Department of Medicine, University of Illinois (M.R.G., S.F.M., R.D.K., L.A.F.), ... Cook County Hospital and Rush Medical College (R.P.G.), Chicago, Illinois ...
0021-972X/99/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1999 by The Endocrine Society
Vol. 84, No. 1 Printed in U.S.A.
Loss of Heterozygosity on Chromosome 11q13 in Two Families with Acromegaly/Gigantism Is Independent of Mutations of the Multiple Endocrine Neoplasia Type I Gene* MONICA R. GADELHA, TONI R. PREZANT, KARINA N. UNE, ROBERTA P. GLICK, STANLEY F. MOSKAL II, MARIO VAISMAN, SHLOMO MELMED, RHONDA D. KINEMAN, AND LAWRENCE A. FROHMAN Department of Medicine, University of Illinois (M.R.G., S.F.M., R.D.K., L.A.F.), Chicago, Illinois 60612; the Division of Endocrinology and Metabolism, Cedars-Sinai Research Institute, University of California (T.R.P., S.M.), Los Angeles, California 90048; the Department of Medicine, Federal University of Rio de Janeiro (M.R.G., K.N.U., M.V.), Rio de Janeiro, Brazil; and the Department of Neurosurgery, Cook County Hospital and Rush Medical College (R.P.G.), Chicago, Illinois 60612 ABSTRACT Familial acromegaly/gigantism occurring in the absence of multiple endocrine neoplasia type I (MEN-1) or the Carney complex has been reported in 18 families since the biochemical diagnosis of GH excess became available, and the genetic defect is unknown. In the present study we examined 2 unrelated families with isolated acromegaly/gigantism. In family A, 3 of 4 siblings were affected, with ages at diagnosis of 19, 21, and 23 yr. In family B, 5 of 13 siblings exhibited the phenotype and were diagnosed at 13, 15, 17, 17, and 24 yr of age. All 8 affected patients had elevated basal GH levels associated with high insulin-like growth factor I levels and/or nonsuppressible serum GH levels during an oral glucose tolerance test. GHRH levels were normal in affected members of family A. An invasive macroadenoma was found in 6 subjects, and a microadenoma was found in 1 subject from family B. The sequence of the GHRH receptor complementary DNA in 1 tumor from family A was normal. There was no history of consanguinity in either family, and the past medical history and laboratory results excluded MEN-1 and the Carney complex in all
affected and unaffected screened subjects. Five of 8 subjects have undergone pituitary surgery to date, and paraffin-embedded pituitary blocks were available for analysis. Loss of heterozygosity on chromosome 11q13 was studied by comparing microsatellite polymorphisms of leukocyte and tumor DNA using PYGM (centromeric) and D11S527 (telomeric), markers closely linked to the MEN-1 tumor suppressor gene. All tumors exhibited a loss of heterozygosity at both markers. Sequencing of the MEN-1 gene revealed no germline mutations in either family, nor was a somatic mutation found in tumor DNA from one subject in family A. The integrity of the MEN-1 gene in this subject was further supported by demonstration of the presence of MEN-1 messenger ribonucleic acid, as assessed by RT-PCR. These data indicate that loss of heterozygosity in these affected family members appears independent of MEN-1 gene changes and suggest that a novel (tissue-specific?) tumor suppressor gene(s) linked to the PYGM marker and expressed in the pituitary is essential for regulation of somatotrope proliferation. (J Clin Endocrinol Metab 84: 249 –256, 1999)
T
HE MAJORITY of pituitary somatotropic adenomas are sporadic, occurring at an annual incidence of 3 cases/ million subjects and a prevalence of 40 – 60 cases/million (1, 2). These tumors are initiated by a somatic mutation in a single somatotrope followed by monoclonal proliferation (3). Subsequent accumulation of additional mutations causes activation of oncogenes and/or inactivation of tumor suppressor genes, leading to the progression of neoplasia (4, 5). It is also thought that hypothalamic and growth factors contribute to the progression of pituitary neoplasia (6). Only a small number of somatotropinomas occur with a familial aggregation, either as isolated acromegaly/gigan-
tism or as a component of a multiple endocrine neoplasia complex, including the Carney complex and the Wermer syndrome [multiple endocrine neoplasia type 1 (MEN-1)]. The Carney complex exhibits an autosomal dominant phenotype characterized by myxomas, spotty skin pigmentation, and endocrine tumors (testicular, adrenal, and pituitary) (7, 8), in which up to 21% of patients have GH-secreting pituitary tumors (9). The Carney complex is genetically heterogeneous (10), with one of the responsible genes linked to chromosome 2p16 (11). MEN-1, in contrast, is an autosomal dominant predisposition to hyperplasia/tumor of the parathyroid glands, endocrine pancreas, and anterior pituitary. Forty-five percent of MEN-1 patients develop pituitary adenomas, of which 10% are somatotropinomas and 60% are prolactinomas (12). In 1988, Larsson et al. (13) mapped the MEN-1 locus to chromosome 11q13, and 9 yr later, Chandrasekharappa et al. (14) cloned the MEN-1 gene, which encodes a 610-amino acid protein (menin) of unknown function. Agarwal et al. (15) reported a high prevalence of germline mutations in the coding region of the MEN-1 gene in 47 members of 50 MEN-1
