Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases

Stuppia, Liborio; Antonucci, Ivana; Palka, Giandomenico; Gatta, Valentina

doi:10.3390/ijms13033245

Cited by 198 publications

(134 citation statements)

References 163 publications

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“…The MLPA technique is a powerful tool for evaluating genetic deletions that cannot be detected using PCRbased sequencing techniques. The MLPA assay has been adapted to detect aberrant methylation (19). Methylation-sensitive (MS)-MLPA is a modification of the MLPA assay with a methylation-sensitive restriction endonuclease that simultaneously detects the methylation state of CpG islands.…”

Section: Introductionmentioning

confidence: 99%

Genetic and epigenetic states of the GNAS complex in pseudohypoparathyroidism type Ib using methylation-specific multiplex ligation-dependent probe amplification assay

Yuno

Usui

Yambe

et al. 2013

European Journal of Endocrinology

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Context: Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare disorder resulting from genetic and epigenetic aberrations in the GNAS complex. PHP-Ib, usually defined by renal resistance to parathyroid hormone, is due to a maternal loss of GNAS exon A/B methylation and leads to decreased expression of the stimulatory G protein a (Gsa) in specific tissues. Objective: To clarify the usefulness of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), we evaluated genetic and epigenetic changes of the GNAS locus in Japanese PHP-Ib patients. Design: Retrospective case series. Patients: We studied 13 subjects with PHP-Ib (three families with eight affected members and one unaffected member and four sporadic cases). Measurements: The methylation status of GNAS differentially methylated regions (DMRs) was evaluated using MS-MLPA. The main outcome measure was the presence of deletion mutations in the GNAS locus and STX16, which were assessed using MLPA. Results: In all familial PHP-Ib cases, a w3 kb deletion of STX16 and demethylation of the A/B domain were identified. In contrast, no deletion was detected throughout the entire GNAS locus region in the sporadic cases. Broad methylation abnormalities were observed in the GNAS DMRs. Conclusions: MS-MLPA allows for precise and rapid analysis of the methylation status in GNAS DMRs as well as the detection of microdeletion mutations in PHP-Ib. Results confirm the previous findings in this disorder and demonstrate that this method is valuable for the genetic evaluation and visualizing the methylation status. The MS-MLPA assay is a useful tool that may facilitate making the molecular diagnosis of PHP-Ib.

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Section: Introductionmentioning

confidence: 99%

Genetic and epigenetic states of the GNAS complex in pseudohypoparathyroidism type Ib using methylation-specific multiplex ligation-dependent probe amplification assay

Yuno

Usui

Yambe

et al. 2013

European Journal of Endocrinology

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“…Thus, MLPA was efficient in accurately confirming mutations in about 67% of all the cases. A number of studies have found MLPA assay to be highly sensitive diagnostic assay to identify mutation in DMD and BMD patients [9]. In the study to evaluate the efficacy of MLPA technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene by Lai et al, MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR [10].…”

Section: Discussionmentioning

confidence: 99%

Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy

Shrestha

Khatiwada³

et al. 2016

Nepal J Biotechnol

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Duchenne muscular dystrophy (DMD) is X-linked recessive neuromuscular disorders caused due to mutation in dystrophin gene, leading to progressive muscle weakness. This study was done to identify mutation in dystrophin gene in Nepalese patients with DMD using Multiplex Ligation Dependent Probe Amplification (MLPA) assay in Nepal. Twenty one patients from different regions of Nepal, who were clinically diagnosed as DMD were enrolled in the study. Peripheral blood samples were collected in EDTA vials, gDNA was extracted, and deletion mutation in the dystrophin gene was analysed using Multiplex Ligation Dependent Probe Amplification (MLPA) assay. Exon deletion mutation in the dystrophin gene was observed in 14 (66.6%) out of 21 DMD cases. The most common exon deletion was observed and confined in exon 7-14 and 45-53 of dystrophin gene. The location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions. Use of MLPA is useful in detecting copy number changes in DMD proband and suspected carriers in Nepal.

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“…The utility of MLPA assay in the testing of acute leukemias and myelodysplastic syndromes has been analyzed, confirming excellent accuracy and specificity of MLPA as compared to FISH (ref. 39 ). The SALSA MLPA MDS kits (P144 and P145, MRC Holland) are used to detect chromosomal abnormalities commonly associated with MDS.…”

Section: Multiplex Ligation-dependent Probe Amplification (Mlpa)mentioning

confidence: 99%

Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review

Lukackova¹,

Bujalkova²,

Majerova³

et al. 2014

BIOMED PAP

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Background. Myelodysplastic syndromes (MDS) represent a heterogeneous group of premalignant hematologic disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of progression to acute leukemia. Cytogenetic analysis still plays a central role in the diagnosis of MDS, as clonal chromosomal abnormalities are observed in 30-50% of MDS patients. Despite their technical limitations, standard karyotyping and fluorescence in situ hybridization (FISH) are routinely used for identifying recurrent chromosomal rearrangements. However, using this approach means that submicroscopic and not targeted chromosomal aberrations, as well as somatic mutations and epigenetic changes remain largely undetected. Methods and Results. Introduction of methods for the analysis of copy-number variations (CNV), including arraybased technologies and Multiplex ligation-dependent probe amplification (MLPA) has provided novel insights into the molecular pathogenesis of MDS and considerably extended possibilities for genetic laboratory testing. Several novel molecular markers have been discovered and used for diagnosis and prognostic evaluation of patients with MDS. At present, mutational analysis is not routinely performed, as the clinical significance of somatic mutations in MDS has only begun to emerge. However, recently introduced Next-generation sequencing (NGS) technologies could help to elucidate the relationship between chromosomal and molecular aberrations in MDS and lead to further improvement in its diagnosis. Conclusion. This review focuses on the advantages, limitations, clinical applications and future perspectives of three molecular methods (array-based analysis, MLPA and NGS) currently used in genetic testing and/ or translational research of MDS. In conclusion, a brief summary for clinicians from the routine diagnostic point of view is given.

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Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases

Cited by 198 publications

References 163 publications

Genetic and epigenetic states of the GNAS complex in pseudohypoparathyroidism type Ib using methylation-specific multiplex ligation-dependent probe amplification assay

Genetic and epigenetic states of the GNAS complex in pseudohypoparathyroidism type Ib using methylation-specific multiplex ligation-dependent probe amplification assay

Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy

Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review

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