Distribution of RET Mutations in Multiple Endocrine Neoplasia 2 in Denmark 1994–2014: A Nationwide Study

Patients

This retrospective cohort study included 1583 unique patients who underwent RET gene testing in one of three centers covering all of Denmark between September 1994 and December 2014. At the end of the study period on January 1, 2015, the population of Denmark was 5,659,715 (www.dst.dk).

RET testing was performed in 1056, 459, and 104 patients at (i) the Center for Genomic Medicine, Copenhagen University Hospital, (ii) the Department of Molecular Medicine, Aarhus University Hospital, and (iii) the Department of Endocrinology and Medicine, Aalborg University Hospital, respectively. If tested in more than one center, patients were considered only once. One patient was tested in all three centers, while 34 patients were tested in two centers. This yielded a total of 1583 unique patients of which 164 were positive and 1419 negative for a RET mutation (Fig. 1).

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FIG. 1.

Flow chart showing patients and families tested for REarranged during Transfection (RET) mutations in Denmark 1994–2014. The Center for Genomic Medicine, Department of Molecular Medicine, and Department of Endocrinology and Medicine are part of Copenhagen University Hospital, Aarhus University Hospital, and Aalborg University Hospital, respectively.

RET testing was performed on genomic DNA extracted from whole blood in 1581 patients. One patient harbored the p.C634Y + p.Y791F tandem germline mutation. The ARUP database classifies the p.Y791F sequence change as uncertain (www.arup.utah.edu/database/) (30). However, because recent evidence has classified the sequence change as neutral, the patient was counted as a p.C634Y mutation alone (31,32).

Two patients with MTC were tested using formalin-fixed paraffin-embedded tissue. Both patients were deceased at the time of testing. One was tested using normal tissue, and one using submandibular tissue suspected of MTC metastasis. The latter proved positive for the L790F mutation, but due to suspicion of a somatic mutation in the local metastasis, the patient was excluded from our RET germline mutation cohort (Fig. 1). The patient has been described elsewhere (33).

A MEN2 family was defined by (i) clinical workup in one of the four MEN2 management centers in Denmark, (ii) a unique mutation, or (iii) a molecular proven de novo mutation (both parents of the index case tested mutation negative).

Indications for testing included MTC, PHEO, HPTH, HD, and relatives at risk of MEN2, among others.

RET testing centers

Approvals

The investigation was approved by the Danish Health Authority (3-3013-395/2) and the Danish Data Protection Agency (13/19275).

Limitations

To estimate the true number of RET mutations in a country, the entire population needs to be tested. However, this would result in both immense socioeconomic and ethical challenges. For the best possible estimate under the given circumstances, this study included all RET tested patients in Denmark since testing became available and for more than two decades onwards.

To capture yet unrecognized MEN2 patients, the Danish MEN taskforce has recommended routine RET screening in all patients with MTC, PHEO, C-cell or parathyroid hyperplasia, familial/recurrent HPTH, and HPTH <40–50 years of age (36,37). Unfortunately, the completeness of RET screening in these patient groups is unknown and will require further studies. In most other studies, only patients with MTC, clinically diagnosed MEN2, or relatives at risk of MEN2 were submitted to mutational analysis of RET (7 –29,38,39).

To establish a nationwide RET positive MEN2 cohort, data were collected in collaboration with the RET testing and MEN2 management centers in Denmark. This might cause issues of inter-variability in regards to methods and interpretation of results. However, methods and temporal expansion of RET testing were roughly similar in the two major RET testing centers, accounting for 94.7% (1499 patients) of the cohort. The remaining 84 patients were tested solely at the Aalborg University Hospital and thus only for the p.C611Y mutation. Of those, 26 were p.C611Y positive, and 45 were related to a p.C611Y patient. This leaves only 13 patients (0.7% of the total cohort) in which a RET mutation different from the p.C611Y might have been missed.

To estimate the true number of unrelated MEN2 families, a haplotype or genealogy study would be ideal. However, 24/36 apparently unrelated MEN2 families were defined by thorough clinical work-up. As in other studies, this workup is based on the index patient's recollection and knowledge of the familial history. Thus, possible relations between families carrying similar mutations might have been missed and may cause an overestimation of the number of families.