Received July 28, 1998. Revision received September 18, 1998. Accepted September 30, 1998. Address all correspondence and requests for reprints to: Lawrence A. Frohman, M.D., Department of Medicine (M/C 787), University of Illinois, 840 South Wood, Chicago, Illinois 60612. E-mail: frohman@ uic.edu. * This work was supported by USPHS Grants DK-30667 (to L.A.F.) and DK-50238 (to S.M.), the Bane Foundation (to L.A.F.), and the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico of the Ministry for Science and Technology of Brazil (to M.R.G.).
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families. This finding, coupled with the fact that loss of heterozygosity (LOH) in chromosome 11q13 occurs in pancreatic (13), parathyroid (16), and pituitary tumors (17) of MEN-1 patients, suggests that this syndrome is initiated by the complete inactivation of a tumor suppressor gene (MEN1). This is in accord with Knudson’s “two-hit” theory, which states that initiation of neoplasia requires independent inactivation events in each copy of the tumor suppressor gene; the first hit is an inherited allelic germline mutation, and the second is a somatic deletion resulting in LOH (18). Isolated somatotropinomas are an extremely rare subset of familial pituitary tumors. This disease is defined as the occurrence of at least 2 cases of acromegaly or gigantism in a family that does not exhibit a multiple endocrine neoplasia syndrome (Carney complex or MEN-1). Only 42 cases of isolated familial acromegaly/gigantism occurring in 18 families have been reported in the literature since the biochemical diagnosis of GH excess became available (Table 1). The pattern of inheritance raises the possibility of multiple genetic defects. In 1997, Yamada et al. (19) reported specific LOH on chromosome 11q13 in pituitary adenomas of 2 brothers with gigantism, suggesting that familial acromegaly/gigantism may be a phenotypic variant of the MEN-1 syndrome. In the present study, we sought to explore this possibility by investigating LOH at 11q13, MEN-1 germline mutations, and MEN-1 gene expression in 2 unrelated kindreds with familial acromegaly/gigantism.
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results confirmed the diagnosis (Table 2). MRI revealed a macroadenoma with suprasellar extension, which was removed in January 1995 by transsphenoidal surgery, followed by pituitary irradiation. The 19yr-old third brother (4A) was also investigated because of physical stigmata of acromegaly. He complained of headaches and gradually enlarging feet (height, 175 cm). A laboratory diagnosis of acromegaly was made (Table 2); however, the patient declined further evaluation or treatment. GH and IGF-I levels of the unaffected parents and sister are presented in Table 2 for comparison. Plasma GHRH levels were normal in all subjects (data not shown). There was no history of consanguinity, and the past medical history and laboratory results excluded MEN-1 and the Carney complex in all affected and unaffected screened subjects.
Subjects and Methods Family A Family A, of Mexican origin, is composed of four siblings, three of whom are affected (Fig. 1, top panel). The index case, 2A, was a 23-yr-old man who sought medical help because of headaches, decreased vision, and progressively enlarging hands and feet. He appeared acromegalic, with a height of 173 cm, and the diagnosis was confirmed by elevated serum GH and insulin-like growth factor I (IGF-I) levels (Table 2). Magnetic resonance imaging (MRI) revealed a macroadenoma with suprasellar extension. He underwent surgical resections in June 1993 (craniotomy) and July 1994 (transsphenoidal), followed by radiotherapy. In 1994, the 21-yr-old second brother (3A) was also suspected of acromegaly when he accompanied his brother (2A) to the hospital for a clinic visit. Subject 3A presented with decreased vision and had physical attributes characteristic of acromegaly (height, 165 cm). His laboratory
FIG. 1. Pedigree of family A (top panel) and family B (bottom panel). Generations available for study are indicated by Roman numerals, I and II. In the second generation, the subjects are represented in an age-descending order. Affected subjects are shown in shaded symbols. Arrows denote index cases. The age at diagnosis or death is shown below the symbols. The question mark denotes those individuals with a clinical history suggestive of a somatotropinoma or pituitary tumor. E, Female; M, male; B or , deceased.
