CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene

Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.     

Expression of Kit in neurofibromin-deficient human Schwann cells: role in Schwann cell hyperplasia associated with type 1 neurofibromatosis

Type 1 Neurofibromatosis (NF1) is characterized by the formation of neurofibromas, benign tumors composed mainly of Schwann cells, which can turn malignant to form neurofibrosarcomas. Neurofibromin, the protein product of the Nf1 gene, is believed to act as a tumor suppressor, accelerating the conversion of the oncogene Ras to its inactive form. The absence of neurofibromin could therefore lead to higher Ras activity in Schwann cells, resulting in uncontrolled growth through a cascade of events not yet elucidated. We describe the abnormal expression of high levels of the Kit tyrosine kinase receptor in both NF1-derived Schwann cell lines and tissue, as compared to primary Schwann cells or schwannoma-derived cells. High levels of Kit expression in the neurofibrosarcoma-derived Schwann cells correlate with a decrease in neurofibromin expression. Using inhibitors of tyrosine kinase receptors, we found that proliferation of the neurofibrosarcoma-derived cells is dependent upon activation of a subclass of tyrosine-kinase receptors. The proliferation of these cells is not dependent upon an autocrine loop involving typical Schwann cell mitogens. Finally, the proliferation of the neurofibrosarcoma-derived Schwann cells can be increased by stimulation with Kit ligand. These data implicate Kit as one of the components leading to the Schwann cell hyperplasia observed in NF1.     

NF2 gene in neurofibromatosis type 2 patients

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (ezrin-radixin-moesin proteins). Even though penetrance of the disease is >95% and no genetic heterogeneity has been described, point mutations in the NF2 gene have been observed in only 34-66% of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients who express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.     

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.     

Correlation of alpha 2 beta 1 integrin expression with histological type and hormonal receptor status in breast carcinomas

Interactions between cells and extracellular matrix are mediated in part by a family of alpha beta heterodimeric molecules known as integrins. Immunohistochemical studies have shown that benign hyperplastic/neoplastic mammary epithelium expressed high levels of alpha 2 beta 1 collagen/laminin receptor. In contrast, malignant cells of breast carcinoma exhibited marked diminuition or loss of the alpha 2 beta 1 integrin. A correlation has been suggested between the loss of the alpha 2 beta 1 expression and the increased invasiveness of neoplastic cells. This study investigated the expression of alpha 2 beta 1 integrin and its extracellular ligand collagen TV by using monoclonal antibodies on the cryostat section of 124 invasive mammary carcinomas. Two patterns of alpha 2 beta 1 immunoreactivity, i.e. pericellular and basolateral, were identified in breast carcinomas and correlated with their histological type. In most invasive ductal carcinomas of no special type (NOS), integrin staining tended to decrease in both pericellular and basolateral aspects. Loss of basolateral staining for alpha 2 beta 1 integrin corresponded closely to the loss of immunoreactivity for collagen IV. Mucinous and medullary carcinomas showed strongly alpha 2 beta 1 pericellular staining, but no basolateral reactivity or collagen IV expression. Only two of the infiltrating lobular carcinomas expressed strong pericellular reactivity. In 82 ductal carcinomas NOS, the abnormally low expression/absence of alpha 2 beta 1 integrin correlated with estrogen and progesterone receptor negativity (p < 0.04 and p < 0.002, respectively). No correlation between integrin expression, histological grade, nodal involvement and proliferative activity was found. The results of the present study suggest that changes in alpha 2 beta 1 expression correlate with the histological type and hormonal receptor status in breast carcinomas. The clinical implications of these findings remain to be elucidated.     

