Henry Ford Hospital Medical Journal Volume 37 Number 3 Article 7 9-1989 Allele Loss on Chromosome 10 and Point Mutation of ras Oncogenes are Infrequent in Tumors of MEN 2 A Makoto Okazaki Akihiro Miya Norifumi Tanaka Tetsuro Miki Masayuki Yamamoto See next page for additional authors Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons Recommended Citation Okazaki, Makoto; Miya, Akihiro; Tanaka, Norifumi; Miki, Tetsuro; Yamamoto, Masayuki; Motomura, Kazuyoshi; Miyauchi, Akira; Mori, Takesada; and Takai, Shin-ichiro (1989) "Allele Loss on Chromosome 10 and Point Mutation of ras Oncogenes are Infrequent in Tumors of MEN 2 A," Henry Ford Hospital Medical Journal : Vol. 37 : No. 3 , 112-115. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol37/iss3/7 This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Allele Loss on Chromosome 10 and Point Mutation of ras Oncogenes are Infrequent in Tumors of MEN 2 A Authors Makoto Okazaki, Akihiro Miya, Norifumi Tanaka, Tetsuro Miki, Masayuki Yamamoto, Kazuyoshi Motomura, Akira Miyauchi, Takesada Mori, and Shin-ichiro Takai This article is available in Henry Ford Hospital Medical Journal: https://scholarlycommons.henryford.com/ hfhmedjournal/vol37/iss3/7 Allele Loss on Chromosome 10 and Point Mutation of ras Oncogenes are Infrequent in Tlimors of MEN 2 A Makoto Okazaki,* Akihiro Miya,* Norifumi Tanaka,* Tetsuro Miki,' Masayuki Yamamoto,* Kazuyoshi Motomura,* Akira Miyauchi,* Takesada Mori,* and Shin-ichiro Takai* The multiple endocrine neoplasia type 2A (MEN 2A) gene has been mapped to the centromeric region of chromosome 10 by linkage analysis. We examined 36 medullary thyroid carcinomas (MTCs) (16 hereditary and 20 sporadic) and ten pheochromocytomas (eight hereditary and two sporadic) to deled loss of alleles on chromosome 10 using seven polymorphic DNA markers mapped to this chromosome. Of 20 informative cases, only one (5%) sporadic MTC showed loss of heterozygosity at the locus RBP3. Allele loss at the RBP3 locus was nol found in pheochromocytomas from six heterozy gates. All tumors retained constitutional heterozygosity at six other loci on chromosome 10 (DI0SI7, D10S34, D10S24 on the short arm, D10S15 in the pericentromeric region, D10S20, and D10S4 on the long arm). Our findings suggest that the second hit for tumorigenesis in MEN 2 A may not be loss of funct ion of the normal allele al the homologous locus on the other copy of chromosome 10. Mutated ras oncogene was found only in one of 18 MTCs al the codon 61 of //-ras. (Henry Ford Hosp Med J 1989;37:112-5) s Knudson (1) first pointed out and Cavenee et al (2) later 2 X SSC for 15 minutes, in 2 x SSC and 0.1% SDS for 30 min­ Aproved in retinoblastoma, two mutational events are neces­ utes, and in 0.1 x SSC for 10 minutes. The filters were exposed sary for carcinogenesis. More than three events are involved in to XRP films (Kodak) at - 80°C for one to five days. colon cancer (3). In the multiple endocrine neoplasia type 2A (MEN 2A) syndrome, tight linkage with the RBP3 gene (4-6) DNA probes indicates that the first mutation for tumorigenesis is at the Probes that detect polymorphisms at the following loci were MEN2A locus in the pericentromeric region of chromosome 10. used; RBP3 (H.4IRBP) (7), and six other loci on chromosome To clarify other mutation(s) necessary for tumorigenesis in to, ie, D10SI7 (pMHZI5) (8), DI0S34 (cTB14.34) (9), DI0S24 MEN 2A, we examined medullary thyroid carcinomas (MTCs) (p7A9)(10), D10S15(pMCK2)(II), DI0S20 (OS-2) (9), and and pheochromocytomas for allele losses on chromosome 10 D10S4 (pl-lOl) (12). Loci on other chromosomes were DIS7 and for point mutations in ras oncogenes. (XMSI), D22S1 (PMS3-18), D22S9 (p22/34), HRASl (pTBB-2). D13S3 (p9A7), GH (C-H800), and DI8S5 (OS-4) Materials and Methods (13). DNA samples Pairs of tumor and blood samples were obtained from 36 patients with MTC (16 hereditary and 20 sporadic) and from Polymerase chain reaction for amplification of ras oncogenes ten patients with pheochromocytoma (eight hereditary and two and detection of mutation sporadic). Both MTC and pheochromocytoma were avaitable in Selective amplification of regions around codons 12/13 and 61 five patients with MEN 2A. ofeach ras oncogene was performed on the DNA samples using DNA Thermal Cycler (Perkin Elmer Cetus). Slot blot analyses DNA analysis were canied out with synthetic oligonucleotide probes for each High molecular weight DNA was extracted from tumor rai oncogene (N-ra.s' 12,61, H-ra^ 12,61, and K-ra.v 12,13,61). tissues or blood samples according to a standard procedure. DNA (5 to to |xg) was digested with appropriate restriction enzymes, etectrophoresed in 0.7%^ agarose gel, transfened to Submitted for publication: September 30. 