Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex.

Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas. CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22-24 (refs 6, 7). Because of its similarities to the McCune-Albright syndrome and other features, such as paradoxical responses to endocrine signals, genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-alpha (RIalpha), including a polymorphic site within its 5' region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.

Trisomy 7-harbouring non-random duplication of the mutant MET allele in hereditary papillary renal carcinomas.

The gene defect for hereditary papillary renal carcinoma (HPRC) has recently been mapped to chromosome 7q, and germline mutations of MET (also known as c-met) at 7q31 have been detected in patients with HPRC (ref. 2). Tumours from these patients commonly show trisomy of chromosome 7 when analysed by cytogenetic studies and comparative genomic hybridization (CGH). However, the relationship between trisomy 7 and MET germline mutations is not clear. We studied 16 renal tumours from two patients with documented germline mutations in exon 16 of MET. Fluorescent in situ hybridization (FISH) analysis showed trisomy 7 in all tumours. To determine whether the chromosome bearing the mutant or wild-type MET gene was duplicated, we performed duplex PCR and phosphoimage densitometry using polymorphic microsatellite markers D7S1801 and D7S1822, which were linked to the disease gene locus, and D1S1646 as an internal control. We determined the parental origin of chromosome alleles by genotyping parental DNA. In all 16 tumours there was an increased signal intensity (2:1 ratio) of the microsatellite allele from the chromosome bearing the mutant MET compared with the allele from the chromosome bearing the wild-type MET. Our study demonstrates a non-random duplication of the chromosome bearing the mutated MET in HPRC and implicates this event in tumorigenesis.

Perivascular cells harboring multiple endocrine neoplasia type 1 alterations are neoplastic cells in angiofibromas.

Although neoplasia is caused by clonal proliferation of cells, the resulting tumors are frequently heterogeneous, being composed of both neoplastic and reactive cells. Therefore, identification of tumors as neoplastic processes is frequently obscured. We studied cutaneous angiofibroma, which is a tumor of unknown etiology. Combined analysis using immunohistochemistry, selective tissue microdissection, fluorescence in situ hybridization, sequencing analysis, and deletion analysis of the multiple endocrine neoplasia type 1 locus succeeded in the identification of a population of genetically altered, neoplastic cells in these tumors. This approach may be valuable in the future in identifying the etiology of other tumors of unknown etiology.

Pluripotent tumor cells in benign pituitary adenomas associated with multiple endocrine neoplasia type 1.

Analysis of human tumor cells in vitro enhances the study of numerous neoplastic conditions. However, it has been difficult to establish long-term cultures of adenoma cells, especially those of neuroendocrine origin, because the endocrine cells survive only briefly in culture, and fibroblasts overgrow the culture dish in 1 or 2 weeks. We describe cells isolated from pituitary adenomas in two patients with multiple endocrine neoplasia type 1 in which cells with a mesenchymal phenotype evolved from pituitary tumor cells. It appears that these poorly differentiated cells arose from multipotent adenoma cells. This represents a path of cell differentiation not observed previously in humans and may help explain the diverse nature of the benign tumors in multiple endocrine neoplasia type 1.

Duplication of the mutant RET allele in trisomy 10 or loss of the wild-type allele in multiple endocrine neoplasia type 2-associated pheochromocytomas.

Inherited mutations of the RET proto-oncogene are tumorigenic in patients with multiple endocrine neoplasia type 2 (MEN 2). However, it is not understood why only few of the affected cells in the target organs develop into tumors. Genetic analysis of nine pheochromocytomas from five unrelated patients with MEN 2 showed either duplication of the mutant RET allele in trisomy 10 or loss of the wild-type RET allele. Our results suggest a "second hit" causing a dominant effect of the mutant RET allele, through either duplication of the mutant allele or loss of the wild-type allele, as a possible mechanism for pheochromocytoma tumorigenesis in patients with MEN 2.

Constitutional von Hippel-Lindau (VHL) gene deletions detected in VHL families by fluorescence in situ hybridization.

