Molecular Diagnosis Vol. 2 No. 4 1997
Case Study Age-Related Disease Penetrance in a Large Medullary Thyroid Cancer Family With a Codon 609 R E T Gene Mutation K E V I N C. H A L L I N G , *t JOSl~ A. B U F I L L , ; M I C H A E L
S T E V E N A. A R T Z , . ~ A. B E T T S C A R P E N T E R , II D A N S C H A I D , # HOLLY HARTMAN-ADAMS,.~ HO-HUANG CHANGf M I C H A E L M. B O U S T A N Y , ~ L I N D A F 1 T H I A N , qLS I S S Y M. J H I A N G , ~ I STEPHEN
N. T H I B O D E A U *
Rochester, Minnesota; Indianapolis, Indiana; Elkhart, Indiana; Charleston, West Virginia; Columbus, Ohio
Background: Familial medullary thyroid cancer (MTC) is a form of type 2 multiple endocrine neoplasia in which individuals develop MTC as the sole phenotypic manifestation of their disease. A previous study has suggested that patients with familial MTC may have a later age of onset (and more indolent course) of MTC than is observed in individuals with multiple endocrine neoplasia type 2A. Methods and Results: The age-related pcnetranec of MTC, C-cell hyperplasia, and a positive pentagastrin test for carriers of a codon 609 mutation of the R K T gone in a large MFC family was determined. Pcntagastrin testing and surgical pathology lindings for patients who had thyroidectomies were correlated with RE7" sequence analysis findings. The penetrancc of this mutation for the development of MTC was 0% at age 20, 10% at age 30, 50% at age 45, and approximately 100% at age 60.The ages of onset of C-cell hyperplasia and a positive pentagastrin stimulation test were similar, and both preceded the age of onset of MTC. Carriers of the mutated gene in this family had a later age of onset of disease than has been reported for families with multiple endocrine neoplasia type 2A and 2B syndromes. Conclusions: These results may have implications for the clinical management of MTC families with a 609 mutation. Key words: multiple endocrine neoplasia (type 2), C-cell hyperplasia, pentagastrin stimulation testing, R E T proto-oncogene.
From the *Department of Laboratoi;v Medicine and Pathoh;gy and ~Department (~/ Biostatistics, Mayo Foundation, Rochestet. Minnesota; "Deparmzent of Pathoh)gy and lmboralory Medicine, Indiana University School qf Medicine, Indianapolis, Indiana; .;llereditary Cancer Program, Elkhart, Indiana; "~Nuclear Medicine Services, lm., and ,,l)el)artment (~/"Pathology, Charleston Area Medical Centez Charleston, West Virginia; and ~D~Tmrtment of lnternal Medicine and Physiology, Ohio State University, Colm~tbus, Ohio. Reprint requests: Stephen N. Thibodeau, PhD, Molecular Genetics, Hilton 97(I, Mayo Clinic, Rochester, MN 55905. Copyright © 1997 by Churchill lJvingstone °o 1084-8592/97/0204-000658.00/0
Molecular Diagnosis Vol. 2 No. 4 December 1997
Medullary thyroid cancer (MTC) is an uncommon malignancy of parafollicular or C cells, neuroendocrine cells that normally produce the hormone calcitonin. Overproduction of calcitonin by neoplastic C cells has served as the basis for presymptomatic detection of the disease in persons deemed to be at risk, based on a positive family history of MTC. Although most MTCs are sporadic, inherited forms have been characterized clinically and account for about 20% of MTCs and 1% to 2% of all thyroid malignancies. Initial observations identified an association between inherited MTC and neoplasms of the adrenal and parathyroid gland and, less often, with a variety of mucosal and skeletal abnormalities [1,2]. These characteristic clinical entities have been termed the multiple endocrine neoplasia (MEN) type 2 syndromes. Three phenotypic variants of the MEN2 syndromes have been recognized. Multiple endocrine neoplasia type 2A is characterized by the development of MTC, pheochromocytoma, and parathyroid hyperplasia/adenoma; MEN2B by MTC, skeletal abnormalities, and mucosal neuromas; and familial MTC by MTC alone . Pheochromocytoma and parathyroid neoplasms develop in approximately 50 and 10% of individuals with MEN2A, respectively . Consequently, small M E N 2 A families may not manifest the full phenotypic spectrum of the disease and may be mistaken as familial MTC families. To avoid misclassification of MEN2 families, Mulligan et al have proposed