Codon 611 mutations

In the present study, mutations in codon 611 of exon 10 were the most prevalent accounting for 36% of the 36 families. This appears to be an unusually high prevalence. Numerous other studies did not detect any mutations in codon 611, despite systematic testing of RET exon 10 (8 –10,16 –19,21 –26,28,29,39). Most of these are single-center studies, and there might be an expectation of capturing more MEN2 patients in a nationwide study such as the one presented here. However, this does not seem to be the explanation. In fact, in three large European predominantly multicenter series based on a total of 232, 191, and 97 families, the prevalence of codon 611 mutations was only 0%, 3%, and 1%, respectively (Table 3) (6,7,12,13). The highest prevalence rates reported have been from Portugal (40%), Saudi Arabia (11%), and Iran (9%). However, these reports were based on small mainly single-center studies with a total of 5, 11, and 9 families (20,27,38).

Mutations highly prevalent in other series

An unusually high prevalence of specific RET mutations has also been described in other populations (6,7,26,28,29). In Cyprus, eight families were studied, and all carried the p.C618R mutation (28). A Sardinian study of seven families found three kindreds (43%) with the p.V804M mutation (29). In a large Italian multicenter study, 22% and 10% of the families had mutations in codons 804 and 891, respectively (Table 3) (7). A large German study identified codon 790 mutations in 14% of all included families, while a recent Greek study of 58 families found that 36% harbored the p.G533C mutation in exon 8 (Table 3) (6,26).

Results from these studies indicate geographical differences in the distribution of RET mutations. On the other hand, if mutations in exon 8 are disregarded in the Greek study, the distribution of mutations would be largely similar to that of other sizeable series where exon 8 was not routinely tested (7,12,13). Also, the high prevalence of codon 804 and 891 mutations in Italy may be caused by an earlier and more extensive testing of exons 14 and 15 compared to other series. A similar explanation for the high prevalence of codon 611 mutations in Denmark seems unlikely, since the traditional RET panel included testing of exon 10 from the very beginning (40). Thus, there seems to be a genuine high prevalence of codon 611 mutations in Denmark. This is supported by the fact that 70% (114/163) of the patients studied carry a codon 611 mutation.

Possible founder effect

Among the 13 apparently unrelated families with codon 611 mutations in the present study, 12 had the p.C611Y mutation. This could indicate a possible founder effect for the p.C611Y mutation. A similar effect has been indicated in other studies (7,26 –29,41,42).

In the Mediterranean islands of Cyprus and Sardinia, the authors suggested that a founder effect for the p.C618R and the p.V804M mutation could be explained by geographical isolation (28,29). However, geographical isolation alone does not seem to explain a possible founder effect in Denmark, since its southern border connects the country to Germany and the rest of Continental Europe.

In Greece, the p.G533C mutation was found especially in the southern part of Central Greece and the southeast region of the Peloponnese (26). In Italy, the p.S891A mutation was mainly present in a well-defined northern area. The Italian authors also found a high prevalence of codon 804 mutations, but excluded a founder effect by single-nucleotide polymorphism analysis (7). Only few and very small studies have proven a founder effect of RET mutations in mainland areas by haplotype analysis (27,41,42). To elaborate on this question in Denmark, further investigations are planned.

Prevalence of other mutations

Codon 634 mutations have been reported as the most predominant in several European (Table 3) (6 –14) and non-European series (Table 4) (17 –25). This might be explained by earlier onset of disease and a presentation frequently involving the full-blown MEN2 syndrome (MTC, PHEO, and HPTH). Mutations of codon 634 were the second most frequent alteration found in the present study, accounting for 17% (Table 3).

In the three largest studies to date, the prevalence of the p.V804L/M mutations ranged from 10% to 22% (Table 3) (6,7,12,13). In the present cohort, only one family (3%) was identified with this mutation. Despite testing for mutations in exon 14 since 1996 and 1997 in the two major RET testing centers, one center did not test the non-cysteine domains until after 2000. Retesting of patients tested before 2000 was performed but not systematically. Consequently, some patients tested for these mutations between 1996 and 2000 might have been missed, and this may result in a reduced number of families with non-cysteine codon 804 mutations. The distribution of the remaining mutations did not differ largely from that found in the literature.