Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin-ezrin-radixin family of cytoskeleton-associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Common pathogenetic mechanism for three tumor types in bilateral acoustic neurofibromatosis.

Bilateral acoustic neurofibromatosis (BANF) is a genetic defect associated with multiple tumors of neural crest origin. Specific loss of alleles from chromosome 22 was detected with polymorphic DNA markers in two acoustic neuromas, two neurofibromas, and one meningioma from BANF patients. This indicates a common pathogenetic mechanism for all three tumor types. The two neurofibromas were among three taken from the same patient, and both showed loss of identical alleles demonstrating that the same chromosome suffered deletion in both tumors. The third neurofibroma from this patient showed no detectable loss of heterozygosity, which suggests the possibility of a more subtle mutational event that affects chromosome 22. In the two acoustic neuromas, only a portion of chromosome 22 was deleted, narrowing the possible chromosomal location of the gene that causes BANF to the region distal to the D22S9 locus in band 22q11. The identification of progressively smaller deletions on chromosome 22 in these tumor types may well provide a means to clone and characterize the defect.

Physical mapping of a translocation breakpoint in neurofibromatosis.

The gene for von Recklinghausen neurofibromatosis (NF1), one of the most common autosomal-dominant disorders of humans, was recently mapped to chromosome 17 by linkage analysis. The identification of two NF1 patients with balanced translocations that involved chromosome 17q11.2 suggests that the disease can arise by gross rearrangement of the NF1 locus, and that the NF1 gene might be identified by cloning the region around these translocation breakpoints. To further define the region of these translocations, a series of chromosome 17 Not I-linking clones has been mapped to proximal 17q and studied by pulsed-field gel electrophoresis. One clone, 17L1 (D17S133), clearly identifies the breakpoint in an NF1 patient with a t(1;17) translocation. A 2.3-megabase pulsed-field map of this region was constructed and indicates that the NF1 breakpoint is only 10 to 240 kilobases away from 17L1. This finding prepares the way for the cloning of NF1.

Progress towards identifying the neurofibromatosis (NF1) gene.

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder of humans. Linkage analysis has recently mapped the NF1 gene to the proximal long arm of chromosome 17. The identification of two NF1 patients with balanced translocations has now allowed the location of the gene to be narrowed to a few hundred kilobases of chromosome band 17q11.2, using a combination of somatic cell hybrid technology, linking clones and pulsed field gel electrophoresis.

P-glycoprotein, HER-2/neu, and mutant p53 expression in human gynecologic tumors.

BACKGROUND: Overexpression of P-glycoprotein has been associated with a worse prognosis for some groups of patients not receiving chemotherapy. Recently, it has been demonstrated that in vitro both c-Ha-Ras overexpression and mutant p53 overexpression do activate the MDR1 gene (also known as PGY1) in murine NIH 3T3 cells. This direct connection between oncogenic activation, antioncogenic malfunctioning (presence of mutant instead of wild-type p53 protein), and MDR1 gene expression constitutes a fundamental conceptual model that could provide an explanation for the obscure prognostic role, in the absence of chemotherapy, of the MDR1 gene. PURPOSE: Our goal was to test whether the relationship between MDR1 (P-glycoprotein) expression, oncogenic activation, and mutant p53 protein expression demonstrated in vitro is also reproducible in vivo for two groups of human gynecologic tumors. METHODS: Fifty tumor specimens (31 mammary, 11 endometrial, and eight cervical) were analyzed. They had been obtained from previously untreated patients. Aliquots of these specimens had been frozen and stored at -70 degrees C since surgical collection or routinely fixed in formalin and embedded in paraffin. DNA was extracted from routinely fixed specimens for single-strand conformation polymorphism (SSCP) analysis. Immunohistochemical techniques were used on frozen material to determine: 1) P-glycoprotein expression using two different monoclonal antibodies (c219 and JSB1); 2) HER-2/neu (c-erb-B2; also known as ERBB2) expression using the NCL-CB11 monoclonal antibody; and 3) mutant p53 protein expression using the PAb 1801 monoclonal antibody. Polymerase chain reaction (PCR)-SSCP was used to confirm recognition of the mutated isoform of p53. Endometrial and cervical carcinomas were studied by both PCR-SSCP DNA analysis and immunohistochemical analysis. Only when there was full concordance between both methods were endometrial and cervical tumors considered to express mutant p53. RESULTS: A statistically significant (P = .009; Fisher's exact test) association between HER-2/neu and MDR1 expression was found for the more aggressive form of inoperable, locally advanced mammary carcinoma. Expression of HER-2/neu or mutant p53 was similar in both tumor groups studied--mammary carcinoma with a low basal expression of P-glycoprotein compared with endometrial and cervical carcinomas with significantly (P = .0002; chi-square test) higher levels of expression. CONCLUSIONS: The highly statistically significant coexpression of P-glycoprotein and HER-2/neu took place only in the subgroup of aggressive, locally advanced, inoperable mammary carcinomas, whereas no statistically significant association could be found for operable tumors. No association between mutant p53 expression and MDR1 activation was found in the human tumors analyzed.