that familial MTC be defined as a family with four or more individuals with MTC, but no pheochromocytoma or parathyroid disease . Remarkable progress in the understanding of the molecular basis of the MEN2 syndromes has occurred in the past decade. In 1987, two groups mapped the gene for M E N 2 A to the pericentromeric region of chromosome 10 [5,6]. Subsequently, MEN2B and familial MTC were m a p p e d to the same region [7,8]. Subsequent investigations demonstrated that mutations of the R E T protooncogene accounted for the phenotypic abnormalities that characterize MEN2A, MEN2B, and familial MTC [4,9-11]. The Ret protein is a transmembrane receptor tyrosine kinase. Most of the known R E T mutations associated with M E N 2 A and familial MTC occur in a cysteine-rich region of the extracellular portion of the Ret protein. Codons 609,611,618, 620, and 634 are the sites of
missense mutations in approximately 95% of M E N 2 A and 85 % of familial MTC families . Familial MTC families with mutations of codon 768 (exon 13) and codon 804 (exon 14) have been described more recently [12,13]. Multiple endocrine neoplasia type 2B families, on the other hand, have missense mutations of codon 918 of the RETprotooncogene, and somatic mutations of codon 918 are present in 30% to 40% of sporadic MTCs [12, 14,15]. Early identification of the manifestations of MEN2 followed by thyroidectomy can prevent metastasis of MTC . Thyroidectomy in children, however, can be associated with complications that adversely affect growth and development . Our understanding of the natural history of familial MTC is evolving. Data from relatively few families studied to date suggest that MTC in affected individuals occurs later in life and may have a more indolent course. Guidelines for clinical screening that have been used for families with MEN2A may not be applicable to families with familial MTC. To address this issue, we studied a large familial MTC family with a TGC-to-TAC mutation of codon 609 of the R E T gene. We performed R E T gene analysis, pentagastrin (PG) stimulation testing, and pathologic examination of resected thyroids in members of this family to determine the age of onset of disease in gene carriers and to evaluate the role of PG stimulation testing in this disease.
Materials and Methods Patient Population Members of this family are located in West Virginia, Indiana, Michigan, and other states. Clinical evaluation of patients consisted of a history and physical, with particular attention to signs and symptoms of the MEN2 syndromes along with PG testing and R E T gene analysis of at-risk individuals. Serum calcium levels and 24-hour urinary catecholamines were determined to exclude parathyroid and adrenal medullary disease, respectively.
PG Stimulation Testing The majority of the patients in this study had PG stimulation testing performed in West Virginia and Indiana. Testing consisted of obtaining a sample
Familial Medullary Thyroid Cancer
for baseline calcitonin determination prior to administering 0.5 p,g/kg P G intravenously. Blood samples were obtained 1.5 and 4.5 minutes after P G administration for the determination of serum calcitonin levels. In most instances, serum calcitonin levels were determined by a radioimmunoassay performed at the Charleston Area Medical Center Nuclear Medicine Endocrine Laboratory, using Diagnostic Products Corporation Calcitonin Double Antibody Radioimmunoassay. Similar to other investigators, we defined a positive P G stimulation test as a test in which the peak calcitonin level is three times the basal calcitonin level or in which any calcitonin level is 200 ng/L or greater [18,19]. Patients with equivocal test results were reevaluated with a follow-up P G stimulation test several months later.
Pathologic Examination MTC was diagnosed if cytologically atypical C cells extended beyond the follicular basement membrane. Patients with equivocal C-cell hyperplasia (CCH) by hematoxylin and eosin staining were further assessed by employing a rabbit antihuman calcitonin antibody (Bio Genix; San Ramon, CA) and the streptavidin technique. In these instances, C C H was defined as greater than 50 C cells in three low-power fields .
