Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene

Dehainault, Catherine; Laugé, Anthony; Caux‐Moncoutier, Virginie; Pagès-Berhouet, Sabine; Doz, François; Desjardins, Laurence; Couturier, Jérôme; Gauthier‐Villars, Marion; Stoppa‐Lyonnet, Dominique; Houdayer, Claude

doi:10.1093/nar/gnh137

Cited by 54 publications

(22 citation statements)

References 34 publications

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“…In addition to undesired mismatches, another factor that could affect the interpretation of semiquantitative methods is the quality of DNA templates. As noted in previous studies with fluorescent semiquantitative assays such as MLPA, QMPSF, and IP-RP-HPLC [Nakagawa et al, 2003;Charbonnier et al, 2000;Dehainault et al, 2004], as well as in the present study with nonfluorescent NFMP-HPLC, the quantity and quality of gDNA templates should be carefully checked to ensure unambiguous results. In our experience, both with MLPA and NFMP-HPLC, the use of low quality gDNA templates may generate anomalous electropherograms and chromatograms (data not shown).…”

Section: Discussionmentioning

confidence: 64%

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

Lellis¹,

Curia²,

Catalano³

et al. 2006

Hum. Mutat.

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Communicated by Jing ChengLarge genomic rearrangements are recognized as playing a pathogenic role in an increasing number of human genetic diseases. It is important to develop efficient methods for the routine detection and confirmation of these germline defects. Multiplex ligation-dependent probe amplification (MLPA) is considered an early step for molecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPA analysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verified by two independent methods. In this study, we developed nonfluorescent multiplex-PCR coupled to highperformance liquid chromatography (NFMP-HPLC) and analyzed whether the use of this method combined with MLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements. A total of nine nonfluorescent multiplex-PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons. Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number of cycles (r25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The results obtained by NFMP-HPLC were highly reproducible. The combined use of MLPA and NFMP-HPLC identified and independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients with hereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and in three cases multiple contiguous exons. These results were in agreement with breakpoint and complementary DNA (cDNA) analyses. Considering that MLPA and NFMP-HPLC are unlikely to be affected by the same artifacts, their combined use could also provide a robust and cost-effective strategy for routine screening and confirmation of putative rearrangements in other genes, especially when a single exon is involved or a precise characterization of breakpoints is not achieved. Hum Mutat 27(10), [1047][1048][1049][1050][1051][1052][1053][1054][1055][1056] 2006.

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Section: Discussionmentioning

confidence: 64%

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

Lellis¹,

Curia²,

Catalano³

et al. 2006

Hum. Mutat.

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“…Lastly, various semi-quantitative multiplex fluorescent PCR protocols have been used to detect large rearrangements culminating in the so-called QMPSF (Casilli et al 2002). Also more recently, some authors described a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (Dehainault et al 2004). Large deletions or duplications can also be assessed by high resolution comparative genome hybridization microarrays methodology (Porchet et al 2004).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene

Crépin¹,

Pigny²,

Escande³

et al. 2006

Journal of Molecular Endocrinology

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The identification of mutations in the MEN1 gene causing MEN1 has represented a challenge since the cloning of the gene in 1997 because of the lack of mutation hot-spots in the gene and the lack of phenotype-genotype correlations. The use of denaturing high performance liquid chromatography (DHPLC), a high throughput, reliable and automated heteroduplex-based technique, is the ideal for mutation detection in MEN1. In this work, DHPLC was optimised for the screening of the nine coding exons and splice junctions of MEN1. Thanks to collaboration between two French laboratories recognised as reference centres for genotypic MEN1 diagnosis (Lyon and Lille), a blind retrospective study conducted in a cohort of 160 unrelated MEN1 probands with (or without) known germline mutations was undertaken to evaluate the sensitivity of DHPLC. We were able to detect 101 different sequence variations by DHPLC, distributed in the 10 analysed DNA fragments and corresponding to 100% of mutation detection compared with direct sequencing. 1·2% of samples were considered as false positive, exhibiting a heterogenous profile. DHPLC did not detect five cases of deletion or duplication of complete exons, neither did direct sequencing, showing the limits of the technique. Nevertheless, the method appeared to allow automated, rapid and low-cost mutation detection with high accuracy. Direct sequencing can be then applied to identify the sequence variations on the targeted DNA fragments showing heterozygous profile by DHPLC. In conclusion, genotypic diagnosis of MEN1 can benefit from DHPLC in terms of efficacy, rapidity and cost.

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“…We utilize one or two genomic references to determine the copy number of the gene to be examined: quantitation of the target gene is relative to reference genes of known copy number (Celi et al 1993;Dehainault et al 2004;Noonan et al 1990). A fragment from within a potentially duplicated or deleted target region is amplified simultaneously with a disomic reference region in an MP system.…”

Section: Discussionmentioning

confidence: 99%

“…All the above show that this method is very well suited to diagnosing a duplication causing CMT1A or a deletion causing HNPP. Dehainault et al (2004) described an application to detect gene rearrangements of the RB1 gene using MP and DHPLC. Their report used the same principles as MP/DHPLC to determine relative gene dosage.…”

Section: Discussionmentioning

confidence: 99%

“…In our study, we introduced a reliable method using DHPLC with non-fluorescent detection to quantify PMP22 gene copy number, which is more cost-effective with same resolution. Furthermore, Dehainault et al (2004) improved the amplification of high GC content DNA using a touchdown protocol. The principles of the touchdown protocol raise a doubt about equitable competition and the results may not be a very precise quantification of gene dosage.…”

Section: Discussionmentioning

confidence: 99%

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A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay

Lin

Lee

et al. 2006

J Hum Genet

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Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2-12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR-restriction fragment length polymorphism analysis. We concluded that the MP/ DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.

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Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene

Cited by 54 publications

References 34 publications

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene

A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay

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