TABLE 1. Previous reports of familial somatotropinomas Family
Patient relationship
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Brothers Brothers Brothers Uncle, nephew Brothers Uncle, nieces, and nephews Brother, sister Mother, son Mother, son 2 brothers, sister Father, son Sisters 3 cases Mother, son Father, daughter Father, son Uncle, 2 brothers Brothers
Age at diagnosis (yr)
22, 20 17, 11 28, 25 62, 50 26, 20, 15 27, 32, 38, 25, 9 58, 48 54, 26 45, 52 29, 17, 17 N/A, 32 14, 10 N/A 22, 10 34, 24 38, 15 18, 26, 22 61, 53
Clinical diagnosis
Reference
Gigantism Gigantism Gigantism Acromegaly Gigantism Acromegaly (3), prolactinoma, gigantism Acromegaly Acromegaly Acromegaly Gigantism, acromegaly (2) Acromegaly and hyperthyroidism, acromegaly Gigantism Acromegaly Acromegaly, gigantism Acromegaly Acromegaly, gigantism Gigantism Acromegaly
Levin et al., 1974 (29) Kurisaka et al., 1981 (30) Prescott et al., 1982 (31) Jones et al., 1984 (32) Abbassioun et al., 1986 (33) Pestell et al., 1989 (34) McCarthy et al., 1990 (35) McCarthy et al., 1990 (35) McCarthy et al. 1990 (35) Tamburrano et al., 1992 (36) Links et al., 1993 (37) Matsuno et al., 1994 (38) Xu et al., 1995 (39) Benlian et al., 1995 (40) Kakiya et al., 1997 (41) Tiulpakov et al., 1997 (42) Yamada et al., 1997 (19) Tanaka et al., 1998 (22)
FAMILIAL ACROMEGALY/GIGANTISM Family B Family B, of Brazilian origin, consisted of 13 siblings, 5 of whom are affected (Fig. 1, lower panel). The index case (4B) presented at 24 yr of age with galactorrhea, headaches, and progressively enlarging hands and feet that had developed over the previous 4 yr. She appeared acromegalic, with a height of 165 cm and a weight of 75.5 kg, and exhibited hyperhydrosis and left temporal hemianopsia. Elevated basal GH levels and lack of GH suppression by glucose administration confirmed the diagnosis (Table 3). The MRI revealed a macroadenoma with infra- and suprasellar extensions. Transsphenoidal surgery was performed in June 1995, followed by radiotherapy. During the clinical history, she reported that a brother (2B) and a sister (10B) were tall in stature. Subject 2B, a 29-yr-old man (height of 224 cm), was diagnosed at 17 yr of age and had subsequently undergone two frontal craniotomies and radiotherapy. Subject 10B, a 15-yr-old female, presented with headaches and primary amenorrhea. Her height was 185 cm, and her weight was 100 kg. Serum basal GH was elevated and was not suppressed by oral glucose (Table 3). The computed tomography scan showed a macroadenoma with suprasellar expansion. Transsphenoidal surgery was performed in August 1995, followed by radiotherapy. Subsequent clinical and laboratory screening of the asymptomatic family members (Table 3) revealed that subjects 11B and 12B also had evidence of GH hypersecretion. Subject 11B, a 17-yr-old male, with a height of 172 cm and a weight of 57.5 kg, had an elevated serum basal GH value that was not suppressed during an oral glucose tolerance test (Table 3). The computed tomography scan
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revealed a microadenoma. Subject 12B, a 13-yr-old female, with a height of 164 cm and a weight of 53 kg, had elevated basal GH and IGF-I levels, and serum GH levels were not suppressed by oral glucose administration (Table 3). A MRI subsequently revealed a 4.0 3 2.8 3 2.4-cm pituitary macroadenoma with suprasellar extension (Fig. 2), confirming the diagnosis of a GH-secreting tumor. Also of interest is subject 8B, who died at the age of 16 yr with a clinical history suggestive of pituitary apoplexy. In addition, the father’s brother was reported to have “coarse” features and died at the age of 18 yr from a “brain tumor”. Subject 9B died at 2 yr of age from an unrelated condition. There was no history of consanguinity, and the past medical history and laboratory results excluded MEN-1 and the Carney complex in all affected and unaffected screened subjects.
