Confirmation of pathogenicity of the MEN1 missense mutations by analysis of protein instability and aberrant splicing
Y. Nagamura1, M. Yamazaki2, S. Shimazu1, K. Sano2, T. Tsukada1 & A. Sakurai2
Multiple endocrine neoplasia type 1 (MEN1) is a relatively rare autosomal dominantly inherited condition characterized by hyperplastic and neoplastic disorders of endocrine organs such as the parathyroid, anterior pituitary and gastroenteropancreatic endocrine tissues. Germline mutation of the causative gene, MEN1, which localizes to human chromosome 11q13 and encodes the 610 amino-acid nuclear protein menin, can be identified in most affected subjects. MEN1 gene mutation analysis is a powerful tool for the early diagnosis of MEN1, but the clinical significance of MEN1 gene mutations is not always obvious. In the case of frameshift mutations, nonsense mutations or large deletions, it is relatively straightforward to consider those lesions as pathogenic. However, when identified mutations are missense mutation or in-frame deletions, molecular diagnosis of MEN1 is not so simple, since the pathogenicity of these mutations is not clear per se. We recently identified previously unreported missense MEN1 gene mutations, c.824G>T and c.1118C>T, from patients with primary hyperparathyroidism, and evaluated for their pathogenicity.
These mutations were predicted to generate putative missense menin proteins, R275M and P373L, respectively. A stability test using a quantitative fluorescent immunohistochemical method (Yaguchi 2004, Shimazu 2011) demonstrated that the P373L mutant is highly unstable, suggesting that this mutation is likely causative for typical MEN1, whereas the stability of the R275M mutant was reduced only slightly. Given that the c.824G>T mutation occurred at an exon-intron junction, this mutation was suspected to act as a splicing mutation rather than a simple missense mutation. Analysis of leukocyte mRNA and minigene experiments indicated that the mutant c.824G>T allele gives rise to abnormally spliced menin mRNA.
We conclude that both mutations are likely causative for typical MEN1. The menin stability test and leukocyte mRNA analysis may be valuable for the initial molecular diagnosis and subsequent management of patients with germline MEN1 mutations, especially in the case of putative missense mutations of ambiguous pathogenicity.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details unavailable.