DNA Sequencing of the RET Proto-Oncogene Individuals were initially examined for mutations of exons 10 and 11. Following the consistent identification of a TGC-to-TAC mutation of codon 609 in several affected family members, the remaining individuals in the family (with the exception of IV-17) were examined only for a mutation of codon 609 in exon 10. Template for sequencing was prepared by polymerase chain reaction amplification of a fragment containing both exons 10 and 11 of the R E T gene. The primers used were R e t A (5' TCC G G G G C A G C A T T G TT 3') and CRT 19A (5' CTT G A A G G C ATC CAC G G A G A 3') . Sanger dideoxy sequencing was performed with the Promega fmol sequencing system kit (Promega, Madison, WI) and either Ret D (5' TTG G G A CCT CAG ATG T G C T G T T 3') for exon 10 or CRT 19B (5' G C A TAC G C A G C C T G T ACC C 3') for exon 11 .
Hailing et al.
Codon 918 Mutation Identification One individual in this family was evaluated for the presence of a codon 918 mutation of the R E T gene in both peripheral blood and tumor. Exon 16 of the R E T gene (containing codon 918) was amplified by polymerase chain reaction using R e t 16F (5' A G G G A T A G G G C C T G G G C T TC 3') and Ret 16R (5' T A A CCT C C A CCC C A A G A G A G 3') primers. Restriction digestion with FokI followed by agarose gel electrophoresis was then used to identify the presence of a codon 918 mutation.
Statistical Analysis The age-specific penetrances of each of the endpoints (positive P G stimulation, CCH, and MTC) were estimated by the Kaplan-Meier method, which censored individuals at their last follow-up examination if they were free from an event . The proband was excluded from these calculations to prevent bias.
Results The Family Figure 1 shows the pedigree of the family and results of R E T gene analysis, PG testing, and pathologic diagnosis for each individual for whom the information is known. There are 120 individuals in four generations. Eighteen individuals have developed MTC, 15 in generation lII and 3 in generation IV. The pattern of transmission of MTC is consistent with an autosomal dominant inheritance pattern. None of the individuals with a germline R E T mutation had stigmata of M E N 2 A or M E N 2 B (ie, pheochromocytoma, hyperparathyroidism, mucosal neuromas, or Marfanoid habitus). Thus, the family fulfills the criteria proposed by Mulligan et al. for familial MTC . No individuals have died from MTC at this time. One patient is alive with evidence of recurrent disease following thyroidectomy and 17 are alive without evidence of recurrent disease.
RET Gene Analysis The proband 1II-37 was shown to have a TGCto-TAC missense mutation of codon 609. Seventyfive individuals have been assessed for the pres-
Molecular Diagnosis Vol. 2 No. 4 December 1997
ence of this mutation. Table 1 summarizes the results of sequence analysis for each generation and the entire family. Two individuals in generation I, five in generation II, and three in generation III are deceased and have not been tested. Thirty-five of the individuals in generations II, III, and IV have either declined testing or have not yet sent samples for testing.
Sixty-seven individuals have had both R E T gene analysis and PG testing. A calculation of the sensitivity and specificity of the PG stimulation test using R E T gene analysis as the "gold standard" for detection of carriers is complicated by the fact that the sensitivity and specificity of PG stimulation increase with age. Nonetheless, with this caveat in mind, we determined that the PG stimulation test had a sensitivity of 62% (20 of 32 carriers had a positive PG test), specificity of 74% (26 of 35 noncarriers had a negative PG test), positive predictive value of 69% (20 of 29 individuals with a positive PG test were carriers), and negative predictive value of 68% (26 of 38 individuals with a negative PG test were not carriers) for this kindred.