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer syndrome predisposing to a variety of tumor types that include retinal hemangioblastomas, hemangioblastomas of the central nervous system, renal cell carcinomas, pancreatic cysts and tumors, pheochromocytomas, endolymphatic sac tumors, and epididymal cystadenomas [W. M. Linehan et al., J. Am. Med. Assoc., 273: 564-570, 1995; E. A. Maher and W. G. Kaelin, Jr., Medicine (Baltimore), 76: 381-391, 1997; W. M. Linehan and R. D. Klausner, In: B. Vogelstein and K. Kinzler (eds.), The Genetic Basis of Human Cancer, pp. 455-473, McGraw-Hill, 1998]. The VHL gene was localized to chromosome 3p25-26 and cloned [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. Germline mutations in the VHL gene have been detected in the majority of VHL kindreds. The reported frequency of detection of VHL germline mutations has varied from 39 to 80% (J. M. Whaley et al., Am. J. Hum. Genet., 55: 1092-1102, 1994; Clinical Research Group for Japan, Hum. Mol. Genet., 4: 2233-2237, 1995; F. Chen et al., Hum. Mutat., 5: 66-75, 1995; E. R. Maher et al., J. Med. Genet., 33: 328-332, 1996; B. Zbar, Cancer Surv., 25: 219-232, 1995). Recently a quantitative Southern blotting procedure was found to improve this frequency (C. Stolle et al., Hum. Mutat., 12: 417-423, 1998). In the present study, we report the use of fluorescence in situ hybridization (FISH) as a method to detect and characterize VHL germline deletions. We reexamined a group of VHL patients shown previously by single-strand conformation and sequencing analysis not to harbor point mutations in the VHL locus. We found constitutional deletions in 29 of 30 VHL patients in this group using cosmid and P1 probes that cover the VHL locus. We then tested six phenotypically normal offspring from four of these VHL families: two were found to carry the deletion and the other four were deletion-free. In addition, germline mosaicism of the VHL gene was identified in one family. In sum, FISH was found to be a simple and reliable method to detect VHL germline deletions and practically useful in cases where other methods of screening have failed to detect a VHL gene abnormality.

Mutations of the MEN1 tumor suppressor gene in pituitary tumors.

Although pituitary adenomas are monoclonal proliferations, somatic mutations involving genes that govern cell proliferation or hormone production have been difficult to identify. The genetic etiology of most pituitary tumors, therefore, remains unknown. Pituitary adenomas can develop sporadically or as a part of multiple endocrine neoplasia type 1 (MEN1). Recently, the gene responsible for MEN1 was cloned. To elucidate the potential etiological role of the MEN1 gene in pituitary tumorigenesis, 39 sporadic pituitary adenomas from 38 patients and 1 pituitary adenoma from a familial MEN1 patient were examined for MEN1 gene mutations and allelic deletions. Four of 39 sporadic pituitary adenomas showed a deletion of one copy of the MEN1 gene, and a specific MEN1 gene mutation in the remaining gene copy was detected in 2 of these tumors. The corresponding germ-line sequence was normal in all sporadic cases. A specific MEN1 mutation was detected in a pituitary adenoma and corresponding germ-line DNA in a patient with familial MEN1. An allelic deletion of the remaining copy of the MEN1 gene was also found in the patient's tumor. Genetic alterations of the MEN1 gene represent a candidate pathogenetic mechanism of pituitary tumorigenesis. The data suggest that somatic MEN1 gene mutations and deletions play a causative role in the development of a subgroup of sporadic pituitary adenomas.

Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas.

Gastrinomas and insulinomas are frequent in multiple endocrine neoplasia type 1 (MEN1). The MEN1 tumor suppressor gene was recently identified. To elucidate the etiological role of the MEN1 gene in sporadic enteropancreatic endocrine tumorigenesis, we analyzed tumors (28 gastrinomas and 12 insulinomas) from 40 patients for MEN1 gene mutations and allelic deletions. One copy of the MEN1 gene was found to be deleted in 25 of 27 (93%) sporadic gastrinomas and in 6 of 12 (50%) sporadic insulinomas. MEN1 gene mutations were identified in 9 of 27 (33%) sporadic gastrinomas and 2 of 12 (17%) insulinomas and were not seen in corresponding germ-line DNA sequence. A specific MEN1 mutation was detected in one gastrinoma and in the corresponding germ-line DNA of a patient who had no family history of MEN1. Somatic MEN1 gene mutations and deletions play a critical role in the tumorigenesis of sporadic gastrinomas and may also contribute to the development of a subgroup of insulinomas.

Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas.

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

Allelic loss of 11q13 as detected by MEN1-FISH is not associated with mutation of the MEN1 gene in lymphoid neoplasms.

