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Activating variants in the receptor tyrosine kinase RE arranged during T ransfection (RET) cause multiple endocrine neoplasia type 2 (MEN 2), an autosomal dominantly inherited cancer-susceptibility syndrome. The variant c.166C>A, p.Leu56Met in RET was recently reported in two patients with medullary thyroid cancer (MTC). The presence of a pheochromocytoma in one of the patients, suggested a possible pathogenic role of the variant in MEN 2A. Here, we present clinical follow up of a Danish RET Leu56Met cohort. Patients were evaluated for signs of MEN 2 according to a set of predefined criteria. None of the seven patients in our cohort exhibited evidence of MEN 2. Furthermore, we found the Leu56Met variant in our in-house diagnostic cohort with an allele frequency of 0.59%, suggesting that it is a common variant in the population. Additionally, none of the patients who harbored the allele were listed in the Danish MTC and MEN 2 registries. In conclusion, our findings do not support a pathogenic role of the Leu56Met variant in MEN 2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hansen, Borgwardt, Rasmussen, Godballe, Poulsen, Vieira, Mathiesen and Rossing.)

Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases.

Purpose: In familial amyloid cardiomyopathy of Danish origin, the amyloid microfibrils contain a mutant transthyretin (TTR) with a methionine-for-leucine substitution at the molecular position 111. We studied the possible occurrence of this variant TTR-Met111 in serum from afflicted as well as nonafflicted family members and their offspring, in order to define its possible role as predictor of the disease and to describe its mode of inheritance., Patients and Methods: Stored, frozen serum samples obtained from 1959 through 1960 from 36 of 40 living members of the kindred were analyzed. The method employed to detect the abnormal TTR was based on the electrophoretic separation of fragments produced by cyanogen bromide cleavage at the two methionine sites., Results: All sera from family members with amyloid cardiomyopathy contained the variant transthyretin TTR-Met111 as did sera from half of their offspring. In contrast, nonafflicted family members and their offspring were seronegative for TTR-Met111. Three cousins from the second generation died between 1980 and 1986 of amyloid cardiomyopathy. The presence of variant TTR-Met111 preceded their deaths by 20 to 26 years., Conclusions: The occurrence in serum of the mutant transthyretin TTR-Met111 is linked to the occurrence of amyloid cardiomyopathy in patients and their offspring, while unafflicted branches of the family are negative for the variant protein. That the occurrence in serum of TTR-Met111 precedes the onset of clinical amyloid cardiomyopathy by several decades makes the variant TTR a marker for the disease. The distribution of afflicted family members and seropositivity for the variant TTR shows an autosomal dominant mode of inheritance., Clinical Significance: The results make possible early detection of potential patients and provide tools for genetic counseling. Cardiac transplantation may provide a new therapeutic option.

Familial amyloid cardiomyopathy in a Danish kindred is associated with a specific mutation (Met for Leu111) in the transthyretin (TTR) gene, causing the loss of a recognition site for the restriction enzyme DdeI in the gene. We describe a diagnostic test for the molecular detection of this mutation. A sequence of the TTR gene containing the mutation was amplified by the polymerase chain reaction from isolated genomic DNA of two affected patients and several controls. DdeI digestion of the amplified DNA from the patients revealed 3 bands by gel-electrophoresis, whereas amplified DNA of the controls showed only 2 bands, consistent with complete digestion. Thus, the assumed heterozygous TTR Met111 mutation was confirmed in the affected patients.

We have previously demonstrated an association between secondary AA type amyloid fibrils and glycosaminoglycans (GAGs) in human liver. The present study was aimed at investigating whether a similar association could be demonstrated in isolated cardiac amyloid fibrils from a unique Danish family with amyloid cardiomyopathy related to variant transthyretin (TTR) with a single amino acid substitution of a methionin for leucine at position 111 (TTR Met 111). Using gel filtration and ion exchange chromatography, significant amounts of GAGs were detected in close association with purified myocardial amyloid fibrils, whereas only trace amounts of polysaccharides were present in the corresponding normal preparation. The GAGs were identified as 50% chondroitin sulfate, 33% heparin/heparan sulfate, and 17% hyaluronan. With the methods used the amyloid associated GAGs appeared as high molecular weight free polysaccharide chains, and not as part of intact proteoglycans (PGs) in the fibril extracts. We conclude that the association between purified amyloid fibrils and GAGs may be a general feature of amyloid deposits. Also, we suggest that the proportion of different GAGs in the amyloid deposits may depend both on the organ or tissues affected and the type of proteins making up the fibrils.