Lipolytic effects of beta1, beta2 and beta3-adrenergic agonists in isolated human fat cells from omental and retroperitoneal adipose tissues

The presence of beta1- and beta2-adrenoceptors has been clearly established in human fat cells. There is some controversy about the presence and function of beta3-adrenoceptors. It is well established that there are marked regional variations in catecholamine-induced lipolysis. In this work the possibility that a beta3-adrenoceptor plays a significant role in the control of lipid mobilization is studied and also its importance in comparison to beta1- and beta2-adrenoceptors in isolated human fat cells, is evaluated, by measuring the in vitro lipolysis induced by dobutamine, salbutamol, metaproterenol, BRL 37344 and CGP 12177A. Human adipocytes from omental and retroperitoneal fat deposits exhibited an "atypical" beta-adrenergic response but, given the small lipolytic effect initiated by BRL 37344 and CGP 12177A, they are probably poorly equipped in functional beta3-adrenoceptors.     

Expression of the ERBB2/neu and neurofibromatosis type 1 gene products in reactive and neoplastic schwann cell proliferation

In the present study we investigated the expression of the ERBB2 protein and neurofibromin in human benign and malignant Schwann cell tumors, traumatic neuromas and peripheral nerves without pathological findings. By immunohistochemistry and Western analysis ERBB2 expression was not detectable in normal nerves but in proliferating Schwann cells of traumatic neuromas. While none of the malignant schwannomas exhibited ERBB2 expression, weak expression was seen in a small proportion of the benign schwannomas. Low levels of neurofibromin were detectable in normal nerves but not in traumatic neuromas or tumors. These data indicate an inverse expression pattern of ERBB2 and neurofibromin in human non-neoplastic Schwann cells, but not in neurinoma cells.     

Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence

The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.     

In situ distribution of integrin alpha 2 beta 1 and alpha-actinin in melanocytic proliferations

Integrin alpha 2 beta 1 is a transmembrane protein receptor for collagen and laminin previously reported as a melanoma tumor progression antigen. alpha-Actinin is an actin-binding protein reported to interact with the cytoplasmic domain of the beta 1-integrin chain of alpha 2 beta 1. In vitro, both alpha 2 beta 1 and alpha-actinin play a role in melanoma cell motility. In turn, increased melanoma cell line motility (measured as mean migration rates), correlates with metastasis. To determine the in situ distribution of these proteins, we used monoclonal antibodies directed against the alpha 2-integrin subunit of alpha 2 beta 1 and alpha-actinin on frozen sections of 33 melanocytic proliferations, which included dermal nevi, primary melanomas, and metastatic melanomas. We found that the superficial portion of all of the melanocytic proliferations tested stained for alpha-actinin. In benign nevi and superficial spreading melanoma, there was a notable loss of staining for alpha-actinin in the cells in the deep reticular dermis. In contrast, alpha-actinin was present on almost all of the tumor cells in the nodular melanomas and the melanoma metastases. Tumors stained either uniformly positive or uniformly negative for alpha 2 beta 1; the expression of this protein correlated with the later stages of melanoma progression. Our findings suggest that alpha-actinin protein levels initially decrease and then increase during melanocytic tumor progression, whereas the alpha 2 subunit protein appears in the later stages of melanoma progression. The variable distribution of these proteins is evidence for the differential adhesive and motile properties of subpopulations of cells in melanocytic proliferations.     

Expression pattern of integrin beta 1 subunit in Purkinje cells of rat and cerebellar mutant mice

We found that integrin beta 1 subunit (INT beta 1)-immunoreactive Purkinje cells first appeared caudally at postnatal day (PD) 6 of rat and most Purkinje cells gradually became positive by PD 12. The expression of INT beta 1 was then suppressed in some of these cells, so that the positive Purkinje cells in the adult were organized into parasagittal bands interposed by negative cells throughout the vermis and hemispheres. When Purkinje cells were deprived of their climbing fiber innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the subsequent patterning of INT beta 1-positive Purkinje cells was not changed. In both reeler and weaver mice, the INT beta 1-positive parasagittal bands were observed, however, the Purkinje cells in the staggerer mice did not express INT beta 1 at any stage. These data suggest that the expression of INT beta 1 in Purkinje cells is genetically programmed in the developing cerebellum, and that the afferent synaptic inputs by climbing and parallel fibers are not prerequisites for INT beta 1 expression in Purkinje cells. Therefore, the unique distribution patterns of INT beta 1-positive Purkinje cells provides a new marker for postnatal development of rodent cerebella.     