Deletions and rearrangements involving the long arm of chromosome 11 are not infrequent occurrences in the non-Hodgkin's lymphomas. Recently, a tumor suppressor gene, the multiple endocrine neoplasia type 1 gene (MEN1) was cloned and mapped to chromosome 11q13. To assess the potential involvement of this gene in lymphomagenesis, we examined 94 primary cases of lymphoma and 12 cell lines by a combination of fluorescent in situ hybridization and PCR-SSCP analysis. In our initial analysis of 41 primary B or T lymphomas, MEN1 FISH analysis revealed allelic deletions in 15 cases (three of four B cell chronic lymphocytic leukemias, six of 15 follicular lymphomas, three of nine diffuse large B cell lymphomas, two of five mantle cell lymphomas, one of four Burkitt's lymphoma). To discern whether the MEN1 gene was in fact the target of the deletions, we assessed 20 of these 41 cases and an additional 74 primary lymphomas and 12 cell lines for MEN1 gene mutations using PCR-SSCP analysis. Abnormal SSCP patterns were found in exon 2 in two of the primary lymphoma cases and in one of the cell lines, but not in any of the original cases that showed MEN1 deletions by FISH. Furthermore, sequencing analysis revealed that the abnormal SSCP patterns in exon 2 were the result of a previously described genetic polymorphism (S145S: AGC --> ACT), and in one sample, the result of this S145S polymorphism associated with a second nucleotide substitution at position 498 which left the encoded amino acid unchanged. Our study indicates that the 11q13 locus is a frequent target of deletion in lymphoid neoplasms, but that there are no associated mutations of the MEN1 gene. This suggests that the 11q deletions either target another gene in lymphomas, or that the MEN1 gene is inactivated through means other than mutation.

11q13 allelic loss in pituitary tumors in patients with multiple endocrine neoplasia syndrome type 1.

Pituitary adenomas may develop sporadically or as part of the multiple endocrine neoplasia type 1 (MEN 1) syndrome. The gene responsible for MEN 1 syndrome was recently identified and cloned. Low rates of MEN 1 gene mutations and deletions have been reported in sporadic pituitary adenomas. To elucidate the role of the MEN 1 gene in the pathogenesis of MEN 1-associated pituitary tumors, we examined pituitary adenomas from 11 MEN 1 patients for the presence of 11q13 allelic loss. Ten of the 11 pituitary tumors were informative by PCR-based loss of heterozygosity analysis. Using a combination of family pedigree analysis and restriction analysis directed at the mutated allele in 8 of the 10 informative cases, it was demonstrated in all 8 cases that it is the wild-type allele that undergoes deletion. All 11 tumors, 4 of which were growth hormone secreting, were additionally analyzed for mutation in the Gs alpha subunit (gsp) gene. None of the tumors (0 of 11 tumors) revealed a gsp gene mutation. Therefore, genetic alterations of the MEN 1 gene seem to play a dominant role in MEN 1-associated pituitary tumorigenesis, whereas gsp gene mutations do not seem to be a frequent event in either growth hormone-secreting or other types of MEN 1-associated pituitary tumors. These results suggest that MEN 1-associated pituitary tumors develop via genetic pathways that differ from those of most sporadic pituitary tumors.

Chromosome 2 (2p16) abnormalities in Carney complex tumours.

Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia and lentiginosis syndrome characterised by spotty skin pigmentation, cardiac, skin, and breast myxomas, and a variety of endocrine and other tumours. The disease is genetically heterogeneous; two loci have been mapped to chromosomes 17q22-24 (the CNC1 locus) and 2p16 (CNC2). Mutations in the PRKAR1A tumour suppressor gene were recently found in CNC1 mapping kindreds, while the CNC2 and perhaps other genes remain unidentified. Analysis of tumour chromosome rearrangements is a useful tool for uncovering genes with a role in tumorigenesis and/or tumour progression. CGH analysis showed a low level 2p amplification recurrently in four of eight CNC tumours; one tumour showed specific amplification of the 2p16-p23 region only. To define more precisely the 2p amplicon in these and other tumours, we completed the genomic mapping of the CNC2 region, and analysed 46 tumour samples from CNC patients with and without PRKAR1A mutations by fluorescence in situ hybridisation (FISH) using bacterial artificial chromosomes (BACs). Consistent cytogenetic changes of the region were detected in 40 (87%) of the samples analysed. Twenty-four samples (60%) showed amplification of the region represented as homogeneously stained regions (HSRs). The size of the amplicon varied from case to case, and frequently from cell to cell in the same tumour. Three tumours (8%) showed both amplification and deletion of the region in their cells. Thirteen tumours (32%) showed deletions only. These molecular cytogenetic changes included the region that is covered by BACs 400-P-14 and 514-O-11 and, in the genetic map, corresponds to an area flanked by polymorphic markers D2S2251 and D2S2292; other BACs on the centromeric and telomeric end of this region were included in varying degrees. We conclude that cytogenetic changes of the 2p16 chromosomal region that harbours the CNC2 locus are frequently observed in tumours from CNC patients, including those with germline, inactivating PRKAR1A mutations. These changes are mostly amplifications of the 2p16 region, that overlap with a previously identified amplicon in sporadic thyroid cancer, and an area often deleted in sporadic adrenal tumours. Both thyroid and adrenal tumours constitute part of CNC indicating that the responsible gene(s) in this area may indeed be involved in both inherited and sporadic endocrine tumour pathogenesis and/or progression.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus.

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

"Molecular rulers" for calibrating phenotypic effects of telomere imbalance.

As a result of the increasing use of genome wide telomere screening, it has become evident that a significant proportion of people with idiopathic mental retardation have subtle abnormalities involving the telomeres of human chromosomes. However, during the course of these studies, there have also been telomeric imbalances identified in normal people that are not associated with any apparent phenotype. We have begun to scrutinize cases from both of these groups by determining the extent of the duplication or deletion associated with the imbalance. Five cases were examined where the telomere rearrangement resulted in trisomy for the 16p telomere. The size of the trisomic segment ranged from approximately 4-7 Mb and the phenotype included mental and growth retardation, brain malformations, heart defects, cleft palate, pancreatic insufficiency, genitourinary abnormalities, and dysmorphic features. Three cases with telomeric deletions without apparent phenotypic effects were also examined, one from 10q and two from 17p. All three deletions were inherited from a phenotypically normal parent carrying the same deletion, thus without apparent phenotypic effect. The largest deletion among these cases was approximately 600 kb on 17p. Similar studies are necessary for all telomeric regions to differentiate between those telomeric rearrangements that are pathogenic and those that are benign variants. Towards this goal, we are developing "molecular rulers" that incorporate multiple clones at each telomere that span the most distal 5 Mb region. While telomere screening has enabled the identification of telomere rearrangements, the use of molecular rulers will allow better phenotype prediction and prognosis related to these findings.

Effect of cardiopulmonary bypass on circulating concentrations of leucocyte elastase and free radical activity.

Circulating concentrations of leucocyte elastase and free radical activity were measured in 11 adults undergoing cardiopulmonary bypass. In all patients the bypass procedure was associated with pronounced changes in plasma elastase concentrations, and peak enzyme concentrations correlated closely with the duration of bypass (r = 0.91, p less than 0.001). Serial measurement of octadeca-9, 11-dienoic acid, a non-peroxide marker of free radical activity, showed significant changes only in the plasma free fatty acid fraction, suggesting a direct relation to the action of heparin rather than to the bypass procedure as such. These studies support the hypothesis that neutrophil activation plays a central role in the organ dysfunction that may complicate cardiopulmonary bypass and suggest that elastase release rather than free radical generation may be the appropriate marker of the event.

The human vitamin D receptor gene (VDR) is localized to region 12cen-q12 by fluorescent in situ hybridization and radiation hybrid mapping: genetic and physical VDR map.

The vitamin D receptor (VDR) is a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The VDR gene was previously mapped to human chromosome 12q13-12q14, but its precise physical and genetic localization are unknown. The present study reports the mapping of the human VDR gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene, and physical mapping of the VDR gene by fluorescent in situ hybridization (FISH). RH analysis placed the VDR gene locus at chromosome 12cen-q12, flanked by Stanford Human Genome Center (SHGC) 30216 and SHGC 9798 (D12S1892) markers. FISH analysis of a BAC containing the VDR gene confirmed its centromeric location. Thus, we have identified a BAC and genetic markers which can be used in the genetic analysis of the VDR gene and investigation of its involvement in osteoporosis and related disorders. We conclude that the VDR gene is centromeric to its previously reported locus on chromosome 12.

