Introduction

The RET proto-oncogene encodes a transmembrane receptor with a tyrosine kinase domain that is expressed in cells derived from neural crest.1,2,3 It is known that its germline mutations develop several human hereditary disorders, including multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung's disease (HSCR).4,5 MEN 2 is an autosomal dominant neoplastic syndrome that is classified into three clinical subtypes, MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC). MEN 2A is characterized by the clinical feature of medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid hyperplasia. MEN 2B patients are affected by a more complex phenotype including MTC, pheochromocytoma, skeletal abnormalities, and mucosal neuroma, whereas FMTC is defined by the sole phenotype of MTC without any other abnormalities. HSCR is a neurocristopathy characterized by the absence of intrinsic ganglion cells in the distal gastrointestinal tract, and is inherited in a non-Mendelian fashion.6

The mechanisms of development of MEN 2 and HSCR by RET mutations are quite different. MEN 2 and HSCR are caused by its ‘gain of function’ and ‘loss of function’ mutations, respectively.4 In MEN 2A patients, mutations are mostly detected in one of the six cysteine residues at codons 609, 611, 618, 620, 630, and 634 in the RET extra-cellular cysteine-rich domain. These cysteine mutations constitutively activate RET by inducing its disulfide-linked homodimerization.7,8,9 On the other hand, MEN 2B mutations are detected at codon 883 or 918 in the RET intracellular kinase domain, leading to RET activation without dimer formation.9,10,11 In FMTC patients, most RET mutations are identified in cysteine residues like the MEN 2A mutations, and some are at codon 768, 804, or 891 in the intracellular kinase domain. HSCR mutations including missense, nonsense, deletion, insertion, and frameshift mutations were reported throughout the whole coding region of RET gene without any hot spots, and impair RET function by various mechanisms. We and others demonstrated that HSCR mutations identified in the RET extracellular domain markedly impair the RET cell surface expression, whereas the mutations in the kinase domain impair the RET kinase activity or downstream signaling.12,13,14,15,16

Interestingly, the development of both MEN 2A/FMTC and HSCR has been reported in some families.17,18,19,20,21,22,23,24,25,26,27,28,29 The screening for germline mutations of the RET proto-oncogene revealed that a single amino-acid substitution in one of the three cysteine residues at codons 609, 618, and 620 is involved in the development of both diseases. In this study, we report the first clinical case to prove the pathogenesis of a cysteine 611 mutation for the development of both MTC and HSCR.

Patients and methods

Patients

Figure 1 shows the pedigree of the family described in this paper. The proband (II-2), a 47-year-old male, underwent left hemithyroidectomy for preoperative diagnosis as papillary thyroid carcinoma of the left thyroid gland in Aichi Cancer Center Hospital (Nagoya, Japan). It was diagnosed as an MTC by the postoperative histopatho-logical examination of his surgical specimens. His family history showed that the proband's father (I-1) died at the age of 56 years from an advanced MTC in Nagoya University Hospital (Nagoya, Japan). In addition, the proband's son (III-3) underwent an operation for HSCR (short segment type) at the age of 1 year in the Central Hospital, Aichi Prefectural Colony (Kasugai, Japan).

Figure 1
figure 1

The pedigree of the family with MTC and HSCR. Individual II-2 is the proband.

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On the basis of these episodes, an inherited disorder was speculated in this family. The proband was admitted to our hospital for further examinations and treatments. The molecular examinations for germline RET mutations in the affected family members were performed under informed consent and permission of ethical committee of Nagoya University School of Medicine. The examination of the proband's father was performed by agreement from his family.

DNA preparation

Genomic DNA of the proband was extracted from peripheral blood leukocytes according to the standard protocols. Genomic DNA of the proband's son was extracted from the paraffin-embedded tissue sections of his ileum that was resected at the operation for adhesive ileus. Three pieces of 10-μm-thick paraffin-embedded tissue sections were incubated in xylene for 30 min in an Eppendorf tube to remove paraffin from the tissues. Xylene was discarded, and the tissues were washed with ethanol and incubated in proteinase K buffer (10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2, 0.45% Tween 20, 0.45% NP-40, 10 mg/ml proteinase K) overnight. The DNAs were purified by phenol/chloroform DNA extraction method. Genomic DNA of the proband's father was also isolated from the paraffin-embedded tissue sections of his autopsied liver.

Mutation analysis

Sets of oligonucleotide primers to amplify the protein coding sequences of the RET gene were designed based on the published genomic sequences.30 PCR reactions were performed using the genomic DNAs of the proband, proband's son, and proband's father as templates, and the PCR products were subjected to sequencing directly, or they were cloned into pGEM-T plasmids (Promega, Madison, WI, USA) and sequenced. Double-strand sequencing of the PCR products and the inserts in pGEM-T plasmids was performed by a cycle sequencing program using the BigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences were determined by an automated Applied Biosystems sequencer model 310 (Applied Biosystems, Foster City, CA, USA).