[si: ) x?,
Forty-one patients have had thyroidectomies. The primary indication for thyroidectomy for patients in this study was the presence of a germline R E T mutation in that individual. Surgical pathology reports were available for 39 of the 41 patients who had thyroidectomies. The 39 patients could be divided into several groups based on R E T and PG testing status. A correlation of the surgical pathology findings with the R E T sequence analysis and PG stimulation testing results is shown in Table 2. Six patients with R E T mutations have not yet had a thyroidectomy. The 5 patients with a R E T muta-
Pedigree of familial MTC family. The presence or absence of a codon 609 (TGC-to-TAC) mutation in an individual is indicated as Pos (mutation present) or Neg (mutation absent) underneath the patient symbol. Individuals who have not been tested for the 609 mutation have N D (not determined) beneath the symbol. The presence or absence of medullary cancer and/or CCH (for patients who have had a thyroidectomy) and results of PG testing are as follows:  M T C ,  = C C H , N = t h y r o i d e c t o m y ,  = positive PG test. F i g . 1.
Familial MedullaryThyroid Cancer • Hailing et al. 281 Table 1. R E T Mutation Analysis Findings Generation I
II III IV All
No. of Individuals in Generation
No. of Individuals Tested
10 46 62 120
0 23 49 72
No. of Individuals With 609 Mutation NA NA
16 (70%) 18 (37%) 34 (47%)
Abbreviation:NA, not applicable.
tion and no pathologic abnormality at thyroidectomy were 9 to 34 years old (average age, 20.2 years). The 6 patients with a R E T mutation and CCH were 15 to 37 years old (average age, 24 years). The 15 patients with MTC were 21 to 59 years of age (average age, 42.3 years). One individual with MTC and parathyroid hyperplasia (IV-17) did not have a germline mutation of codon 609. Hematoxylin and eosin-stained slides from the MTC were shown to five different pathologists (including two endocrine pathologists) with complete consensus as to the diagnosis of MTC. To exclude the possibility of laboratory error or a specimen mixup, a second specimen was obtained from the individual and it also lacked the 609 mutation present in this family. In addition, DNA extracted from paraffin-embedded normal lymph node resected at the time of the thyroidectomy also lacked the 609 mutation. DNA sequencing of exon 11 also revealed the absence of any germlinc mutation. Interestingly, analysis of DNA extracted from the paraffin-embedded MTC from this patient revealed an ATG-to-ATC mutation at codon 918 that was not present in the DNA from paraffin-embedded lymph node.
Disease Penetrance Figure 2 shows the age-related penetrance of MTC, CCH, or a positive PG stimulation test for those patients with the 609 TGC-to-TCC R K T mutation. The median age of individuals with MTC was 44 years; with CCH, 41 years; and with a positive PG stimulation test, 44 years. As is evident from Figure 2, the interval between age at diagnosis of either CCH or a positive PG test and age at MTC diagnosis was at least 5 years for individuals less than 40 years old.
Age-Related Penetrance One of the decisions facing clinicians caring for MEN2 kindreds is when to recommend thyroidectomy. As Thomas and Gagel have pointed out, the decision to perform early thyroidectomy requires a risk/benefit analysis "that considers the risks of the surgical procedure, general anesthesia, and thyroid hormone replacement therapy in children" . Previous studies have suggested that early thyroidectomy of MEN2 carriers can prevent the de-
Table 2. Surgical Pathology Findings: Correlation With R E T Status and Pentagastrin Stimulation Test Results Surgical Pathology Findings RET Status/PG Testing Results RET+/PG+ RET+/PG RET-/PG+ RET-/PG RETND/PG-
No Pathologic Abnormality
MTC and CCH
4 5 3
RET ÷, RET mutationpresent:R E T - , RET mutationabsent;PG+, positivepenlagastrin test; PG , negativepentagastrintest; R E T N D, R E T sequenceanalysisnot done.