Mutational analysis of N-RAS and GAP-related domain of the neurofibromatosis type 1 gene in chronic myelogenous leukemia

RAS mutations can be detected in a variable number of patients with myeloproliferative disorders such as myelodysplastic syndromes and acute myeloid leukemia, but are rare events in chronic myelogenous leukemia in chronic phase. However, there is good evidence supporting the involvement of RAS signalling pathway in CML and this could be due to alterations in RAS activity regulatory proteins. The neurofibromatosis (NF1) gene down-regulates the RAS signal transduction pathway through the inhibitory function of its GAP-related domain (GRD) on RAS protein. The loss or alteration of neurofibromin (the NF1 protein) may produce a disfunction similar to point mutations in the RAS gene resulting in the permanent stimulation of the RAS signal transduction pathway. Mutations involving the GRD region of the NF1 gene (GRD-NF1) have been described in a variety of tumors such as colon carcinoma and astrocytoma. Germline mutations and deletions in the NF1 gene, as seen in neurofibromatosis type 1, are also associated with certain myeloid disorders. In the present work, we sought to identify mutations in the codons 12/13 and 61 of RAS gene and in the Lys-1423 codon of GRD-NF1, which are well known hot spots in these genes, in a group of 36 adults and ten children with chronic myelogenous leukemia in chronic phase and blast crisis. Using the PCR-SSCP and the allele-specific restriction assay (ASRA) techniques, we were not able to observe any RAS or NF1 detectable mutation. These findings suggest that RAS and GRD-NF1 mutations are not involved either in chronic phase or in the progression to blast crisis in chronic myelogenous leukemia in adults and children.     

Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells

Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers.     

CD9, but not other tetraspans, associates with the beta1 integrin precursor

Insulin resistance is common in patients with angina pectoris, a positive exercise electrocardiogram, and normal coronary angiograms (syndrome X). It is still not known whether insulin resistance affects the cardiac muscle itself and, if so, whether insulin resistance involves myocardial hemodynamics and energy metabolism. We investigated hemodynamics as well as metabolite exchanges across the heart and the forearm in eight patients with syndrome X and eight control subjects during a baseline period after an overnight fast and during a hyperinsulinemic-euglycemic clamp. Myocardial hemodynamics and metabolism were studied at rest, during pace stress, and in the recovery period after pacing. Neither coronary sinus blood flow nor forearm blood flow differed between the groups before and during the clamp. Whole body insulin-stimulated glucose uptake was decreased in the patients (15.6+/-2.1 vs. 23.1+/-2.0 micromol x kg-1 x min-1). Insulin-stimulated glucose uptake in the forearm and the cardiac muscle was equally reduced in the patients (46+/-5 and 48+/-5%). Myocardial glucose uptake correlated with total arterial delivery in the control subjects (r = 0.63, P < 0.01), but not in patients (r = 0.22, P = 0.13). Carbohydrate and lipid oxidation was similar in the two groups at rest, and changes during the clamp were not different in control subjects and patients either at rest, during pacing, or in the recovery period. Patients with syndrome X exhibit myocardial insulin resistance, but cardiac energy metabolism remains unaffected. In patients with syndrome X, insulin-stimulated glucose uptake is independent from myocardial blood flow.     