Tumor histology Paraffin tumor blocks were sectioned (5 mm) and stained with hematoxylin-eosin or subjected to immunocytochemistry for GH and PRL using the standard avidin-biotin-peroxidase complex technique. The primary antibodies used in the reaction have been previously characterized [GH, 1:5000 (20); PRL, 1:5000 (NIH)].
Analysis of LOH on chromosome 11q13 The study protocol was approved by the institutional review board of the University of Illinois at Chicago with the informed consent of all
TABLE 2. Family A: clinical and laboratory values
Subject
Affected 2A 3A 4A Unaffected Father Mother 1A
Sex
Age (yr)a
Ht (cm)a
M M M
23 21 19
173 165 175
34
N/A N/A 152
M F F
Laboratory resultsb PTH (pg/mL)
Ca (mg/dL)
Gastrin (pg/mL)
GH (ng/mL)
IGF-I (ng/mL)
PRL (ng/mL)
38 25 52
9.1 9.4
15 19 11
75 24 3.2
490 570 571
16 6.0 7.1
,0.1 ,0.1 2
50 20 83
a
Age and height at diagnosis for affected subjects or at time of clinical screening of unaffected subjects. b Normal values: PTH, 10 – 65 pg/mL; calcium, 8.5–10.5 mg/dL; gastrin, less than 41 pg/mL; GH, less than 5 ng/mL; IGF-I, less than 534 ng/mL (adults); PRL, less than 25 ng/mL. TABLE 3. Family B: clinical and laboratory values
Subject
Affected 2Bc 4B 10B 11B 12B Unaffected Father Mother 1B 3B 5B 6B 7B 13B a
Sex
Age (yr)a
Ht (cm)a
M F F M F
17 24 15 17 13
M F M F F F M F
62 54 33 29 23 23 21 10
Laboratory resultsb PTH (pg/mL)
Ca/P (mg/dL)
Gastrin (pg/mL)
Glucose (mg/dL)
GH (ng/mL)
224d 165 185 172 164
600e 21 25 25 39
9.0/4.4 8.9/5.6 8.4/4.0 9.0/3.6 9.7/6.6
30 123 36 61 104
99 94 136 84 97
34 74 65 8.3 24
169 157 N/A 165 159 159 170 142
24 27 35 39 32 33 12 12
8.9/3.2 9.1/3.1 9.2/3.7 9.3/3.4 9.4/3.1 9.0/3.5 9.5/2.8 8.8/3.6
118 62 48 76 83 115 75 26
84 97 90 89 80 77 81 72
6.6 2.5 2.6 70 8.3 2.6 2.4 4.0
OGTT (nadir) GH (ng/mL)
IGF-I (ng/mL)
PRL (ng/mL)
26 65 100 3.2 20
767 500c 495c 542 965
40 14 11 9.0 16
0.71 N/A N/A 0.91 ,0.05 0.06 ,0.05 0.24
80 209 246 258 321 345 410 220
3.0 5.3 5.0 18 15 14 6.3 9.6
Age and height at diagnosis for affected subjects or at time of clinical screening for unaffected subjects. Normal values: PTH, 12–70 pg/mL; Ca, 8.8 –11 mg/dL; P, 3–7 mg/dL (3–17 yr), 2.5– 4.8 (adults); gastrin, less than 90 pg/mL; glucose, 70 –110 mg/dL; GH, less than 5 ng/mL; IGF-I 95– 400 (6 –10 yr), 130 – 850 (11–18 yr), 50 – 400 (adults) ng/mL; PRL, less than 25 ng/mL. c Posttreatment values. d Height at time of diagnosis unknown. Value shown was at age 29 yr. e Measurement performed in a different laboratory with a normal range of 230 – 680 pg/mL. b
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with DNAzol (Life Technologies). The concentration of leukocyte DNA was determined spectrophotometrically. Tumor DNA (3 mL) or leukocyte DNA (500 ng) was amplified using primers specific for microsatellite markers, PYGM (CA GA) and D11S527 (Research Genetics, Inc., Huntsville, AL), both of which are on chromosome 11q13 and closely linked to the MEN-1 gene. The repeat CA GA is located within the muscle glycogen phosphorylase gene (PYGM), which is centromeric to the MEN-1 gene, whereas D11S527 is telomeric. The reverse primer of each oligonucleotide pair was end labeled with [g-32P]ATP using the 59 DNA Terminus Labeling System kit (Life Technologies). The PCR reaction mixture (20 mL) contained 1 3 PCR buffer (Perkin Elmer, Norwalk, CT), 