Results

A 47-year-old male (II-2 in Figure 1) underwent a completion thyroidectomy with bilateral lymph node dissection. Small lesions of the remnant thyroid were diagnosed as MTC, but no pathological changes such as adenoma or hyperplasia were detected in the biopsy specimens of the parathyroid glands. It was also pointed out that he had a small tumor (2 cm in diameter) in the left adrenal gland by a screening computed tomography (CT) examination. His serum and urinary catecholamine levels and other adrenal hormones were within normal range, and he has not undergone an operation to resect the adrenal tumor to date. However, it seems likely that the left adrenal tumor is pheochromocytoma, thus, the possibility that he may be a MEN 2A patient is not excluded.

The sequences of RET exons 10 and 11, where mutations are usually present in MEN 2A/FMTC patients, were examined, and a TGC to TCT heterozygous germline mutation was detected in codon 611 of the RET gene, resulting in the amino-acid change from cysteine to serine (C611S) (Figure 2). The proband's son developed HSCR and was also screened for RET mutations. The same heterozygous mutation in codon 611 was detected. No other nucleotide alterations were found in the whole coding sequence of the RET gene of the proband's son. The same C611S mutation was present in the proband's father who died of MTC.

Figure 2
figure 2

C611S mutation detected in the family. Sequences of RET exon 10 of the proband were determined by direct sequencing of the PCR products (a). RET exon 10 was also sequenced after subcloning of the PCR products into the pGEM-T plasmid (b, c). A TGC to TCT heterozygous mutation was detected at codon 611. (b) wild-type allele, (c) mutant allele.

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Discussion

A total of 22 families with RET mutations that developed both MEN 2A/FMTC and HSCR were previously reported.18,19,20,21,22,23,24,25,26,27,28,29 The RET mutations identified in these families include two C609Y, five C618R, three C618S, eight C620R, three C620S, and one C620W mutations. In the present report, we described a new family affected with both MTC and HSCR. The mutation analyses of the RET gene revealed that a substitution of serine for cysteine at codon 611 was detected in affected members of the family. This family did not fulfill the definition of MEN 2A or FMTC, because MTC developed in only two members so far. Despite this fact, this is the first case of a family that suggests the pathogenesity of RET codon 611 mutation for the development of MTC and HSCR that are caused by gain-of-function and loss-of-function mutations of RET, respectively.

We previously proposed a possible mechanism for the development of both diseases by a single cysteine mutation in RET.4,31,32 In NIH3T3 cells, the transforming activities of the RET proteins harboring the mutations in cysteine 609, 611, 618, or 620 were significantly lower than those harboring the mutations in cysteine 634.31,33 The low transforming activities of the former mutant proteins were caused by impairment of their cell surface expression. Cysteine 609, 611, 618 or 620 mutations may alter the correct folding of RET as observed with the HSCR mutations identified in the RET extracellular domain,13,14 thereby interfering with RET maturation, intracellular trafficking or stable expression on the cell surface. The signal through the reduced RET proteins on the cell surface would not be sufficient for the differentiation of the intestinal ganglion cells, resulting in the development of HSCR.

In addition, cysteine 609, 611, 618, or 620 mutations can induce ligand-independent RET dimerization at low levels responsible for the transforming activity in NIH3T3 cells.31,33 Since these mutations were also identified in many MEN 2A/FMTC families, the low transforming activity appears to be sufficient to trigger the development of MTC. Based on these data, we predicted that cysteine 611 mutations have the potential to develop both HSCR and MTC.31 Mutation analysis of the family reported here supported our prediction.

Another interesting point of the present case is that two nucleotides of RET codon 611 were substituted (from TGC to TCT). No case that shows two-nucleotide alteration in one codon of the RET gene was previously reported, although some cases that have two codons with one-nucleotide alteration in the RET gene were reported.27 In addition, the present case showed no nucleotide alterations at polymorphic codons in the RET gene that may affect the development of HSCR.34,35,36,37,38

Germline mutations in several genes including GDNF, neurturin, endothelin receptor B, endothelin-3, SOX10, endothelin-converting enzyme 1, and ZFHX1B were reported in HSCR patients. However, the mutation frequencies of these genes in HSCR patients are significantly low compared with that of the RET gene.39,40,41,42,43,44,45,46,47 Furthermore, some other loci were proposed to be associated with the risk of HSCR.48 Although the possibility that the mutations of these genes are present and affect the development of HSCR in this case is not excluded, it is not practical to screen all the genes. Among the genes described above, the RET status is the most important for follow-up of HSCR patients because the patients who have a mutation at codon 609, 611, 618, or 620 in the RET gene are at risk for the development of MTC. Thus, the screening for these codons in the RET gene should be performed in all cases of HSCR routinely.