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Familial medullary thyroid carcinoma and prominent corneal nerves associated with the germline V804M and V778I mutations on the same allele of RET

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Editor—Germline mutations in theRET proto-oncogene are associated with multiple endocrine neoplasia (MEN) type 2A, 2B, and familial medullary thyroid carcinoma (FMTC). MEN 2A is characterised primarily by medullary thyroid carcinoma (MTC), parathyroid hyperplasia, and phaeochromocytoma. MEN 2B has additional stigmata including a marfanoid habitus, mucosal neuromas, and corneal nerve thickening. In FMTC, the only lesion present is MTC. Although characteristic of MEN 2B, prominent corneal nerves have been noted in subjects with MEN 2A.

Case reports

We have studied a family presenting with MTC and corneal nerve thickening (CNT), but no evidence of MEN 2 related disease outside the thyroid gland (fig 1). The proband (III.1) was found to have prominent corneal nerves on routine ophthalmological examination in his late 30s. No further action was taken and he presented with a palpable thyroid mass at the age of 53.

Figure 1

A truncated pedigree of the family is shown. Filled symbols indicate affected subjects. The letters below the symbols refer to the site of cancer, the numbers following the abbreviations indicate age at diagnosis. MTC, medullary thyroid cancer; PSU, primary site unknown; d, age at death; CNT, corneal nerve thickening.

MEN 2B was suspected and he was referred for laboratory screening tests for MTC and phaeochromocytoma. Serum calcitonin was raised at 4090 ng/l (normal range <60 ng/l). Serum calcium and phosphorus were normal. Twenty four hour urinary total catecholamine, metanephrine, and vanillylmandelic acid levels were within normal limits. A thoracic and abdominal CT scan performed before surgery was normal and scanning of the neck confirmed the presence of the right thyroid mass. The patient underwent a total thyroidectomy and pathological examination indicated bilateral MTC with extension beyond the capsule. His endocrinologist referred him for a genetic evaluation with a diagnosis of MEN 2B. However, other than CNT, he and his relatives did not have any of the other stigmata of this subtype. Family history was notable for thyroid carcinoma in the maternal cousin (III.4) and her son (IV.2). Interestingly, his mother (II.1), who appeared to be an obligate carrier, remained unaffected and died at 93 years of age related causes. The proband was encouraged to inform his family in Spain about the option of genetic counselling through the centre in Barcelona. Subsequently, IV.2 and all of his children were assessed and offered genetic testing. Patient IV.2 first noticed a right cervical enlargement at the age of 35 and later was diagnosed with multiple nodules and subjected to total thyroidectomy. The histological examination showed a multicentric medullary thyroid carcinoma with bilateral thyroid involvement. Lymph node and bone metastases were identified. Catecholamines and metanephrines were within normal limits. He was also found to have prominent corneal nerves. His children were screened for serum calcitonin. One of the three offspring (V.2) had had raised basal and pentagastrin stimulated levels (CT 15 pg/ml, basal and 23 pg/ml, five minutes).

The proband's genomic DNA was screened for mutations in exons 10, 11, and 13-16 of the RET gene. Direct sequencing identified two heterozygous missense mutations. Analysis of exon 13 showed a single base pair substitution of G to A at codon 778 resulting in a change from valine to isoleucine in the predicted amino acid sequence. Sequence analysis of exon 14 showed a heterozygous single base pair substitution of G to A at codon 804 resulting in a change from valine to methionine. Only exons 13 and 14 were analysed in the proband's mother, sister, and niece. Both mutations were subsequently identified in II.1 (an obligate carrier) and neither was found in III.2 or IV.1. Testing in Spain was performed concurrently on the affected subject IV.2, who was found to be positive for both V778I and V804M. All of his children were tested and one had the same genotype as her father while two were negative for both exon 13 and 14 missense mutations. Of note, the previously undescribed variant in exon 13 was present only in subjects carrying the V804M mutation. No mutation was found in RET codon 778 in four unaffected (V804M negative) family members, or in 73 and 20 controls from Montreal and Barcelona, respectively. Furthermore, we have amplified the proband's DNA by PCR, encompassing exon 13/intron 13/exon 14 of RET, by using an upstream primer localised at the end of intron 12 and a downstream primer localised at the beginning of intron 14. The PCR product is 1497 bp long. Since the G to A at position 778 destroys a singleMaeII restriction site (ACGT) on the PCR product, only the normal homozygous allele is cut by theMaeII enzyme. After an overnight incubation with MaeII to assure complete digestion, we performed a second round PCR on the MaeII digestion product. Only an allele with the A (rather than the G) nucleotide in codon 778 can be amplified since this amplified PCR product is left intact by MaeII. AnotherMaeII digestion on this second PCR product confirmed the absence of a MaeII site. Restriction analysis with NlaIII (CATG), which cuts the mutated allele (G to A) at codon 804, indicated that the 778I variant and the 804M mutation are on the same allele. Moreover, we performed direct sequencing of the second PCR amplification product and we were able to confirm the presence of the 778I variant on the same 804M mutated allele.

