Simple detection of genomic microdeletions and microduplications using QMPSF in patients with idiopathic mental retardation

Department of Genetics, Rouen University Hospital, Rouen, France.
European Journal of HumanGenetics (Impact Factor: 4.35). 10/2006; 14(9):1009-17. DOI: 10.1038/sj.ejhg.5201661
Source: PubMed


In contrast to the numerous well-known microdeletion syndromes, only a few microduplications have been described, and this discrepancy may be due in part to methodological bias. In order to facilitate the detection of genomic microdeletions and microduplications, we developed a new assay based on QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) able to explore simultaneously 12 candidate loci involved in mental retardation (MR) and known to be the target of genomic rearrangements. We first screened 153 patients with MR and facial dysmorphism associated with malformations, or growth anomalies, or familial history, with cytogenetically normal chromosomes, and the absence of FRAXA mutation and subtelomeric rearrangements. In this series, we found a 5q35 deletion removing the NSD1 gene in a patient with severe epilepsy, profound MR and, retrospectively, craniofacial features of Sotos syndrome. In a second series, we screened 140 patients with MR and behaviour disturbance who did not fulfil the de Vries criteria for subtelomeric rearrangements and who had a normal karyotype and no detectable FRAXA mutation. We detected a 22q11 deletion in a patient with moderate MR, obesity, and facial dysmorphism and a 4 Mb 17p11 duplication in a patient with moderate MR, behaviour disturbance, strabismus, and aspecific facial features. This new QMPSF assay can be gradually upgraded to include additional loci involved in newly recognised microduplication/microdeletion syndromes, and should facilitate wide screenings of patients with idiopathic MR and provide better estimates of the microduplication frequency in the MR population.

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    • "This step was performed to ensure that the primers amplified only the genomic region of interest and also to guarantee that the forward and reverse primers were free of single nucleotide polymorphisms (SNPs). The HMBS marker (hydroxymethylbilane synthase) was chosen as reference gene, and the primer sequences used for its amplification were derived from Saugier-Veber et al. [22], without the addition of the universal extension cited by the authors. The primer sequences and the sizes of the amplicons are shown in Table 1. "
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    ABSTRACT: Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.
    Disease markers 04/2014; 2014. DOI:10.1155/2014/836082 · 1.56 Impact Factor
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    • "In the present study, we showed that copy numbers measured by mrcPCR are highly consistent with the standard or the most sensitive method currently available. In addition, mrcPCR requires as little as 10 ng DNA and about three hours of assay time, so it could be competitive with other currently available molecular methods for measuring copy numbers, including multiplex-ligation-dependent probe amplification (MLPA) [34], multiplex amplification and probe hybridization (MAPH) [35], and quantitative multiplex PCR of short fluorescent fragment (QMPSF) [36]. Furthermore, mrcPCR promises to be an invaluable method for measurement of copy-number variation in genes with variants of similar structures, including FCGR3 [23], CCL3L1 [5], and C4 [37], owing to the facts that, unlike the PRT method, extra-methodology such as REDVR is not necessary [7], [23], and that more versatile application to many genes without dispersed repeat sequences similar to the target gene is possible. "
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    ABSTRACT: Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.
    PLoS ONE 07/2013; 8(7):e69414. DOI:10.1371/journal.pone.0069414 · 3.23 Impact Factor
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    • "Features reported in the literature include: significant delay in motor and mental development, hypotonia, large ears/low set ears, hyperextensible joints, cutaneous syndactyly, short neck, failure to thrive, epicanthal folds, hypertelorism, flat nasal bridge, club foot, hip dysplasia, cardiac anomalies, humoral immunodeficiency and gastroschisis. Saugier-Veber et al. [24] detected a 22q11 deletion in a patient with moderate MR, obesity, and facial dysmorphism. A significant delay in motor and mental development was observed by almost all. "
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    ABSTRACT: Thanatophoric dysplasia (TD) is the most lethal and most severe type of dysplasia. It has distinct features, the most important of which is short tubular bones and short ribs with platyspondyly, allowing a precise radiologic and prenatal ultrasonographic diagnosis. It has been reported to be caused by mutations in the FGFR3 gene, but exactly how cytogenetic abnormalities might lead to TD is unclear. We report a case of TD with different prenatal sonographic features compatible with the classification of type I. In the result of cytogenetic examination, we found de novo CAs in 28% of cells analyzed from the affected infant; 75% of the abnormalities were numerical, and of those, 25% were structural aberrations; 21% of cells revealed predominantly numerical aberrations. Monosomy 18, 21 and 22 was observed in 4% of cells, monosomy 20 in 2%, and monosomy 7, 8, 14, 17 and 19 in 1%. Structural changes were observed in 7% of cells. It appears that these chromosomes may be preferentially involved in and important for TD development.
    American Journal of Case Reports 06/2012; 13:109-13. DOI:10.12659/AJCR.883026
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