Molecular cloning and characterization of alternatively spliced transcripts of the mouse neurofibromatosis 2 gene.

The human neurofibromatosis 2 (NF2) gene has recently been isolated and predicted to encode a novel protein named merlin. Based on its high homology to the moesin-ezrin-radixin family of proteins, it may be involved in mediating interactions between the plasma membrane and the cytoskeleton. Here we report the isolation and characterization of multiple transcript isoforms of the mouse NF2 gene. The full length coding complementary DNA sequence of transcript isoform I is 1788 base pairs in length, shares 90% sequence identity with the human NF2 complementary DNA, and encodes a putative protein of 596 amino acids sharing 98% homology with the human merlin protein. Transcript isoforms II and III carry a 45- and 16-base pair insertion, respectively, at nucleotide 1740 at the 3' end, generated by two different modes of alternative splicing; both insertions introduce premature termination codons. Thus, transcript isoforms II and III predict proteins of 591 and 584 amino acids with altered COOH-termini of more hydrophilic character as compared to isoform I. Northern blot analysis and reverse transcription-polymerase chain reaction analysis indicate that the mouse NF2 gene is widely expressed in different tissue types and that the alternative transcripts are variantly expressed. The results presented here indicate high conservation of the NF2 gene during evolution and suggest a possible role for the COOH-terminus in mouse merlin function.

Farnesyltransferase inhibitors block the neurofibromatosis type I (NF1) malignant phenotype.

Neurofibromatosis type I (NF1) is a hereditary tumor and developmental disorder whose defective gene was cloned previously. The protein product of the NF1 gene, neurofibromin, contains a domain that shows significant sequence homology to the known catalytic domains of mammalian Ras GTPase-activating proteins (GAP) and the yeast IRA1 and IRA2 proteins. This homologous region of neurofibromin has been shown to exhibit GAP activity toward Ras proteins. Malignant schwannoma cell lines from NF1 patients contain normal levels of GAP and nonmutated Ras proteins but barely detectable levels of neurofibromin, based on genetic mutations in the NF1 gene. Because these cells contain constitutively activated Ras.GTP, it has been proposed that neurofibromin may be the sole negative regulator of Ras in these cells. Overall, these results have implied an important role of the Ras signaling pathway in NF1 malignant schwannomas. Recently, several laboratories have developed small molecule inhibitors of Ras function that inhibit the enzyme farnesyltransferase (FT). FT-mediated post-translational farnesylation of Ras proteins is absolutely necessary for Ras function since this modification is required for the anchoring of Ras proteins to the plasma cell membrane. Although previous studies have shown that FT inhibitors can block the growth of tumor cells carrying mutant Ras proteins, it remained unclear how this class of inhibitors would affect tumor cells such as in NF1, whose malignant growth appears to be mediated by up-regulation of wild-type Ras activity. Thus, in the current study, we investigated whether BMS-186511, a bisubstrate analogue inhibitor of FT, would inhibit the malignant growth properties of a cell line established from malignant schwannoma of an NF1 patient. Our results indicate that the malignant growth properties of ST88-14 cells, the most malignant cell line among several well-characterized NF1 cells, are inhibited by BMS-186511 in a concentration-dependent manner. Following treatment with BMS-186511, ST88-14 cells became flat, nonrefractile, were contact-inhibited, and lost their ability to grow in soft agar. In the drug-exposed cells, Ras proteins were prevented from FT-mediated membrane association. BMS-186511 was found to specifically inhibit FT, but not geranylgeranyltransferase I, a closely related enzyme. Thus, it is conceivable that FT inhibitors may ultimately become the first generation of drugs against the malignant phenotype in NF1 based on rational insights into the mechanism of action of neurofibromin.

