Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2525-2528, April 1984 Medical Sciences

Chromosome 20 deletion in multiple endocrine neoplasia types 2A and 2B: A double-blind study (medullary thyroid cancer/pheochromocytoma/calcitonin/oncogenes/ mapping) V. R. BABU*, DANIEL L. VAN DYKEt, AND CHARLES E. JACKSON Medical Genetics and Birth Defects Center and Division of Clinical Genetics, Department of Medicine, Henry Ford Hospital, Detroit, MI 48202 Communicated by James V. Neel, January 3, 1984

ABSTRACT Multiple endocrine neoplasia type 2A and 2B non-MEN-2 control subjects. All of the MEN families and (MEN-2A and MEN-2B) are autosomal dominantly inherited controls were of European ancestry except for one Black syndromes in which medullary thyroid cancers are associated MEN-2A family. All MEN-2 patients had normal intelli- with adrenal pheochromocytomas. A double-blind analysis of gence and an otherwise normal phenotype. Males and fe- high-resolution G-banded was performed on males were about equally represented among the MEN-2 blood specimens from patients in four MEN-2A families and and control subjects. In one of the four MEN-2A families, five MEN-2B (mucosal neuroma phenotype) families and from only medullary thyroid cancers and parathyroid tumors have control subjects. Excluding studies on duplicate blood speci- been detected; in another family, only medullary thyroid mens, 9 of 11 control subjects were scored as having normal cancers and pheochromocytomas have been detected; and in chromosomes 20, and 11 of 14 MEN-2 patients were scored as two families, all three tumor types have been detected. having chromosomal deletion: del(20)(p12.2p12.2) (X2 = 9.00; Three of the MEN-2B (medullary thyroid cancers, mucosal P < 0.001). Two new mutant MEN-2B patients had apparently neuromas, and pheochromocytomas) subjects are members normal chromosomes 20. These findings demonstrate that the of two families, and three are probably new mutants be- dominant mutation in most MEN-2A and MEN-2B families is cause, except for two children of one of them, no relatives a visible deletion within band 20pl2.2. are known or suspected of having MEN-2B syndrome. The 11 non-MEN-2 control subjects included 4 medical person- Improvements in cytogenetic methodology to obtain high- nel, 2 spouses of MEN patients, 2 definitely unaffected resolution (1-3) have helped delineate two syn- MEN-2 family members, 1 subject with MEN-1 syndrome dromes that cause congenital malformations and predispose (parathyroid and pituitary tumors and suspected pancreatic the host to specific neoplasia: (i) a chromosome 13q deletion islet tumors), 1 subject with a pheochromocytoma asso- in the retinoblastoma-mental retardation-congenital malfor- ciated with von Hipple-Lindau disease, and her normal sib- mation complex (4-6) and (ii) a chromosome lip deletion in ling. the aniridia-Wilms tumor association (7-9). In mendelian Cytogenetic Analysis. Chromosome preparations were dominant retinoblastoma or Wilms tumor without mental re- made from amethopterin-synchronized, phytohemaggluti- tardation or other malformations, the tumor cells may have a nin-stimulated lymphocyte cultures with minor modifica- abnormality, but the peripheral blood chromo- tions of the method detailed by Yunis et al. (1). In our tech- somes of the patient appear normal (10-12). In two families nique, vinblastine sulfate (0.0625 pug/ml) replaced colchi- with autosomal dominant predisposition to renal-cell carci- cine, and the slides were G-banded by trypsin with Giemsa. noma, the affected individuals had either an inherited (13) or During the first several years of our investigations of high- a tumor-acquired (14) balanced translocation. Many other resolution karyotypes in MEN-2 (16-18), neurofibromatosis autosomal dominant conditions have a predisposition to can- (16), XX males (21), Prader Willi and William syndromes, cer (15). Most are probably due to point mutations, but the familial breast cancer, and other disorders (unpublished findings in retinoblastoma, Wilms tumor, and renal-cell car- data), a protocol for investigating suspected minute chromo- cinoma suggest that some may exhibit visible chromosome some abnormalities was developed, which was used in the aberrations with high-resolution cytogenetic