Cutaneous tumors in patients with multiple endocrine neoplasia type 1 show allelic deletion of the MEN1 gene.

Multiple endocrine neoplasia type 1 (MEN1), the heritable tendency to develop tumors of the parathyroid, pituitary, and entero-pancreatic endocrine tissues, is the consequence of a germline mutation in the MEN1 gene. Endocrine tumors in these patients result when the mutant MEN1 allele is accompanied by loss of the normal MEN1 allele. Recently it was reported that MEN1 patients also exhibit several cutaneous tumors, including multiple angiofibromas, collagenomas, and lipomas. The purpose of this study was to examine skin lesions from patients with MEN1 for allelic loss of the MEN1 gene. Skin lesions from five patients with MEN1 were examined using fluorescence in situ hybridization. Six angiofibromas, three collagenomas, and one lipoma showed allelic deletion of the MEN1 gene. Allelic deletion was not observed in a melanocytic nevus or acrochordon from patients with MEN1. It was also not observed in an angiofibroma from a patient with tuberous sclerosis. These results suggest that loss of function of the wild-type MEN1 gene product plays a role in the development of angiofibromas, collagenomas, and lipomas in patients with MEN1.

Genetic and histologic studies of somatomammotropic pituitary tumors in patients with the "complex of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas" (Carney complex).

Carney complex (CNC) is a familial multiple neoplasia and lentiginosis syndrome with features overlapping those of McCune-Albright syndrome (MAS) and other multiple endocrine neoplasia (MEN) syndromes, MEN type 1 (MEN 1), in particular. GH-producing pituitary tumors have been described in individual reports and in at least two large CNC patient series. It has been suggested that the evolution of acromegaly in CNC resembles that of the other endocrine manifestations of CNC in its chronic, often indolent, progressive nature. However, histologic and molecular evidence has not been presented in support of this hypothesis. In this investigation, the pituitary glands of eight patients with CNC and acromegaly [age, 22.9+/-11.6 yr (mean +/- SD)] were studied histologically. Tumor DNA was used for comparative genomic hybridization (CGH) (four tumors). All tumors stained for both GH and prolactin PRL (eight of eight), and some for other hormones, including alpha-subunit. Evidence for somatomammotroph hyperplasia was present in five of the eight patients in proximity to adenoma tissue; in the remaining three only adenoma tissue was available for study. CGH showed multiple changes involving losses of chromosomal regions 6q, 7q, 11p, and 11q, and gains of 1pter-p32, 2q35-qter, 9q33-qter, 12q24-qter, 16, 17, 19p, 20p, 20q, 22p and 22q in the most aggressive tumor, an invasive macroadenoma; no chromosomal changes were seen in the microadenomas diagnosed prospectively (3 tumors). We conclude that, in at least some patients with CNC, the pituitary gland is characterized by somatotroph hyperplasia, which precedes GH-producing tumor formation, in a pathway similar to that suggested for MAS-related pituitary tumors. Three GH-producing microadenomas showed no genetic changes by CGH, whereas a macroadenoma in a patient, whose advanced acromegaly was not cured by surgery, showed extensive CGH changes. We speculate that these changes represent secondary and tertiary genetic "hits" involved in pituitary oncogenesis. The data (1) underline the need for early investigation for acromegaly in patients with CNC; (2) provide a molecular hypothesis for its clinical progression; and (3) suggest a model for MAS- and, perhaps, MEN 1-related GH-producing tumor formation.

Human CYP11B2 (aldosterone synthase) maps to chromosome 8q24.3.

Aldosterone synthase (AS) is encoded by the CYP11B2 gene, a candidate for familial hypertension. CYP11B2 was previously mapped to chromosome 8q but its precise localization is necessary for genetic studies of hypertension. The present study reports the genetic mapping of the human CYP11B2 gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene and its physical mapping by fluorescent in situ hybridization (FISH). The CYP11B2 locus is on the most distal segment of the long arm of chromosome 8, proximal to the microsatellite polymorphic marker D8S1704. This location, which was confirmed by FISH, is approximately 60cM telomeric to the currently listed human gene locus (chromosome 8q21-22) and corresponds to cytogenetic band 8q24.3. The BACs containing the gene and a high-resolution map of the CYP11B2 locus are useful for genetic studies of hypertension and other endocrine disorders.