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.
References
[1]
Moline J, Eng C (2011) Multiple endocrine neoplasia type 2: an overview. Genet Med 13: 755–764.
[2]
Akerstrom G, Stalberg P (2009) Surgical management of MEN-1 and -2: state of the art. Surg Clin N Am 89: 1047–1068.
[3]
Quayle FJ, Fialkowski EA, Benveniste R, Moley JF (2007) Pheochromocytoma penetrance varies by RET mutation in MEN 2A. Surgery 142: 800–805.
[4]
Raue F, Kraimps JL, Dralle H, Cougard P, Proye C, et al. (1995) Primary hyperparathyroidism in multiple endocrine neoplasia type 2A. J Int Med 238: 369–373.
[5]
Marini F, Falchetti A, DelMonte F, Carbonell-Sala S, Tognarini I, et al. (2006) Multiple endocrine neoplasia type 2. Orphanet J Rare Dis 1: 45.
[6]
Gagel RF, Melvin KE, Tashjian AH Jr, Miller HH, Feldman ZT, et al. (1975) Natural history of the familial medullary thyroid carcinoma-pheochromocytoma syndrome and the identification of preneoplastic stages by screening studies: a five-year report. Trans Assoc Am Physicians 88: 177–191.
[7]
Punales MK, Graf H, Gross JL, Maia AL (2003) RET codon 634 mutations in multiple endocrine neoplasia type 2: variable clinical features and clinical outcome. J Clin Endocrinol Metab 88: 2644–2649.
[8]
Norbury G, Norbury CJ (2008) Non-invasive prenatal diagnosis of single gene disorders: How close are we? Semin Fetal Neonatal Med 13: 76–83.
[9]
Pinzani P, Salvianti F, Pazzagli M, Orlando C (2010) Circulating nucleic acids in cancer and pregnancy. Methods 50: 302–307.
[10]
Gormally E, Caboux E, Vineis P, Hainaut P (2007) Circulating free DNA in plasma or serum as biomarker of carcinogenesis: Practical aspects and biological significance. Mutat Res 635: 105–117.
[11]
Stroun M, Lyautey J, Lederrey C, Olson-Sand A, Anker P (2001) About the possible origin and mechanism of circulating DNA: Apoptosis and active DNA release. Clin Chim Acta 313: 139–142.
[12]
García-Olmo DC, Ruiz-Piqueras R, García-Olmo D (2004) Circulating nucleic acids in plasma and serum (CNAPS) and its relation to stem cells and cancer metastasis: State of the issue. Histol Histopathol 19: 575–583.
[13]
García-Olmo D, García-Olmo DC (2001) Functionality of circulating DNA: The hypothesis of genometastasis. Ann NY Acad Sci 945: 265–275.
[14]
Schwarzenbach H, Alix-Panabières C, Müller I, Letang N, Vendrell JP, et al. (2009) Cell-free tumor DNA in blood plasma as a marker for circulating tumor cells in prostate cancer. Clin Cancer Res 153: 1032–1038.
[15]
Chiu RWK, Lo YMD (2011) Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age. Semin Fetal Neonatal Med 16: 88–93.
[16]
Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, et al. (1998) Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis. Am J Hum Genet 62: 768–775.
[17]
Lun FMF, Chiu RWK, Chan KCA, Leung TK, Lau TK, et al. (2008) Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma. Clin Chem 54: 1664–1672.
[18]
Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, et al. (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64: 218–224.
[19]
Milbury CA, Li J, Makrigiorgos GM (2009) COLD-PCR-Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations. Clin Chem 55: 2130–2143.
[20]
Li J, Wang L, Mamon H, Kulke MH, Berbeco R, et al. (2008) Replacing PCR with COLDPCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14: 579–584.
[21]
Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, et al. (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61: 1659–1665.
[22]
Zhong XY, von Muhlenen I, Li Y, Kang A, Gupta AK, et al. (2007) Increased concentrations of antibody-bound circulatory cell-free DNA in rheumatoid arthritis. Clin Chem 53: 1609–1614.
[23]
Rainer TH, Wong LK, Lam W, Yuen E, Lam NY, et al. (2003) Prognostic use of circulating plasma nucleic acid concentrations in patients with acute stroke. Clin Chem 49: 562–569.
[24]
Antonatos D, Patsilinakos S, Spanodimos S, Korkonikitas P, Tsigas D (2006) Cell-free DNA levels as a prognostic marker in acute myocardial infarction. Ann N Y Acad Sci 1075: 278–281.
[25]
Macher HC, Egea-Guerrero JJ, Revuelto-Rey J, Gordillo-Escobar E, Enamorado-Enamorado J, et al. (2012) Role of early cell-free DNA levels decrease as a predictive marker of fatal outcome after severe traumatic brain injury. Clin Chim Acta 414: 12–17.
[26]
Garc？a-Moreira V, Prieto-Garc？a B, Baltar-Mart？n JM, Ortega-Suarez F, Alvarez FV (2009) Cell-free DNA as a noninvasive acute rejection marker in renal transplantation. Clin Chem 55: 1958–1966.
[27]
Garc？a-Moreira V, de la Cera-Mart？nez T, Gago-Gonzalez E, Prieto-Garc？a B, Alvarez-Menendez FV (2008) Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR. Clin Chem Lab Med 44: 1410–1415.
[28]
Saukkonen K, Lakkisto P, Pettila V, Varpula M, Karlsson S, et al. (2008) Cell-free plasma DNA as a predictor of outcome in severe sepsis and septic shock. Clin Chem 54: 1000–1007.
[29]
Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, et al. (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350: 485–487.
[30]
Illanes S, Denbow M, Kailasam C, Finning K, Soothill PW (2007) Early detection of cell-free fetal DNA in maternal plasma. Early Hum Dev 83: 563–566.
[31]
Macher HC, Noguerol P, Medrano-Campillo P, Garrido-Márquez MR, Rubio-Calvo A, et al. (2012) Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: Results in clinical benefits and cost saving. Clin Chim Acta 413: 490–494.
[32]
Finning K, Martin P, Summers J, Massey E, Poole G, et al. (2008) Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study. Br Med J 336: 816–818.
[33]
Minon JM, Gerard C, Senterre JM, Schaaps JP, Foidart JM (2008) Routine fetal RHD genotyping with maternal plasma: a four-year experience in Belgium. Transfusion 48: 373–381.
[34]
Lo YMD, Lun FMF, Chan KCA, Tsui NBY, Chong KC, et al. (2007) From the cover: digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci USA 104: 13116–13121.
[35]
Wright CF, Burton H (2009) The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis. Human Reprod Update 15: 139–151.
[36]
Margraf RL, Mao R, Highsmith WE, Holtegaard LM, Wittwer CT (2006) Mutation scanning of the RET protooncogene using High-Resolution Melting analysis. Clin Chem 52: 138–141.
[37]
Reed GH, Wittwer CT (2004) Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 50: 1748–1754.
[38]
Loeb LA, Loeb KR, Anderson JP (2003) Multiple mutations and cancer. Proc Nat Acad Sci USA 100: 776–781.
[39]
Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, et al. (2005) Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diag 7: 413–421.
[40]
Milbury CA, Li J, Liu P, Makrigiorgos GM (2011) COLD-PCR: Improving the sensitivity of molecular diagnostics assays. Expert Rev Mol Diagn 11: 159–169.
