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Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders.

ABSTRACT: Mutations in the PAX6 gene have been implicated in aniridia, a congenital malformation of the eye with severe hypoplasia of the iris. However, not all aniridia cases can be explained by mutations in the PAX6 gene. The purpose of this study was to enhance the molecular diagnosis of aniridia using multiplex ligation-dependent probe amplification (MLPA). Total genomic DNA was isolated from peripheral blood of 70 unrelated probands affected with aniridia. Polymerase chain reaction (PCR) was performed followed by automated bidirectional sequencing. Additionally, MLPA was performed. We identified 24 different point mutations in the PAX6 gene in 34 patients after sequencing. In eight additional patients, we identified a deletion of one or more exons of the PAX6 gene or in the 3' regulatory region of the PAX6 gene using MLPA. This work demonstrates the necessity to screen for larger deletions in the region of the PAX6 gene in addition to the sequencing of exons in the PAX6 gene. The mutation detection rate will increase from 49% to 60%. This shows that MLPA substantially enhances the molecular diagnosis of aniridia.

SUBMITTER: Redeker EJ

PROVIDER: S-EPMC2375324 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders.

Redeker Egbert J W EJ de Visser Annette S H AS Bergen Arthur A B AA Mannens Marcel M A M MM

Molecular vision 20080507

Mutations in the PAX6 gene have been implicated in aniridia, a congenital malformation of the eye with severe hypoplasia of the iris. However, not all aniridia cases can be explained by mutations in the PAX6 gene. The purpose of this study was to enhance the molecular diagnosis of aniridia using multiplex ligation-dependent probe amplification (MLPA). Total genomic DNA was isolated from peripheral blood of 70 unrelated probands affected with aniridia. Polymerase chain reaction (PCR) was performe ...[more]

PMID: 18483559

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Project description:Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like(aCGH) profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1(aCGH) profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like(aCGH) profile.The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-like(aCGH) breast cancers and 45 non-BRCA1-like(aCGH) breast cancers, which had previously been analyzed by aCGH. The BRCA1-like(aCGH) group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-like(aCGH) breast cancers and 32 non-BRCA1-like(aCGH) breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours.BRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay.Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues.

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Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders.

Publications

Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders.

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