1.0 mmol/L MgCl2, 0.2 mmol/L deoxy (d)-NTPs (Boehringer Mannheim, Indianapolis, IN), 0.25 mmol/L primers, and 1.5 U AmpliTaq (Perkin Elmer). A “hot start” was performed at 95 C for 90 s, and each of the 39 subsequent cycles consisted of denaturation at 94 C for 40 s, annealing at 62 C for 40 s, and extension at 72 C for 60 s, followed by a final extension at 72 C for 5 min. Leukocyte and tumor DNA products were electrophoresed in a 6% polyacrylamide-8.3 mol/L urea gel. The PCR products were visualized by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) or autoradiography. The predicted sizes of PYGM and D11S527 alleles were 156 –190 and 142–166 bp, respectively; the markers were considered informative if leukocyte DNA showed heterozygosity. The intensity of the alleles was measured by image analysis of the PhosphorImager (ImageQuant, Molecular Dynamics, Inc., Sunnyvale, CA). A greater than 50% decrease or the absence of one of the expected PCR products in the tumor sample compared with the leukocyte sample was interpreted as LOH.
MEN-1 gene sequencing
FIG. 2. Preoperative, gadolinium-enhanced, T1-weighted MRI of patient 12B (13 yr old) whose leukocyte DNA allelic pattern suggested a predisposition for pituitary neoplasia. An oral glucose tolerance test confirmed the diagnosis of GH hypersecretion. A coronal view is shown in the upper panel, and a sagittal view is shown in the lower panel. A macroadenoma (4.0 3 2.8 3 2.4 cm) is present with suprasellar extension and compression of the optic chiasm.
Tumor DNA was obtained from 15–20 sections (7 mm) from paraffinembedded tissue. Sections were deparaffinized and treated with 0.5 mg/mL proteinase K (Ambion, Inc., Austin, TX) in 200 mL 1 3 Perkin Elmer PCR buffer without MgCl2 (37 C). The proteinase K was heat inactivated at 95 C for 5 min, and the DNA was further purified using the QIAamp tissue kit (Qiagen, Santa Clarita, CA) and eluted with 100 mL 0.5 3 kit buffer (AE). Leukocyte DNA of one affected and one unaffected member of each family (3A, 1A, 4B, and 1B) and tumor DNA from subjects 3A and 4B were used for sequencing. Nine coding exons and 17 splice junctions of the MEN-1 gene were amplified by PCR in 50-mL reactions containing 150 ng leukocyte DNA or 5 mL tumor DNA, 1 3 PCR buffer with 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 10 pmol primer, and 2.5 U AmpliTaq Gold (Perkin Elmer). Dimethylsulfoxide (10%) was included for the amplification of exon 10. PCR conditions were 94 C for 10 min, followed by 40 cycles of 94 C for 30 s, 55– 60 C for 30 s, and 72 C for 1–2 min. The annealing temperature depended on primers, whereas the extension time varied with product length. PCR products are shown in Table 4 with their associated exon coordinates. The PCR products were either used directly or gel isolated (Qiagen) for sequence analysis with the [a-33P]dideoxy-NTP ThermoSequenase kit (Amersham, Arlington Heights, IL). Reactions were electrophoresed in 6% acrylamide-7 mol/L urea gels and autoradiographed. Dimethylsulfoxide (10%) was included for exon 2 (2652R) and exon 10 sequence reactions. The genomic DNA coordinates for the primers used in the sequencing reactions are provided in Table 4. Sequencing of the MEN-1 gene from tumor DNA required PCR amplification of shorter sequence motifs, also shown in Table 4.
