Hum Genet (1995) 95:187-190

9 Springer-Verlag 1995

Syuya Takiguchi-Shirahama 9 Kumiko Koyama Akira Miyauchi. Takanobu Wakasugi - Seiichi Oishi Hiroshi Takami 9 Kazumasa Hikiji - Yusuke Nakamura

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A)

Received: 20 June 1994 / Revised: 9 August 1994

Abstract Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10ql 1.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (RsaI, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic

S. Takiguchi-Shirahama 9K. Hikiji SRL Inc., Hachioji, Tokyo, Japan S. Takiguchi-Shirahama 9K. Koyama 9Y. Nakamura (1~) Department of Biochemistry, Cancer Institute, 1-37-1, Kami-Ikebukuro, Toshima, Tokyo 170, Japan A. Miyauchi 2nd Department of Surgery, Kagawa Medical School, Kagawa, Japan T. Wakasugi Division of Internal Medicine, Fukui Prefectural Hospital, Fukui, Japan

alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.

Introduction Multiple endocrine neoplasia (MEN2A) characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia, is a dominantly inherited cancer syndrome (Schimke 1984). The gene(s) responsible for multiple endocrine neoplasia types 2A and 2B (MEN2A and 2B) and familial medullary thyroid carcinoma (FMTC) were mapped to chromosome 10ql 1.2 by genetic linkage analyses in a large number of families carrying these diseases (Mathew et al. 1987; Simpson et al. 1987; Lairmore et al. 1991). Recently, alteration of a proto-oncogene (RET) in the candidate region was shown to be responsible for all three syndromes, when point mutations were detected in MEN2 and FMTC patients (Mulligan et al. 1994; Donis-Keller et al. 1993; Hofstra et al. 1994). The RET gene product contains Ca2+-binding motifs, a cadherin-like sequence, a cysteine-rich domain, an extracellular domain, a transmembrane domain, and a tyrosine kinase domain (Kwok et al. 1993; Takahashi et al. 1988). The germline RET mutations found so far in MEN2A patients have occurred at sites encoding the cysteine residues within the extracellular and transmembrane domain. To characterize germline mutations in Japanese MEN2A patients for accurate presymptomatic diagnosis, we analyzed the DNA sequences of exons 10 and 11 of the RET gene in affected representatives from eight families carrying MEN2A.

Materials and methods

S. Oishi 3rd Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto, Japan

Families and DNA samples

H. Takami 1st Department of Surgery, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Genomic DNAs were extracted from the peripheral lymphocytes of 38 individuals in eight families carrying MEN2A, according to methods described previously (Tokino et al. 1992).

188 Determination of intronic sequences DNA sequence including introns 9 and 10 was amplified using the following set of primers: 9FCE, 5"-CATCGAATTCCATCAACATTTGCCCTCAGG-3" and I1RDE, 5"-GTAGGAATTCTAGCAGTGGATGCAGAAGGC-3". DNA sequences of intron-exon boundaries were determined by dideoxy sequencing of subcloned polymerase chain reaction (PCR) products, using primers corresponding to DNA sequences in exons 10 (5"-GCTCCCCAGGCTCGTGTC-3") and 11 (5"-AAGAGGACAGCGGCTGCGAT3") (Kwok et al. 1993; Takahashi et at. 1988).

'd d

d,. 797.20;:::. [ ] ( ~ ; m~ulllry lhyrotec l r c l n o m l

Mutation analysis

+

Exons l0 and I I of the RET gene were amplified separately from genomic DNAs of affected individuals. 100ng of each DNA was used as template under the following conditions: each 25 bt] reaction contained 10 mM TRIS-HC1, pH 8.3, 50mM KC1, 0.75 mM MgC12, 0.01% gelatin, 200M dNTPs and 0.5 U of Taq polymerase, with 1MM each of PCR primers 10FFE (5"-GTAGGAATTCGACACGAGCCTGGGGAGC-3"), 10RLE (5"-CATCGAATTCTTGTTGGACCTCAGATGTGC-3") and 1I FJE (5"-GTAGGAATTCGCAGCCTGTACCCAGTGGTG-3"), 11RHE (5"-CATCGAATTCACCGGAAGAGGAGTAGCTG-3").PCR conditions consisted of 30 s at 94~ 60s at 60~ and 60s at 72~ for 35 cycles. Amplified products were completely digested with EcoRl and subcloned into the EcoRI site of pBluescript KS(-). A mixture of at least 50 subclones was used as the template for DNA sequencing with primers 10 K (5 "-CTGGGAGGTGGTGGTGGTC-3")or 111 (5 "- TGCCGAGCCTCTGCGGTG-3"), using a T7 sequencing kit (Boehringer Mannheim), according to the manufacturer's instructions.

; dllKI

Family KM2

Segregation analysis by PCR-restriction enzyme digestion Point mutations found in our MEN2A patients affected recognition sites of three restriction enzymes, RsaI, AluI, or q b l . Exons 10 and 11 of the RET gene were each amplified using genomic DNAs from members of the MEN2A families as templates with oligonucleotide primers 10FFE and 10RLE, or 1 IFJE and 1IRHE. Subsequently, each PCR product was digested with the restriction enzyme appropriate to the particular mutation and separated by electrophoresis on 2% agarose gels.