methodology. double-blind study. Each blood sample was delivered to the In 1981 we suggested that individuals with mendelian dom- laboratory identified by a code number known inant multiple endocrine neoplasia type 2A syndrome carry only to the clinical geneticist. Several high-resolution GTG- an interstitial 20p deletion (16-18). However, Hsu et al. (19) banded slides were prepared, and the location was noted of and Emmertsen et al. (20) were not able to distinguish the all prometaphase and late prophase spreads (approximately deletion in affected members of one MEN-2 family studied 850 and 1,000 bands per haploid set, respectively). Two or by each group. We describe here our findings in a double- three cytogeneticists independently scored each chromo- blind study of MEN-2 patients and controls, scoring high- some 20 pair from these cells for the 20p deletion. All scoring resolution G-banded cells for the presence or absence of the was done through the microscope to assure optimal resolu- 20p deletion. tion. The majority of cells located were not considered scor- able because one or both 20p arms overlapped with other MATERIALS AND METHODS chromosomes, the critical band 20pl2 was bent or twisted or Study Subjects. The blind study included nine indepen- had overlapped, or the homo- dently scored blood samples from eight affected members of logs clearly differed in their degree of condensation. After four MEN-2A families, eight independently scored blood both (or all three) cytogeneticists concurred that a subject samples from six affected members of five MEN-2B fam- had the deletion or not, the 'code' was broken. Agreement ilies, and 13 independently scored blood samples from 11 among the cytogeneticists has been attained in every case for

The Abbreviation: MEN, multiple endocrine neoplasia. publication costs of this article were defrayed in part by page charge *Present address: Arkansas Children's Hospital, 804 Wolfe Street, payment. This article must therefore be hereby marked "advertisement" Little Rock, AR 72202. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2525 Downloaded by guest on September 26, 2021 2526 Medical Sciences: Babu et al. Proc. Natl. Acad Sci. USA 81 (1984) which a sufficient number (at least 9-20) of adequate chro- MEN-2 patients carry a 20p deletion and all non-MEN-2 sub- mosome 20 pairs was available for scoring. jects have normal chromosomes 20, there were 25 correct and 5 incorrect assignments to phenotype based on the RESULTS karyotype scores (X2 = 13.25; P << 0.001). Likewise, 20 of The double-blind study included 30 independently scored 25 blood samples from different individuals (subsequent blood samples from 25 subjects (Table 1). Nine samples from samples excluded) were identified correctly in the double eight non-MEN-2 control subjects were scored as having blind study (X2 = 9.00; P < 0.001). normal chromosomes 20. Two control samples were scored A nonblind reassessment was carried out on the samples as having a 20p deletion (karyotype 20p-), clearly in error. that had scores inconsistent with the phenotype of the sub- Unbeknownst to the cytogeneticists, a second blood sample ject. The two new mutant MEN-2B patients scored as having from each of these two individuals was added to the double- normal chromosomes 20 (Table 1, ALS II-1 and MAR II-4) blind study, and both samples were scored as having normal still appeared to have normal chromosomes 20 on indepen- chromosomes 20. Nine blood samples from eight MEN-2A dent reevaluation by all three cytogeneticists. The other patients were scored as having the 20p deletion (Fig. 1). three inconsistent results (Table 1, MEN-2B patient TRU Three samples from MEN-2B patients were scored as having III-1 and controls LIGOR and EST II-3) had been scored the 20p deletion, and three were scored as having normal concurrently, and the errors in scoring were discovered in chromosomes 20. One sample that scored as normal and one the same code-breaking ceremony. It is our subjective inter- that scored as karyotype 20p- were from siblings in the TRU pretation that these errors occurred because we had inadver- kindred. Unbeknownst to the cytogeneticists, a second tently relaxed our scoring criteria for these samples by at- blood sample from these two patients