1989. Hybond-N nylon filters (Amersham), and hybridized with Accepted for publication: October 20. 1989. ' Second Deparimeni of Surgery. Osaka University Medical Schtxil. Japan. '-P-labeled probes at 65°C for 15 to 20 hours in 5 x Denhaldt's tDepartment of Geriatric Medicine. Osaka University Medical Schtxil. Japan. solution, 6 X SSC, 0.5% SDS, and 0.1 mg denatured sonicated tSecond Department of Surgery. Kagawa Medical School. Japan. Address correspondence to Dr Takai. Second Department of Surgery. Osaka University salmon sperm DNA per mL. The filters were washed at 65°C in Medical SchtMil. 1-1-,S0 Fukushima. Fukushima-ku. Osaka 5.S3. Japan. 112 Henry Ford Hosp Med J —Vol 37. Nos 3 & 4, 1989 Allele Loss and ras Mutation in MEN 2A—Okazaki ct al Results (MS 12) lost an allele at the RBP3 locus (Table 1) (Figure). This Allele loss on chromosome 10 in MTC and tumor (MS 12) maintained the constitutional heterozygosity at pheochromocytoma the locus D10S20. At the RBP3 locus, 20 patients with MTC were hetero- The other MTCs and all pheochromocytomas did not show zygotes. Of these 20 cases, only one (5%) sporadic MTC any allele toss at atl loci tested on chromosome 10 (Table 2). Table 1 Loss of Heterozygosity on Chromosomes 1,10, and 22 in Medullary Thyroid Carcinoma and Pheochromocytoma Locus DIOS17 DI0S34 D10S24 RBP3 D10S15 DI0S20 D10S4 DIS7 D22S1 D22S< Probe pMHZIS cTB14.34 p7A9 H.4IRBP MCK2 OS-2 pl-101 \MSI PMS3-I8 p22/3' Location I Op lOp lOp 10pll.2-qll.2 10pll-q21.2 10q21-q26 lOq Ip33 22q 22q Enzyme Bg 1 II Taq I Taq 1 Bgl II or Mspl Pvu II Hind III Taq 1 Hinf I Bgl II Taq I MTC MFI — — 1/2 1/2 — 12 — 1 MF2 — — 1/2 — — 1/2 1/2 — 1/2 MF3 — — 1 '2 1/2 — 1/2 MF4 — l'2 — — — 1/2 — 1/2 MFS 1/2 — — — — 1/2 1/2 1 /2 1/2 MF6 — — — 1/2 1/2 MF7 — — — 1/2 — 1/2 — 1.2 1/2 — MFS — — — 1/2 1 2 1.2 1/2 1/2 — 1/2 MF9 — — 1/2 1/2 1/2 — 1/2 MFIO — — 1/2 MSll 1/2 — 12 — — 1/2 1/2 1/2 MS12 — — — 2 — 12 — 1/2 1/2 1/2 MS13 1/2 — — — — — MS 14 1/3 1/2 1/2 MSlS — — 1/2 — 1/2 — 2 1/2 1/2 MS16 — — 1/2 — — l'2 1/2 MS17 — — — — — I.'2 1/2 1/2 — MSI8 — — — — 1/2 I.'2 I,'2 1/2 — MS 19 — I.O — — 1/2 12 1 MS20 — — 1/2 — 1/2 MS21 1/2 1/2 — 2 12 MS22 — — 1/2 — 1/2 MS23 1/2 — 1/2 MS24 — 1/2 MS25 — 1/2 — 1/2 MS26 1/2 — — 1/2 MS27 — 1 — 1/2 MF28 1/2 12 1/2 1 2 MS29 1/2 1/2 1/2 — MS30 1/2 |/2 1/2 MS31 1/2 1/2 1 '2 Pheochromocytoma PFI — 1/2 1 2 1.2 — 1 2 PF2 1/2 — — 1/2 — 1 PF3 — 1/2 — 1/2 PS4 — 1/2 — 2 PS5 — 1/2 1/2 1/2 1/2 MTC and Pheochromocytoma MPIM 1/2 1/2 — — — 12 — 2 — 1/2 P 1/2 1/2 — — — 1/2 — 1/2 1/2 MP2M 1/2 — 12 — — 1/2 1/2 1/2 P 1/2 — — — — 1 1 2 MP3M — 1/2 — — — 1/2 1/2 — — p MP4M — — 1/2 P — 12 MP5M I.'2 1/2 1/2 P 1/2 2 1/2 Note: " 1/2" indicates that the patient was constitutionally heterozygous for the marker, and the heterozygosity was retained in thc tumor DNA. or "2" indicates that the allele was retained, and the other allele was lost in thc tumor DNA. "—" indicates that the patient was constitutionally homozygous and not informative. The absence of entry indicates that this marker was not tested. Henry Ford Hosp Med J—Vol 37, Nos 3 & 4, 1989 Allele Loss and ras Mutation in MEN 2A—Okazaki et al 113 Table 2 Loss of Heterozygosity on Chromosomes 1,10, and 22 in Medullary Thyroid Carcinoma and Pheochromocytoma Number of Tumor Tumors D10S17 D10S34 DI0S24 RBP3 DI0SI5 DI0S20 D10S4 D1S7 D22S1 D22S9 MTL Hereditary 16 0/2 0/3 0/0 0/9 O/l 0/7 (1/5 1/16 0/3 1/9 Sporadic 20 0/3 0/1 O/l 1/11 0/0 0/4 (),./3 4/18 0/9 0/9 Tolal 36 0/5 0/4 O/l 1/20 o/l 0/11 O/X 5/34 0/14 I/I8 Pheochromocytoma Hereditary 8 0/2 0/2 O/l 0/3 o/l 0/3 0/0 4/7 2/3 1/2 Sporadic 2 0/0 0/2 O/l 1/2 0/1 Total 10 0/2 0/2 0/1 0/5 O/I 0/4 0/0 5/9 2/4 1/2 Note: Data in columns 3-12 indicate loss of heterozygosity/heterozygotes.