Both beta 1- and beta 2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

The involvement of beta 1- and beta 2-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with beta blockers (usually beta 1-selective) and 9 patients not treated with beta blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. beta 1-adrenoceptors were activated by (-)-noradrenaline during beta 2-adrenoceptor blockade with 50 nmol/l ICI 118551. beta 2-adrenoceptors were activated by (-)-adrenaline during beta 1-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (-)-noradrenaline and (-)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (-)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a beta blocker and 17/17 atrial strips from 15/15 patients chronically treated with beta blockers. ICI 118551 (50 nmol/l) blocked the (-)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with beta blockers and 17/20 atrial strips from 15/18 patients chronically treated with beta blockers. The incidence of arrhythmic contractions evoked by both (-)-noradrenaline and (-)-adrenaline was higher in chronically beta blocked patients than in non beta blocked patients. We conclude that both beta 1- and beta 2-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic beta 1-adrenoceptor blockade.     

Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.     

The thrombospondin receptor CD47 (IAP) modulates and associates with alpha2 beta1 integrin in vascular smooth muscle cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antialphav beta3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP was detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function.     

A mutation in the neurofibromatosis type 2 tumor-suppressor gene, giving rise to widely different clinical phenotypes in two unrelated individuals

Polychlorinated biphenyls (PCBs) are industrial chemicals that are long-lasting global environmental contaminants. PCBs have been reported to adversely affect reproduction in laboratory and wild animals by reducing the incidence of breeding and the survival rate of young. The present study was undertaken to determine the toxic potential of PCBs on in vitro fertilization (IVF) in the mouse. Aroclor 1221, 1254, and 1268, and 3, 3', 4, 4'-tetrachlorobiphenyl (TCB), a PCB congener, were added to IVF medium at various concentrations (0.01, 0.1, 1, and 10 micrograms/mL). Cumulus masses containing oocytes were obtained from superovulated B6D2F1 mice and cultured in medium containing PCB to which capacitated sperm were added. Oocytes were assessed for fertilization 20 to 24 h after insemination. A-1221, A-1268, and TCB reduced the fertilization rate at the 1 microgram/mL and 10 micrograms/mL doses, while inhibition of fertilization by A-1254 reached significance at 0.1 microgram/ml. Furthermore, all of these chemicals caused an increased incidence of degenerative ova and abnormal 2-cell embryos at the higher dose levels (1 microgram/mL and 10 micrograms/mL). The results suggest that higher dosages of PCB and TCB adversely affect fertilization and cause an increased incidence of degeneration of oocytes and abnormality in the early mouse embryos.     

Mutation analysis of the MEN1 gene in multiple endocrine neoplasia type 1, familial acromegaly and familial isolated hyperparathyroidism

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disease characterized by neoplasia of the parathyroid glands, the endocrine pancreas, and the anterior pituitary gland. In addition, families with isolated endocrine neoplasia, notably familial isolated hyperparathyroidism (FIHP) and familial acromegaly, have also been reported. However, whether these families constitute MEN 1 variants or separate entities remains speculative as the genetic bases for these diseases are unclear. The gene for MEN 1 has recently been cloned and characterized. Using single strand conformation analysis (SSCA) and sequencing, we performed mutation analysis in: a) a total of 55 MEN 1 families from 7 countries, b) 13 isolated MEN 1 cases without family history of the disease, c) 8 acromegaly families, and d) 4 FIHP families. Mutations were identified in 27 MEN 1 families and 9 isolated cases. The 22 different mutations spread across most of the 9 translated exons and included frameshift (11), nonsense (6), splice (2), missense mutations (2), and in-frame deletions (1). Among the 19 Finnish MEN 1 probands, a 1466del12 mutation was identified in 6 families with identical 11q13 haplotypes and in 2 isolated cases indicating a common founder. One frameshift mutation caused by 359del4 (GTCT) was found in 1 isolated case and 4 kindreds of different origin and haplotypes; this mutation therefore represents a common "warm" spot in the MEN1 gene. By analyzing the DNA of the parents of an isolated case one mutation was confirmed to be de novo. No mutation was found in any of the acromegaly and small FIHP families, suggesting that genetic defects other than the MEN1 gene might be involved and that additional such families need to be analyzed.