MEN-1 gene expression subjects. Pituitary adenoma tissue was obtained from paraffin-embedded blocks (patients 2A, 3A, 2B, 4B, and 10B). Fifteen sections (5 mm) were deparaffinized and incubated with 100 –300 mL digestion buffer containing 50 mmol/L Tris-HCl (pH 8.5), 1 mmol/L ethylenediamine tetraacetate, 0.5% Tween-20 (Sigma Chemical Co., St. Louis, MO), and 0.1– 0.2 mg/mL proteinase K (Life Technologies, Grand Island, NY). Samples were incubated at 37 or 55 C for 120 h. After centrifugation, the supernatant was transferred to a fresh tube and proteinase K heat inactivated (95 C, 8 min). Organic extraction (phenol-chloroform) was necessary to obtain PCR products from tumor sample 10B. Peripheral blood samples were collected from all eight patients, their parents, and unaffected siblings. DNA was extracted from leukocytes
A small fragment of tumor from patient 3A was collected at surgery and stored at 270 C. Total ribonucleic acid (RNA) was extracted according to the Tri Reagent protocol (Molecular Research Center, Inc., Cincinnati, OH) with the exception that the aqueous phase was further purified by extraction with phenol-chloroform-isoamyl alcohol (25:24:1; pH 5.2; Fisher Scientific International, Inc., Pittsburgh, PA). RNA was resuspended in 10 mmol/L Tris-HCl (pH 7.6) and 1 mmol/L ethylenediamine tetraacetate, and RNA concentration was determined spectrophotometrically. Total RNA (1 mg) was reverse transcribed using the Superscript Preamplification System for First Strand Complementary DNA (cDNA) Synthesis Protocol (Life Technologies), primed with random hexamers
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TABLE 4. Genomic coordinates of PCR products and sequencing primers
Exon
Genomic coordinatesa
2
2265–2732
3 4 5 6 7 8 9 10
4297– 4505 4716– 4844 5177–5217 5298–5385 6025– 6161 6623– 6758 7196–7360 7578– 8874
Leukocyte DNA
2226–2473 1932–2652c 2429–2833 4096–5520 4096–5520 4096–5520 4096–5520 5911–7426 5911–7426 5911–7426 7339– 8090
Tumor DNA
Sequencing primersb
PCR products
2226F, 2226F, 2429F, 4259F, 4904R 5138F 5138F 5911F 6577F 7426R 7445F,
2473R 2473R 2652F, 2833R 4637R
7775F
PCR products
Sequencing primers
2226–2652 2429–2833
2226F 2429F, 2833R
4096– 4637 4480– 4904 5138–5520 5138–5520 5911– 6200 6577– 6801 7160–7426 7544–7862 7775– 8090
4259F, 4637R 4904R 5138F 5138F, 5520R 5911F 6577F 7160F, 7426R 7544F 7775F
a
All numbers correspond to the genomic sequence of MEN-1 (GenBank accession no. U93237). F, Forward; R, reverse. c This PCR product was used to sequence the 59-region of exon 2 in leukocyte DNA from patients 3A and 4B. b
(0.2 mg). One tenth (2 mL) of the RT reaction was amplified by PCR in a final volume of 50 mL that included 1 3 PCR buffer (Perkin Elmer), 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs (Boehringer Mannheim), 0.2 mmol/L primers, and 2.5 U AmpliTaq (Perkin Elmer). The PCR conditions were denaturation at 95 C for 3 min, followed by 40 cycles of 94 C for 1 min, 62 C for 1 min, 72 C for 2 min, and a final extension at 72 C for 15 min. A 25-mL aliquot of the PCR product was analyzed by gel electrophoresis using a 1.5% agarose gel containing ethidium bromide (0.5 mg/mL). The primers amplified a 382-bp product spanning nucleotides 252– 633 of the MEN-1 cDNA (GenBank accession no. U93236).