was added to the dou- tempting to analyze too many cells with substandard G- ble-blind study, and both samples were scored as having the banding and by being insufficiently attuned to the variability 20p deletion. The other two MEN-2B patients whose blood between homologs caused by differential chromosome con- samples were scored as having normal chromosomes 20 ap- densation. pear by pedigree analysis to represent new mutants. samples from 11 DISCUSSION In summary, 11 of 13 non-MEN-2 control Based on our double-blind study, we interpret the chromo- subjects were scored as having normal chromosomes 20, and dominant MEN-2A and 14 of 17 blood samples from 14 MEN-2 patients were scored some abnormality in the autosomal If one assumes that all MEN-2B syndromes as an interstitial deletion involving part as having the 20p deletion (Table 2). of the light-staining subband 20pl2.2 and designate the de- Table 1. Findings in the double-blind chromosome study of the fect as 46,XX or XY,del(20)(p12.2p12.2). Three less likely chromosome 20 deletion in MEN-2A and MEN-2B syndromes interpretations of our observation are that the 20p deletion is a normal chromosome variant, part of a chromosome 20 in- Subject Karyotype Phenotype version, or part of an insertional translocation. First, the ab- MUR 11-2 20p- MEN-2A normality is unlikely to represent a normal chromosome MUR III-1 20p- MEN-2A variant because it was not observed in any of the control SLA 11-7 20p- MEN-2A subjects in the present study or in any of the ethnically di- SLA 111-5 20p- MEN-2A verse control subjects in our preliminary investigations (16- SLA 111-6 20p- MEN-2A 18) and because normal variation in euchromatic regions to STI 111-4 20p- MEN-2A our knowledge has not been observed. If the 20p deletion STR 11-1 20p- MEN-2A were a rare normal variant, even if linked in disequilibrium STR III-1 20p- MEN-2A with the MEN-2 gene, it should not have been observed in 11 OGR 11-3 20p- MEN-2B of the 13 MEN-2 families studied by us to date. Second, the TRU III-1 Normal* MEN-2B abnormality is unlikely to be part of an inversion or other TRU 111-2 20p-* MEN-2B intrachromosomal rearrangement because the rest of chro- ALS II-1 Normal* MEN-2B new mutant mosome 20 is normal-appearing in late prophase prepara- BRU 11-1 20p-* MEN-2B new mutant tions. Third, the abnormality is unlikely to be part of an in- MAR 11-4 Normal* MEN-2B new mutant sertional translocation for two reasons. The remainder of the SLI 11-2 Normal Control (MEN-1) karyotype appears normal, although an insertion of part of EST 11-3 20p-* Control (von Hipple-Lindau patient) band 20pl2.2 into a light-staining region on another chromo- EST 11-1 Normal Control (normal sibling of EST 11-3) some might not have been detected, Furthermore, if the ab- GEAYA Normal Control (medical student) normality were an insertional translocation, the MEN-2 pa- LIGOR 20p-* Control (nurse) tients would have been balanced translocation carriers, with MUR 11-3 Normal Control (spouse of MUR 11-2) a proportion of their gametes unbalanced segregants. This NANIE Normal Control (technologist) would have led to phenotypically abnormal liveborns, re- NOJAC Normal Control (unaffected MEN-2A family duced fertility, or increased pregnancy loss for which there member) is no evidence in our families. ROJAR Normal Control (spouse of an MEN-1 patient) In the present study two control samples were erroneously SLA 111-7 Normal Control (unaffected MEN-2A family scored as having the 20p deletion and three MEN-2B sam- member) ples were scored as having normal chromosomes 20. Subse- SUMAI Normal Control (secretary) quent double-blind samples from the two controls were scored as having normal chromosomes 20, and we regard the MEN-2A results from the first two samples as diagnostic errors. One MUR III-li 20p- 20 TRU III-lt 20p- MEN-2B MEN-2B sample scored as having normal chromosomes TRU III-2t 20p- MEN-2B was evaluated as having the 20p deletion in a second speci- EST II-3t Normal Control men added to the double-blind series. Furthermore, an af- LIGORt Normal Control fected sibling of this patient was scored as having the 20p in two in the double-blind based on deletion independent samples *Karyotype score is not consistent with expectations phe- and their affected mother has been scored as having notype. study, included in the double-blind study from sub- the 20p deletion in a nonblind chromosome analysis (unpub- tSubsequent samples we MEN-2B as a jects previously karyotyped. lished data). Thus, regard this sample diag- Downloaded by guest on September 26, 2021 Medical Sciences: Babu et aL Proc. NatL. Acad Sci. USA 81 (1984) 2527