Discussion

The point mutations in exon 14 leading to a substitution of valine 804 by either leucine or methionine have been reported in several families with FMTC.1-3 The prevalence of this particular alteration in codon 804 seems to be quite low in FMTC families (frequency of 0.03 according to Hansford and Mulligan4) and there have been only three reports of the germline V804M in apparently sporadic MTC cases.5-7 Nilssonet al 8 showed that the V804L mutation is not exclusively associated with familial MTC as it has been identified in the MEN 2A pedigree. It appears that certain germline sequence variants identified in exons 13 and 14 are seen at a higher frequency in patients with sporadic MTC as compared with that of the general population. Two of the reported polymorphic variants (L769L in exon 13 and S836S in exon 14) have been implicated as having a potentially promoting effect in the aetiology of MTC.9 10The restriction of the V778I mutation to V804M carriers was noted and we have confirmed that V778I is in cis with the disease allele in the proband. The novel V778I germline alteration may be a low penetrance allele contributing to the development of the CNT in this pedigree. Notwithstanding, codon 804 mutations have been initially associated with a later onset and less aggressive disease phenotype suggesting lower penetrance7; however, there are several reports indicating a quite variable clinical picture including anticipation.3 11 The specific V778I sequence variant may be responsible for significantly earlier age of onset of MTC and more aggressive disease course in this family. Even though the replacement of valine by isoleucine in the predicted amino acid sequence results in a rather benign change (hydrophobic, branched chain to hydrophobic, branched chain amino acid) at the level of primary protein structure, it is not clear how it might alter the tertiary conformation. In addition, it should be noted that both exon 13 and 14 mutations affect the intracellular domain of the receptor tyrosine kinase encoded by theRET proto-oncogene. Miyauchiet al 12 reported a patient with a MEN 2B phenotype owing to a germline V804M mutation and a novel Y806C mutation occurring on the same allele of theRET gene. The authors speculated that the presence of the apparently non-pathogenic Y806C mutation in thecis position might have resulted in a structural change of the RET protein potentiating the significance of V804M alone. This particular case presented at the age of 23 with bilateral MTC with multiple foci and lymph node metastasis. Alternatively, Y806C could represent a low penetrance variant which affects the phenotype because of an additive effect in the presence of the second mutation. Subsequent comparison of the biological activity of the V804M/Y806C combination with that of the MEN 2B exclusive mutations M918T or A883F suggests similar biological properties of these three mutant proteins.13 Furthermore, the transforming activity of RET with the V804/Y806C compound was apparently eight to 13 times that ofRET with each mutation analysed separately. Further studies of the RET alleles in this and other kindreds may identify a unique effect of the known disease causing mutations in the presence of rare variants on the same chromosome 10, resulting in a different phenotype than observed in the presence of the mutation alone. Prominent corneal nerves which can be visualised by slit lamp examination are present in a large proportion of the MEN 2B patients14 and are less frequently observed in those with MEN 2A.15 As there are few such cases, it is not clear whether CNT in association with MEN 2A can be correlated with specific mutations in the RET gene. The significance of CNT was overlooked in our proband whose affected status might have been established earlier if genetic consultation had been considered by the ophthalmologist.

  • A family with MTC and CNT was assessed through a 53 year old affected subject.

  • RET exon 13 and 14 mutations in cis (V778I and V804M) were identified in four family members.

  • A novel variant V778I was found, potentially affecting the phenotype through an additive effect in the presence of V804M.

Acknowledgments

We thank Dr W Gregory for referring the proband to the Hereditary Cancer Clinic at McGill University and the family for their continued cooperation.

References

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