Concordant Inactivated X-Chromosome Results from Early Monoclonal Expansions of Neural Crest Precursors in MEN-2A

<p>Background: The histologic definition of precursor lesions in MEN is controversial for both C-Cell Hyperplasia (CCH) and Adrenal Medullary Hyperplasia (AMH). They are multifocal and the clonality pattern of each focus remains unknown.<br>Methods: We found 10 females with bilateral CCH and another 11 with AMH in a MEN-2A kindred known to carry RET point mutation in codon 634. DNA was extracted from microdissected samples of CCH from each thyroid lobe and from 34 AMH nodules (2 nodules in 3 patients, 3 nodules in 4, and 4 nodules in 4). The samples were then used to study the methylation pattern of X-chromosome inactivation (HUMARA test using Hha-I digested and undigested samples). Appropriate and multiple tissue controls were run in every case. The inactivation pattern of X-chromosome of each sample was compared for any given patient, including only informative cases (2 alleles in both undigested and digested control samples) in the final analysis.<br>Results: Nine CCH patients were informative, 8 of them revealing monoclonal pattern with the same androgen receptor allele preferentially methylated in both lobes. Twenty-seven of 30 AMH nodules from 9 informative patients also revealed monoclonal unbalanced methylation of androgen receptor alleles and the same X-chromosome inactivated in nodules from a given patient. The remaining 2 CCH foci (1 patient) and 3 AMH nodules (2 patients) revealed balanced methylation of androgen receptor alleles. Under the hypothesis of independent allele methylation<br>in different lesions from the same patient, the combined probability of randomly finding the observed monoclonal results was less than 0.0001 for CCH and less than 0.000000001 for AMH.<br>Conclusions: MEN-2A-related CCH and nodular AMH are mainly monoclonal, and show the same X-chromosome inactivated in both thyroid lobes and in adrenal nodules from a given patient. This suggests a multifocal origin for both conditions related to early clonal expansions of neural crest precursors and may represent a paradigm for other germline mutations during embryogenesis.</p>