Results Tumor histology
Immunocytochemistry revealed that all tumors were strongly positive for GH (.90% of tumor cells). Tumors 2A, 3A, 2B, and 4B did not exhibit immunoreactivity for PRL, whereas tumor 10B showed scattered PRL-positive cells (,3% of cells). Analysis of LOH on chromosome 11q13
The microsatellite markers, PYGM and D11S527, were informative in both families. LOH on 11q13 was observed using PYGM in all five tumors studied (Figs. 3 and 4). In addition, all adenomas showed LOH at 11q13 using the D11S527 (data not shown), demonstrating the extent of chromosomal loss. The chromosomal segment that was deleted (transmitted by the mother in both families) is presumed to contain the wild-type copy of the tumor suppressor gene. Consequently, the germline mutation that predisposed these individuals to neoplasia was transmitted paternally (allele C; Figs. 3 and 4). Individual 13B, a 10-yr-old female, has inherited the putative mutant chromosome and is, therefore, at risk of developing a pituitary adenoma. MEN-1 gene sequencing
Sequencing of PCR-amplified constitutional DNA from an affected individual in each family (3A and 4B) revealed no germline mutations within the nine coding exons and the exon-intron boundaries of the MEN-1 gene. However, leukocyte DNA of two unaffected subjects had a sequence change in intron 7. Subject 1A was homozygous with an A to G change at position 6276, whereas 1B was heterozygous
FIG. 3. Analysis of LOH in chromosome 11q13 using the microsatellite marker PYGM in family A. Shown is a phosphorimage of a gel containing PCR-amplified genomic DNA from leukocytes (L) and tumors (T). The mother (M), father (F), and subjects 1A– 4A correspond to the pedigree presented in Fig. 1 (top panel). Lane 1 contains molecular size markers (base pairs). The position of each allele (A, B, and C) is designated to the right of the image. The allele presumed to be associated with the germline mutation (C) is designated by the asterisk.
at the same position (6276 A/G). These changes would not modify the splicing process and are presumed to be benign polymorphisms. In addition, no somatic mutation of the MEN-1 gene was detected in the pituitary adenoma of subject 3A, whereas sequencing of tumor DNA from subject 4B was not successful. MEN-1 gene expression
RNA from tumor 3A and a normal pituitary (autopsy) was subjected to RT-PCR using primers from exons 2 and 3 of the MEN-1 gene. PCR products from both the tumor and normal RNA showed a 382-bp band (Fig. 5), which corresponded to that predicted from the MEN-1 cDNA sequence (GenBank accession no. U93236). These results demonstrate that the MEN-1 gene is effectively transcribed in the pituitary adenoma. Discussion
The present report describes two unrelated families in which acromegaly/gigantism has been diagnosed in a total
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FIG. 4. Analysis of LOH in chromosome 11q13 using the microsatellite marker PYGM in family B. Shown is an autoradiogram of a gel containing PCRamplified genomic DNA from leukocytes (L) and tumors (T). The mother (M), father (F), and subjects 1B–13B correspond to the pedigree presented in Fig. 1 (bottom panel). The position of each allele (A, B, C, and D) is designated to the left of the image. Two other unaffected siblings (family B, subjects 5 and 7; Fig. 1) have alleles A/B and B/D, respectively (data not shown). The allele presumed associated with the germline mutation (C) is designated by the asterisk.
FIG. 5. RT-PCR of MEN-1 messenger RNA from a normal and a neoplastic (Tumor, 3A) pituitary. One microgram of RNA was reversed transcribed, and the cDNA was amplified by PCR using primers that spanned exons 2 and 3 of the MEN-1 gene. PCR products were separated on a 1.5% agarose gel containing ethidium bromide. The 382-bp PCR product was consistent with that predicted from the MEN-1 cDNA sequence.