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Ai _ _~~~~~~:rk~ _ N4- VIx c ow e z!/ W *g FIG. 1. The chromosome 20 pairs depict the appearance of the del(20)(p12.2p12.2) in MEN-2A (Fig. 1 a-c. e. and /lf and MEN-2B (Fig. 1 cl. f. and g) patients. The deletion appears to be similar in both disorders. The single arrows point to band 2Opl2: double arrows point to subbands 2Opl2.1 and 20p12.3. The deletion appears to involve part of the light-staining subband 20pl2.2 and does not appear to affect the bordering dark- staining subbands. When fewer than 550 bands per haploid set are present (early to midmetaphase). both members of the chromosome 20 pair appear normal (not shown). At approximately the 850-band stage (Fig. 1 b-h). the normal light-staining band 20pl?.2 is clearly visible in the normal homolog (on the right) between the dark-staining subbands 20pl2.1 and 2Opl2.3: but in the deletion homolog (on the left). only band 2Op12 is evident. At about the 1.000-band stage (Fig. 1a) band 2Op1r.2 is often visible in both homologs but appears narrower in one. The karyotype nomenclature (22) can be written as 46.XX or XYzdel(20)(p12.2p12.2). nostic error. The other two MEN-2B samples scored as hav- only one dysmorphic child (now deceased) who was report- ing normal chromosomes 20 were from new mutant MEN-2B ed (28) to have a deletion involving most of the short arm of patients, so we are uncertain whether or not they represent chromosome 20. No endocrine abnormalities or tumors were diagnostic errors. These two patients could, for example, be noted in her. Should any other such patients be identified, somatic mosaics with karyotypically normal white blood they should be followed for signs of MEN-2 by utilizing cal- cells. Alternatively, they could carry a point mutation, a citonin and catecholamine testing procedures. frame-shift mutation, another mutation at the same or some Combining the blind study findings of a deletion in four other , or a chromosome deletion that remains beyond MEN-2A families and three of five MEN-2B families with the resolution of present cytogenetic techniques; these ex- our preliminary findings (16-18) and our nonblind identifica- planations might also account for the reportedly normal tion of the 20p deletion in two MEN-2A and one MEN-2B karyotypes in five members of one MEN-2A family in Den- patients (unpublished data), we have observed the del(20) mark (20) and in one MEN sib-pair in Texas (19). The mid- (pl2.2pl2.2) deletion in a total of 18 MEN-2A patients from 8 metaphase chromosomes of MEN-2 patients from our fam- families and 5 MEN-2B patients from 3 of 5 families studied. ilies (L. Weiss and M. A. Block, personal communication) The observation of thus-far indistinguishable deletions in pa- and from other families (23, 24, t) have shown no consistent tients with MEN-2A and MEN-2B suggests that these are abnormalities. closely related entities. Some families with MEN-2A syn- The potential practical applications are evident for high- drome have only thyroid cancer. Other families also have resolution banding studies in the early identification of ge- parathyroid tumors or pheochromocytomas or all three tu- netically affected individuals in MEN-2 families carrying the mor types (27). This variable expressivity among MEN-2A deletion. However, because of present technical limitations and MEN-2B families may reflect etiologic heterogeneity be- (25, 26), some caution is advised in using high-resolution cause of slightly different breakpoints on chromosome 20 banding for early diagnosis of the MEN-2 syndrome. We among families (29). We found a normal karyotype in one would emphasize that the provocative calcitonin (27) and patient with the sporadic form of medullary thyroid cancer other clinical procedures remain the definitive tests for the (18) and in a patient with MEN-1 syndrome (parathyroid- early detection of the tumors of this syndrome. We