Molecular Diagnosis Vol. 2 No. 4 December 1997
-'..... l - 40
; ..... ~j ' I
30 j ...... m
-' j . . ~ , j
; n--,,a ,.,[-:.'1 J
•........ : II !..... f----'l II
Age (years) Fig. 2. A g e - r e l a t e d p e n e t r a n c e of a positive P G test result, CCH, and M T C for carriers of the R E T germline 609 mutation. .... CCH,--= PG test, = MTC.
velopment of MTC . Nonetheless, thyroidectomy of young children is not without its potential complications, including permanent hypocalcemia as a result of injury of the parathyroid glands, damage to the recurrent laryngeal nerve, risks of general anesthesia and postoperative recovery (increased in children relative to adults), and risk of hypothyroidism (affecting normal growth and development) . Wells and Donis-Keller have recommended that M E N 2 A and familial M T C carriers have a thyroidectomy at 5 years of age or any time thereafter once the diagnosis is established . This recommendation appears to be based on the observation that the youngest M E N 2 A patient with histologically confirmed M T C in their studies was only 5 years old. There are no published stud-
ies of the age-related penetrance of M T C in familial M T C kindreds, but Farndon et al. observed that the mean age of M T C diagnosis was significantly later in familial M T C than in M E N 2 A kindreds (43 years v 21 years) . They suggested that MTC in families with familial M T C either develops at a later age or grows more slowly. Could familial MTC carriers delay thyroidectomy until a later age? The answer to this question hinges not so much on the mean age of onset of M T C but more on the earliest age of onset of MTC. As can be seen in Figure 2, none of the individuals in this family developed M T C before age 20, whereas approximately 50% of the carriers had developed MTC by age 45. The mean age of MTC development in the kindred we have studied (41.7 years) is quite similar to that ob-
Familial Medullary Thyroid Cancer
served in the two kindreds studied by Farndon et al. . These findings are interesting when compared with those of Easton et al., who found that a small proportion of M E N 2 A individuals have onset of disease in their second decade of life and positive PG testing in the first decade of life, and support the contention that familial kindreds develop M T C at a later age than M E N 2 A kindreds . The relatively delayed age-related penetrance of M T C in the family we have studied might suggest that it is reasonable to delay thyroidectomy until the late teens for familial M T C carriers. It is important to be cautious before making definitive recommendations, however, as our findings represent the age-related penetrance for a single familial M T C family. The age-related penetrance of MTC for other familial MTC families may be affected by factors such as the site of the R E T mutation (eg, 609 v 768), environment, and possibly modifier genes. Given this, it seems prudent to recommend early thyroidectomy until the role of these factors in determining age-related penetrance can be studied in other familial MTC families. In larger families, recommendations regarding the timing of thyroidectomy may be tailored to the specific penetrance data obtained through the study of the extended pedigree.
Natural History of MTC in Familial MTC Although the clinical follow-up period for the patients in our study (mean follow-up period, approximately 3 years) is relatively short, all but a few of the individuals with MTC have had a favorable clinical course.This suggests, as Farndon et al.  have previously noted, that familial MTC-associated MTC may be less aggressive than that associated with M E N 2 A or MEN2B. Alternatively, MTC may be diagnosed at an earlier stage in familial MTC families than in MEN2A or MEN2B families.
Genotype-Phenotype Correlation The finding of a 609 mutation in this large familial MTC family is interesting because it provides further support for the suggestion by Mulligan et al. that mutations of codon 634 strongly predispose to the development of pheochromocytoma . As Mulligan et al. have shown, however, there are M E N 2 A kindreds with mutations of each of the other four codons (ie, codons 609, 611,618, and 620) and several familial MTC families with codon
Hailing et al.
634 mutations. Thus, although the association of codon 634 mutations with the development of pheochromocytoma is strong, it is not absolute. The possibility that familial MTC families with codon 634 mutations may not have been large enough to manifest the full phenotypic spectrum appears unlikely, as one of the familial M T C families in the Mulligan et al. study had eight individuals with M T C and no evidence of pheochromocytoma or parathyroid disease.
