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
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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.
Table 3 Allele Losses on Chromosome 10 T N Landsvater et al Nelkin et al Present Study (18)1989 (19)1989 1989 MTC 1/28 2/16 1/29 Pheochromocytoma 0/14 0/2 0/8 Note: Data indicate loss of heterozygosity/heterozygotes. -6.3kb
-4.3kb found at D22St in MTCs from 14 informarive cases. In pheo chromocytomas, altel,e loss was found in two (50%) of four at D22St and in one (50%) of two informative cases at D22S9. Both pheochromocytomas (PFI and MP2P) with attete loss on ctiromosome 22 atso tost an attete on chromosome 1, Allele toss was not found at four loci on chromosomes 11, 13, 17, and 18 (data not shown).
Point mutation of ras oncogenes in MTC Of the 18 cases (eight hereditary and ten sporadic), onty one (5.6%) sporadic MTC had mutated ras oncogenes (A to G tran sition at position 2 of codon 61 of W-ras). Bgl I Discussion MSi2 Two or more genetic changes are necessary for car cinogenesis (1-3). In hereditary tumors such as refinobtastoma (14) and MEN 1 (15), the first hit is a mutation of the predispos ing gene, RB 1 and MEN 1, respectivety, on one copy of the chro Figure—Allele loss at the RBP3 locus in a sporadic medullary mosome. In these tumors, it has been demonstrated that the thyroid carcinoma. DNA from medullary thyroid carcinoma of second hit is a toss of normal function of the wild type allele at the case MS 12 lost the longer allele at the RBP3 locus. the homologous locus on the other copy of the chromosome (2,16,17). It woutd be interesting to learn whether a simitar genetic event is responsibte for tumorigenesis in MEN 2A. Landsvater et at (18) and Nelkin et at (19) reported that the Allele loss at loci on other chromosomes frequency of allele tosses on chromosome 10 was tow in MTC Of 34 patients heterozygous at the DtS7 locus, five (15%) and pheochromocytoma. Our results are in accord with their MTCs tost an allele. Of nine heterozygous patients, five data (Tabte 3). (55,6%) pheochromocytomas atso tost an allele at the D1S7 As discussed by these investigators (18,19), toss of normal locus. function of the attete opposite the germ tine defect by subtle At loci on chromosome 22, onty one (5.6%) MTC of 18 infor changes undetectable with Southem btot analysis cannot be ex mative cases tost an allele at D22S9, but allele toss was not cluded. However, we believe it is more likely that genetic events
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