know of pituitary-pancreatic islet cell tumot syndrome)* but more such patients need to be studied to assure that they have fAl Saadi, A. & Lieberman, L. American Thyroid Association normal karyotypes. Meeting, Oct. 9-12, 1968, Washington, DC, p. 69 (abstr.). Few have been localized to chromosome 20. We be- lieve the MEN-2A and MEN-2B loci reside within band Table 2. Summary of the cytogenetic findings in the blind study 20pl2.2. The inosine triphosphatase locus is mapped to the chromosome 20 short arm (30) but is unlikely to reside within Chromosome 20 the deleted region because erythrocytes of four of our MEN- Normal Deletion 20p 2A patients who carry the deletion had quantitatively normal MEN-2A 9 ITP activity (R. S. and M. S. Sparkes, personal communica- MEN-2B 3 5 tion). The locus (30) is on 20q, not Control 11 2 closely linked to MEN-2 (31). The gene for S-adenosylhomo- cysteine hydrolase is reported to reside on chromosome 20 A chromosome 20p deletion was observed in nine-blood samples (32), as is the cellular c-src proto-oncogene (33). The present from eight members of four MEN-2A families and in five blood sam- ples from four members of three MEN-2B families. Normal chromo- findings may facilitate the identification of additional loci somes 20 were observed in blood samples from 11 non-MEN-2 con- and the linear order of genes on chromosome 20 by MEN-2 trol subjects. Two erroneous assignments of control blood samples family studies or by application of restriction endonuclease as having the 20p deletion and one to three erroneous assignments of technology. MEN-2B blood samples as having normal chromosomes 20 are all It is of theoretical importance to extend Knudson's two- discussed in the text. mutation-event theory on the initiation of neoplasia to the Downloaded by guest on September 26, 2021 2528 Medical Sciences: Babu et al. Proc. NatL Acad Sci. USA 81 (1984) MEN-2 syndromes (34-36). Under this theory, the heritable 17. Van Dyke, D. L., Jackson, C. E. & Babu, V. R. (1981) Am. J. chromosome 20 deletion would be postulated as representing Hum. Genet. 33, 69a (abstr.). the first mutational event. Its dominant expression would in- 18. Van Dyke, D. L., Jackson, C. E. & Babu, V. R. (1982) Cyto- clude hyperplasia of thyroid C cells (27, 37, 38) and adrenal genet. Cell Genet. 32, 324 (abstr.). 19. Hsu, T. C., Pathak, S., Samaan, N. & Hickey, R. C. (1981) J. medullary cells (39, 40). This cellular hyperplasia would in- Am. Med. Assoc. 246, 2046-2048. crease the number of cells in which a second mutation might 20. Emmertsen, K., Lamm, L. U., Rasmussen, K. Z., Elbrond, occur. A second mutation in a susceptible cell (41) could, O., Hansen, H. H., Henningson, K., Jorgensen, J. & Petersen, under this theory, lead directly or indirectly to transforma- G. B. (1983) Cancer Genet. Cytogenet. 9, 251-259. tion into medullary thyroid cancer or pheochromocytoma. 21. Magenis, R. E., Webb, M. J., McKean, R. S., Tomar, D., Al- Thus, although MEN-2 is a mendelian dominant predisposi- len, L. J., Kammer, H., Van Dyke, D. L. & Lovrien, E. (1982) tion to neoplasia, the gene is another recessive cancer gene, Hum. Genet. 62, 271-276. similar to that found for retinoblastoma (42, 43). These re- 22. ISCN (1981) Birth Defects: Orig. Artic. Ser. XVII, No. 5, An cessive mechanisms in MEN-2 and chromosome 20 may be International System for Human Cytogenetic Nomenclature: High Resolution Banding (March ofDimes Birth Defects Foun- clarified by studies of polymorphic restriction endonuclease dation, New York). recognition sequences, just as they have with retinoblastoma 23. Nankin, H., Hydovitz, J. & Sapira, J. J. (1970) J. Med. Genet. and (43). 7, 374-378. 24. Levan, G., Mitelman, F. & Telenius, M. (1973) Lancet i, 1510. 25. Schwartz, S. & Palmer, C. G. (1981) Am. J. Hum. Genet. 33, The authors thank K. Mayeda, L. Weiss, R. A. Norum, W. Stan- 121a (abstr.). ley, P. Cornett, M. Worsham, W. L. Flejter, and J. B. Yott. Access 26. Wulfsberg, E. A., Klisak, I. J. & Sparkes, R. S. (1983) Clin. to the MEN-2B patients was generously provided by J. D. Fachnie, Genet. 23, 35-37. J. C. Sisson, and M. R. A. Khairi. This work was supported in part 27. Block, M. A., Jackson, C. E., Greenawald, K. A., Yott, J. 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