MTC in Patient Without a Codon 609 Mutation One of the affected individuals in this family (IV-17) had an unequivocally positive PG stimulation test (basal, 1.5-minute, 4.5-minute calcitonin levels of 37,500, 56,176, and >69,000 ng/L, respectively) but did not have a codon 609 mutation of the R E T gene. Individual IV-17 represents the first individual in a MEN2 kindred who has developed MTC but lacked the R E T mutation present in the other affected family members. This patient's father (1II-21) had a marginally positive P G test (basal, 1.5-minute, and 4.5-minute calcitonin levels of 77,256, 187 ng/L) and extensive C C H at thyroidectomy but also lacked the 609 mutation. Germline mutations occurring in exons 10 and 11 and codon 918 have been excluded for IV-17. Interestingly, the MTC from patient IV-17 had a somatic mutation of codon 918, a mutation frequently found in sporadic MTC [14,15]. This suggests but does not prove that his MTC is of sporadic origin.
Efficacy of PG Stimulation Testing in the Identification of Carriers Patients with MEN2A, MEN2B, and familial MTC develop extensive CCH with age. Medullary thyroid cancer arises from one or more foci of C C H in these patients. The PG stimulation test detects increased levels of calcitonin in patients with C C H and has served as an important screening tool for identification of carriers of R E T mutations in families with familial MTC, M E N 2 A , and MEN2B. Unfortunately, a lack of specificity and sensitivity limits the usefulness of this assay. False positive PG stimulation tests have been observed in individuals lacking germline R E T mutations in certain families including our own [18,
Molecular Diagnosis Vol. 2 No. 4 December 1997
21]. CCH is known to occur with aging, follicular thyroid neoplasms, and thyroiditis [25,26], and approximately 5 % of normal individuals may develop CCH [26-29]. This may account for some of the false positive tests observed. In this study, the lack of specificity of positive PG testing for carriers is demonstrated by the 9 patients with a positive PG test who did not have the 609 mutation. Thyroidectomies on 7 of these individuals revealed no pathologic abnormality in 1 patient, CCH in 5, and MTC in 1 (Table 2). The patient with MTC (IV-17) who did not have a germline mutation at codon 609 was discussed previously. Because PG stimulation testing induces diagnostic calcitonin increments in persons with histopathologic CCH, persons at risk for MTC are generally identified in more advanced stages of the natural history of their illness. R E T gene analysis presumably allows for the identification of at-risk persons well before either biochemical or clinical evidence of the disease is apparent. In our series, for example, 12 patients with a germline R E T mutation had negative PG tests. Thyroidectomy performed on 9 of these individuals revealed MTC and CCH in 2, CCH in 4, and no pathologic abnormality in 3 (Table 2). Individuals with a negative PG test and R E T mutation were, on average, 13 years younger than those who had a positive PG test and R E T mutation (23.5 years vs 36.5 years). This suggests that the relatively large number of "false positive" PG tests for carrier status is in part attributable to having tested individuals before CCH had developed. The two patients with MTC (III-8 and III-9), R E T mutations, and negative PG stimulation tests had unequivocally negative PG stimulation tests (basal, 1.5-minute, and 4.5-minute calcitonin levels of 51,104, 63 and <16, 33, <16 ng/ L, respectively). One of the patients with a R E T mutation, negative PG stimulation test, and CCH had a marginally negative test with basal, 1.5minute, and 4.5-minute calcitonin levels of 31, 92, and 57 ng/L, respectively. Thus, our experience confirms that of other groups, that PG testing is an imperfect screening tool because of a high false negative rate and inability to diagnose carriers prior to the development of CCH.
Conclusions We report on a large family with a germline R E T mutation affecting codon 609 whose members ex-
press MTC as the sole phenotypic manifestation. None of the carriers of the mutation in this family developed MTC before age 20. The age-related penetrance for development of the abnormal phenotype was 50% at age 44 and 100% by age 60. Pentagastrin stimulation testing is a helpful but imperfect screening test for this disease because of the potential for false positive and false negative results. When used in combination with R E T gene analysis, early detection of clinically inapparent disease is possible. Early treatment of gene carriers provides an opportunity for cure. Clinical followup evaluation of our family continues. Prolonged survival of carriers of germline mutations may result in detection of other phenotypic manifestations associated with R E T mutations that would not have otherwise been detected because of early death from MTC. Although other investigators have reported abnormal PG stimulation and CCH in family members not carrying the germline R E T mutation, we observed what appears to be a sporadic MTC in this familial MTC family.