of eight members. The clinical manifestations of excessive GH secretion became apparent between the ages of 13–24 yr in each of the affected members of these two kindreds. Pituitary tumors in at least six of the eight subjects were invasive somatotrope macroadenomas, whereas a microadenoma was detected in subject 11B (aged 17 yr). Only one adenoma also exhibited PRL immunoreactivity. In both families, the disease was expressed among members of a single generation, although in one, an earlier generation may also have been affected. None of the affected subjects in the present report exhibited features of either the MEN-1 syndrome or the Carney complex. Furthermore, the majority of pituitary adenomas associated with the MEN-1 syndrome are usually diagnosed in the third to fifth decade and are most commonly prolactinomas (12). The pedigree in both families is similar to that reported by Yamada et al. (19), who observed two brothers with GH-secreting tumors, whereas
the parent transmitting the gene (mother) was unaffected. Thus, the penetrance is incomplete and may reflect differences in genetic background, environmental factors, and/or the absence of LOH, which is a stochastic event. Because of the limited size of each kindred and the number of affected subjects in the present study, it was not possible to conclusively establish the mode of transmission. We, therefore, initially considered the possibility of an activating mutation of the GHRH receptor, as hyperactivity of the GHRH signal transduction system can lead to somatotrope hyperplasia and/or adenoma formation (21). However, sequencing of leukocyte DNA and tumor cDNA from members of family A failed to reveal mutations in the GHRH receptor gene (data not shown). The possibility of a loss of a tumor suppressor gene was considered by comparing the polymorphic markers, PYGM and D11S527, in leukocyte and tumor DNA. Both revealed LOH of 11q13 in all pituitary tumors tested, suggesting that a tumor suppressor gene important for somatotrope tumor formation is present at this locus. Despite the phenotypic dissimilarities between isolated familial acromegaly/gigantism and MEN-1 syndrome, the MEN-1 gene was considered a candidate because it has been shown to contain inactivating germline mutations in the majority of patients with pituitary tumors associated with the MEN-1 syndrome (14, 15). However, sequencing of the coding regions and the intron/exon boundaries of the MEN-1 gene showed no germline mutation in either family despite the presence of LOH at the 11q13 locus. These results are consistent with the recent report by Tanaka et al. (22) that detected no germline mutations within the coding region of the MEN-1 gene in two kindreds with familial acromegaly. We also explored the possibility that the progression of neoplasia in each kindred could be due to a secondary somatic mutation in the MEN-1 gene. To investigate this possibility, tumor DNA (3A) was sequenced, but was found to be normal. This is in contrast to the report by Tanaka et al. (22) of a nonsense somatic mutation in the MEN-1 gene in one of two brothers with somatotropinomas that exhibited LOH on 11q13. We did not perform complete intron and promoter region sequencing of the MEN-1 gene or investigate methylation-dependent gene silencing (23). Therefore, we cannot be certain that the level of MEN-1 gene expression is normal
FAMILIAL ACROMEGALY/GIGANTISM
in these families. However, the integrity of the MEN-1 gene in this subject (3A) is supported by our observation that the tumor expressed the MEN-1 gene, as assessed by RT-PCR. Taken together, these observations implicate a tumor suppressor gene located within chromosome 11q13 that is independent of MEN-1. LOH of 11q13 in sporadic pituitary adenomas is not associated with genetic mutations (24, 25) or abnormal expression of the MEN-1 gene (25). When combining the results of three published studies (24 –26), it appears that 11q13 allelic loss occurs more frequently in somatotropinomas (19%; 8 of 43) than in corticotropinomas (12%; 3 of 25), nonfunctioning or gonadotropinomas (11%; 8 of 72), prolactinomas (10%; 3 of 30), or thyrotropinomas (0 of 2). A recent report by Farrell et al. (27) suggests that the frequency of LOH within 11q13 in sporadic GH-producing tumors is as high as 38%. Mutational analysis of 13 GHsecreting tumors with allelic loss (24, 25, 27) revealed only 1 with an inactivating mutation of the MEN-1 gene (24). These findings together with the present report strongly suggest that a novel (tissue-specific?) tumor suppressor gene(s) is expressed in the pituitary that is essential for the modulation of somatotrope proliferation. The very high penetrance occurring at an early age in our subjects along with the fact that the majority (63%) of reported cases of familial somatotropinomas are diagnosed before the age of 30 yr imply that the loss of this putative tumor suppressor gene, located within 11q13, is the initiating event in somatotrope tumorigenesis. Furthermore, genetic analysis of sporadic pituitary adenomas has revealed a higher frequency of invasive compared to noninvasive tumors associated with a single allelic loss at 11q13 (5). This observation coupled with the fact that the adenomas in this report were aggressive suggest that this tumor suppressor gene is not only the initiator of tumorigenesis, but exerts an effect on neoplastic progression. Genes already identified within the 11q13 locus code for a variety of proteins that have important roles in cell cycle regulation and intracellular signaling, including BclI, galanin, vascular endothelial growth factor, B56 b-subunit of protein phosphatase 2A, fas-associating protein with death domain, b-adrenergic receptor kinase, phospholipase C, and fibroblast growth factor-4 [which has been associated with invasive prolactinomas (28)]. Which, if any, of these genes are involved in neoplastic transformation of the somatotrope is still unclear. Acknowledgments The authors thank Dr. Clicerio Gonzalez-Villalpando (Mexico City), Drs. Rosane Silva and Joel Grego Filho (Daflon Laboratory), Romulo Cortes Domingues and Lilian Sznajder (Rio de Janeiro), and Fran Norris (Chicago) for technical assistance.
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