Results F r o m PCR experiments using oligonucleotide primers corresponding to exons 9 and 11 to amplify g e n o m i c DNA, the c o m b i n e d size of introns 9 and 10 was estimated to be 1.4 kb (about 8 0 0 b p of intron 9 and 6 0 0 b p of intron 10). After determination of the intronic sequences, PCR primers were designed. For amplification of exon 10 and surrounding intronic sequences, primers corresponding to flanking intronic sequences (10FFE and 10RLE) were used. For amplification of exon 11, primers corresponding to the 5" intronic sequence ( I l F J E ) and the 3" exonic sequence (11RHE) were used. The PCR products derived from these primer sets were 199bp for exon 10 and 2 7 9 b p for exon 11. D N A seq u e n c i n g of subcloned P C R products revealed mutations in all eight M E N 2 A patients. Five were mutations at codon 634 (TGC to TAC in four families, T G C to C G C in one family) that resulted in a m i n o acid changes from cysteine to tyrosine or arginine. Family K M 2 (Fig. 1A) is one in which the mutation occurred at codon 634 (Fig. 1B). A

Fig. 1 A Pedigree of family KM2. Arabic numerals at upper right of some symbols identify family members whose DNA is represented in C. B DNA sequence of the germline mutation in family KM2: left a normal individual; right the affected individual indicated by 1 in A. Codon 634 in exon 11 of the RET gene was mutated from TGC (Cys) to TAC (Tyr), resulting in generation of an RsaI site. C Rsal digestion of polymerase chain reaction (PCR) products of DNAs fiom members of this family; lane numbers correspond to individuals indicated in A. The size of the PCR product was 279bp; affected individuals revealed additional 184-bp and 95-bp fragments. M DNA size marker (~))~174 DNA digested with HaellI)

mutation at codon 618 occurred in families T Y F (Fig. 2A) and TAF; affected individuals in these two families carry a base pair substitution (T to A) that results in an a m i n o acid change from cysteine to serine (Fig,2B). All of the mutations found are missense mutations that affect cysteine residues (Table 1). To investigate cosegregation of these mutations in affected individuals and to establish a simple screening method for presymptomatic diagnosis in seven of our M E N 2 A families, we designed a method based on PCR amplification of g e n o m i c D N A c o m b i n e d with digestion by the restriction e n z y m e s whose cleavage sites were generated by the identified germline mutations. Figures l C and 2C reveal such examinations. As the mutation from T G C to TAC at codon 634 had generated an RsaI site,

189 Table 1 Germline mutations of the RET gene in eight Japanese MEN2A families

A

[]

m.,.

[] ~) (~ +

9

Exon a Codon Nucleotide position changes

Amino acid changes

Families

10 10 11 11

Cys to Ser Cys to Ser Cys to Arg Cys to Tyr

TYF, TAF KM1 TDF KM2, KKMI FWM, KMM3

618 620 634 634

TGC TGC TGC TGC

to AGC to TCC to CGC to TAC

; pheochromocytornlll ; medullarythyroid9 ; died Family TYF

a Numbering of exons and codons refers to their positions in the full-length RET coding sequence defined by Takahashi et al. (1988) and Kwok et al. (1993)

presence or absence of the CfoI site in the P C R product (data not shown).

Discussion

Fig.2 A Pedigree of family TYF. B DNA sequence of the germline mutation in this family; left a normal individual; right the affected individual indicated by 1 in A. Codon 618 in exon 10 of the RET gene was mutated from TGC (Cys) to AGC (Ser), resulting in generation of an AluI site. C AluI digestion of PCR products of DNAs from four family members; lane numbers correspond to the individuals indicated in A. The size of the PCR product was 199 bp. AluI digestion cut the PCR product of normal alleles to two fragments of 156bp and 43 bp, and that of the mutant allele in affected individuals to one l l3-bp fragment and two 43-bp fragments. M DNA size marker (~)Z174 DNA digested with HaelII)

P C R products including this codon produced extra bands in all the affected individuals in family K M 2 (Fig. 1C) and three other families upon digestion with RsaI. Similarly, the mutation from T G C to A G C at codon 618 generated an AluI site; digestion o f the P C R fragment that included this codon with this enzyme, generated a l l3-bp band in the D N A o f disease-allele carriers in families T Y F (Fig. 2C) and TAF. The mutation at codon 634 in family T D F had generated a novel CfoI site, and presymptomatic diagnosis in this family could be judged according to the

The RET oncogene has been proven to be the gene responsible for MEN2A, and MEN2B, and for F M T C as well as Hirschsprung's disease (HSCR) (Romeo et al. 1994; Edery et al. 1994). All point mutations found so far in M E N 2 A and F M T C patients have occurred at one of five codons (609, 6 1 1 , 6 1 8 , 6 2 0 , and 634), each corresponding to a cysteine residue encoded in exons 10 or 11 of the RET gene (Mulligan et al. 1994). Since cysteine residues serve important roles in preserving stable three-dimensional structure, the extracellular domain of the RET proto-oncogene may be structurally altered by the loss of cysteine. The mutated protein may generate an inappropriate signal for cell growth, which could lead to malignant transformation. Furthermore, rearrangements that probably activate the RET gene are frequently found in papillary thyroid carcinoma (Fusco et al. 1987). Although papillary thyroid carcinomas and medullary thyroid carcinomas are derived from different types of progenitor cells, activation of the RET oncogene is likely to play a significant role in malignant transformation of different cell types in the thyroid. Our analyses revealed that germline point mutations in M E N 2 A patients from eight Japanese families had also occurred in sequences within exons 10 or 11 o f the R E T gene that encode cysteine residues. Codon 634 seems to be the same "hot spot" of RET mutation in the Japanese population as that reported in Caucasians (Mulligan et al. 1994). Furthermore, as previously indicated, parathyroid hyperplasia was observed only in the two families (KM2 and K K M 1) carrying the mutation at codon 634.

Acknowledgements This work was supported by a Grant-in-Aid from the Ministry of Science, Culture, and Education, Japan.

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