cell contamination and mosaicism. In addition, there are 24-h, high-throughput demands on centres offering such a service. The extent to which existing and proposed strategies, including different PCR-based assays, a multiplex ligation-dependent probe amplification approach, and microarrays, fulfil the requirements of rapid prenatal testing is discussed. In the past 3 years, we have tested 7720 prenatal samples for trisomies 13, 18 and 21 using a quantitative fluorescence-PCR (QF-PCR) approach. The abnormality rate was 5.7%. There were no misdiagnoses for nonmosaic trisomy, the amplification failure rate was 0.09% of samples, and 97% of samples received a report on the working day following sample receipt. Maternal cell contamination and mosaicism were also detected. Our data recommend a QF-PCR approach as the current method of choice for rapid aneuploidy testing.

Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach.

Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer.British Journal of Cancer (2002) 87, 892–897. doi:10.1038/sj.bjc.6600565 www.bjcancer.com© 2002 Cancer Research UK