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. 2012;7(12):e51024.
doi: 10.1371/journal.pone.0051024. Epub 2012 Dec 7.

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum

Affiliations

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum

Hada C Macher et al. PLoS One. 2012.

Abstract

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genotyping of MEN2A in serum circulating DNA.
HRM analysis of 25 MEN2A patients and 5 healthy controls (red) represented as the normalized and temperature-shifted curves converted to fluorescence different plot for analysis by the LC 480 software. HRM analysis reveals differences in melting curve shape that correlates with patients diagnosed as MEN2A vs all controls.
Figure 2
Figure 2. Comparison of COLD PCR and conventional HRM for non-invasive prenatal diagnosis of MEN2A.
Conventional HRM (A) and Fast COLD PCR (B) analysis of one pregnant woman with a fetus carrying the MEN2A mutation, analyzed together with negative controls and serial dilutions of positive controls. Mutation-containing samples differ appreciably from wild-type melting curves. LC 480 software differences the pregnant woman carrying the MEN2A fetus from the healthy controls after conventional HRM (A, red) and COLD PCR (B, green) analysis.
Figure 3
Figure 3. Comparison of conventional HRM and COLD PCR sequences of PCR products (G>A mutation).
Sequence analysis of both HRM and COLD PCR products from a positive MEN2A with C634Y heterozygous mutation control (A), a negative healthy control (B), and the pregnant woman carrying a fetus with the C634Y mutation (C).

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