Acknowledgments The authors gratefully acknowledge David Deram, Genetic Data Systems, for his work in preparing the pedigree drawing with Progeny software, and Paul Pribaz, Hereditary Cancer Program, Elkhart, Indiana, for assistance in preparing the manuscript.
Received July 16, 1997. Received in revised form August 22, 1997. Accepted August 22, 1997.
References 1. Sipple JH: The association of pheochromocytoma with carcinoma of the thyroid gland. Am J Med 1961;31:163-166 2. Williams ED, Pollock D J: Multiple mucosal neuromata with endocrine tumors: a syndrome allied to von Recklinghausen's disease. J Pathol Bacteriol 1966;91:71-80 3. Farndon JR, Leight GS, Dilley WG, Baylin SB, Smallridge RC, Harrison TS, Wells SA: Familial medullary thyroid carcinoma without associated endocrinopathies: a distinct clinical entity. Br J Surg 1986;73:278-281
Familial Medullary Thyroid Cancer
4. Mulligan LM, Eng C, Healey CS, Clayton D, Kwok J J, Gardner E, Ponder MA, Frilling A, Jackson CE, Lehnert H, Neumann HPH, Thibodeau SN, Ponder BAJ: Specific mutations of the R E T proto-oncogene are related to disease phenotype in MEN2A and FMTC. Nature Genet 1994;6:7074 5. Mathew CGR Chin KS, Easton DE Thorpe K, Carter C, Liou GI, Fong SL, Bridges CD, Haak H, Nieuwenhuijzen Kruseman AC, Shifter S, Hansen HH, Telenius H, Telenius-Berg M, Ponder BAJ: A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature 1987;328:527-528 6. Simpson NE, Kidd KK, Goodfellow P J, McDermid H, Myers S, Kidd JR, Jackson CE, Duncan AMV, Farrer LA, Brasch K, Castiglione C, Genel M, Gertner J, Greenberg CR, Gusella JF, Holden JJA, White BN: Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage. Nature 1987;328:528-530 7. Norum RA, Lafreniere RG, O'Neal LW, Nikola TF, Delaney JR Sisson JC, Sobel H, Lenoir GM, Ponder BAJ, Willard HI:, Jackson CE: Linkage of the multiple endocrine neoplasia type 2B gene (MEN2B) to chromosome 10 markers linked to MEN2A. Genomics 1990;8:313-317 8. Lairmore TC, Howe JR, Korte JA, Dilley WG, Aine L, Aine E, Wells, SA Jr, Donis-Keller H: Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to the same region of chromosome 10 as multiple endocrine neoplasia type 2A. Genomics 1991;9:181-192 9. Quadro L, Panariello L, Salvatore D, Carlomagno F, Del Prete M, Nunziata V, Colantuoni V, Di Giovanni G, Brandi ML, Mannelli M, Gheri R, Verga U, Libroia A, Berger N, Fusco A, Gricco M, Santoro M: Frequent R E T proto-oncogene mutations in multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 1994;72:590-594 10. Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lairmore TC, Howe JR, Moley JF, Goodfellow R Wells SA Jr: Mutations in the R E T proto-oncogene are associated with MEN2A and FMTC. Hum Mol Genet 1993;2:851-856 11. Mulligan LM, Kwok JBJ, Healey CS, Elsdon M J, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BAJ: Germ-line mutations of the R E T proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-460 12. Eng C, Smith DR Mulligan LM, Healey CS, Zvelebil MJ, Stonehouse T J, Ponder MA, Jackson CE, Waterfield MD, Ponder BAJ: A novel point mutation in the tyrosine kinase domain of the R E T proto-oncogene in sporadic medullary thyroid car-
Hailing et al.
cinoma and in a family with FMTC. Oncogene 1995;10:509-513 Bolino A, Schuffenecker I, Luo Y, Seri M, Silengo M, Tocco T, Chabrier G, Houdent C, Murat A, Schlumberger M, Tourniare J, Lenoir GM, Romeo G: R E T mutations in exons 13 and 14 of FMTC patients. Oncogene 1995;10:2415-2419 Hofstra RMW, Landsvater RM, Ceccherini I, Stulp RR Stelwagen T, Luo Y, Pasini B, H6ppener JWM, van Amstel HKR Romeo G, Lips CJM, Buys CHCM: A mutation in the R E T proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 1994;367:375-376 Eng C, Smith DR Mulligan LM, Nagia MA, Healey CS, Ponder MA, Gardner E, Scheumann GFW, Jackson CE, Tunnacliffe A, Ponder BAJ: Point mutation within the tyrosine kinase domain of the R E T proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum Mol Genet 1994;3:237-241 Gagel RF, Tashjian AH, Cummings T, Papathanasopoulos, N, Kaplan MM, DeLellis RA, Wolfe HJ, Reichlin S: The clinical outcome of prospective screening for multiple endocrine neoplasia type 2A. N Engl J Med 1988;318:478-484 Thomas PM, Gagel RF: Advances in genetic screening for multiple endocrine neoplasia type 2 and thc implications for management of children at risk. Endocrinologist 1994;4:140-146 Lips CJM, Landsvater RM, H6ppener JWM, Geerdink RA, Blijham G, Jansen-Schillhorn van Veen JM, Berends MJH, Beemer FA, Brouwers-Smalbraak J, Jansen RPM, van Amstel PK, van Vroonhoven TJMV, Vroom TM: Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A. N Engl J Med 1994;331:828-835 Lips CJM, Minder WH, Leo JR, Alleman A, Hackeng WHL: Evidence of multicentric origin of the multiple endocrine neoplasia syndrome type 2A (Sipple's syndrome) in a large family in The Netherlands. Am J Med 1978;64:569-578 Guyetant S, Wion-Barbot N, Rousselet M, Bigorgne J-C, Sainte-Andre J-P: C-cell hyperplasia associated with chronic lymphocytic thyroiditis: a retrospective quantitative study of 112 cases. Hum Pathol 1994;25:514-521 Tsai M-S, Ledger GA, Khosla S, Gharib H, Thibodeau SN: Identification of multiple endocrine neoplasia, type 2 gene carriers using linkage analysis and analysis of the R E T proto-oncogene. J Clin Endocrinol Metab 1994;78:1261-1264 Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Star Assoc 1958;53:457-481
Molecular Diagnosis Vol. 2 No. 4 December 1997
23. Wells SA, Donis-Keller H: Current perspectives on the diagnosis and management of patients with multiple endocrine neoplasia type 2 syndromes. Endocrinol Metab Clinics North Am 1994;23:215-228 24. Easton DE Ponder MA, Cummings T, Gagel RE Hansen HH, Reichlin S, Tashjian AH, TeleniusBerg M, Ponder BAJ, the Cancer Reasearch Campaign Medullary Thyroid Group: The clinical and screening age-at-onset distribution for the MEN-2 syndrome. Am J Hum Genet 1989;44:208-215 25. Scopsi L, Di Palma S, Ferrari C, Holst JJ, Rehfeld JF, Rilke F: C-cell hyperplasia accompanying thyroid diseases other than medullary carcinoma: an immunocytochemical study by means of antibodies
to calcitonin and somatostatin. Mod Pathol 1991; 4:297-304 Gibson WGH, Peng T-C, Croker BP: Age-associated C-cell hyperplasia in the human thyroid. Am J Pathol 1982;106:388-393 Gibson WCH, Peng T-C, Croker BP: C-cell nodules in adult human thyroid. Am J Clin Pathol 1981;75: 347-350 O'Toole K, Fenoglio-Preiser C, Pushparaj N: Endocrine changes associated with the human aging process. Hum Pathol 1985;16:991-999 Wolfe HJ, DeLellis RA: Familial medullary thyroid carcinoma and C-cell hyperplasia. Clini Endocrinol Metab 1981;10:351-365