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

Evidence for a tumor suppressor gene on chromosome 19q associated with human astrocytomas, oligodendrogliomas, and mixed gliomas.

Previous studies have shown frequent allelic losses of chromosomes 9p, 10, 17p, and 22q in glial tumors. Other researchers have briefly reported that glial tumors may also show allelic losses of chromosome 19, suggesting a putative tumor suppressor gene locus on this chromosome (D. T. Ransom et al., Proc. Am. Assoc. Cancer Res., 32:302, 1991). To evaluate whether loss of chromosome 19 alleles is common in glial tumors of different types and grades, we performed Southern blot restriction fragment length polymorphism analysis for multiple chromosome 19 loci in 122 gliomas from 116 patients. Twenty-nine tumors had loss of constitutional heterozygosity of 19q, and four tumors had partial deletions of 19q. Allelic losses on 19q were restricted to grade III anaplastic astrocytomas (4/9) and grade IV glioblastomas (11/46), grade II oligodendrogliomas (2/5) and grade III anaplastic oligodendrogliomas (2/2), and grade II (5/8) and grade III (5/7) mixed oligoastrocytomas. These data demonstrate genetic similarities between astrocytomas, oligodendrogliomas, and mixed glial tumors and indicate the presence of a glial tumor suppressor gene on chromosome 19q.

p53 mutations are associated with 17p allelic loss in grade II and grade III astrocytoma.

Loss of genetic material on the short arm of chromosome 17 is observed in approximately 40% of human astrocytomas (WHO grades II and III) and in approximately 30% of cases of glioblastoma multiforme (WHO grade IV). Previous studies of glioblastoma multiforme have shown that the p53 gene, located on the short arm of chromosome 17, is frequently mutated in these glioblastomas. To explore whether lower-grade astrocytomas are also associated with corresponding mutations of the p53 gene, we have investigated a series of 22 human astrocytomas of WHO grades II and III both for loss of heterozygosity on chromosome 17p and for p53 mutations. Mutations in the conserved regions of the p53 gene were identified by single strand conformation polymorphism analysis of exons 5, 6, 7, and 8 and were verified by direct DNA sequencing of the polymerase chain reaction products. p53 mutations were observed in 3 of 8 grade II astrocytomas and 4 of 14 grade II astrocytomas. In all 22 tumors, allelic loss of the short arm of chromosome 17 was investigated by restriction fragment length polymorphism analysis. One-half of the grade II astrocytomas (4 of 8) and grade III astrocytomas (7 of 14) exhibited allelic loss on chromosome 17p. Mutations in the p53 gene were exclusively observed in tumors with allelic loss on 17p. Our results show that p53 mutations are not restricted to glioblastoma multiforme and may be important in the tumorigenesis of lower-grade astrocytomas and that p53 mutations in lower-grade astrocytomas are associated with loss of chromosome 17p. These findings are consistent with a recessive mechanism of action of p53 in WHO grade II and III astrocytoma tumorigenesis.

Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.

Recent results from several laboratories including ours strongly suggest that farnesyltransferase (FT) inhibitors belonging to distinct chemical classes block growth of oncogenic Ras transformed cells at concentrations that do not affect the growth and viability of normal cells. This is despite blocking the farnesylation and thus the membrane association of Ras in both cell types. This is a paradox given the requirement for Ras function in normal cell growth. Recent evidence that R-Ras2/TC21 utilizes components of Ras signal transduction pathways to trigger cellular transformation (Graham et al., MCB 14, 4108-4115, 1994) prompted us to consider the possibility that R-Ras2/TC21 is involved in some aspects of the growth regulation of normal cells. If so, R-Ras2/TC21 may be compensating for Ras function in untransformed cells treated with FT inhibitors. In this study, we demonstrated that a cell active bisubstrate analog FT inhibitor, BMS-186511, completely blocked the function of oncogenic Ras, but did not affect the function of oncogenic R-Ras2/TC21, as determined by several criteria including inhibition of anchorage dependent and independent growth, reversal of transformed morphology and restoration of actin cytoskeleton. While it is known that TC21 protein becomes prenylated, it is not known whether it is farnesylated or geranylgeranylated. Our in vitro prenylation experiments indicate that R-Ras2/TC21 protein serves as a good substrate for FT as well as geranylgeranyltransferase I (GGTI) and thus provide the apparent molecular basis for these differences. Overall, these results, coupled with the ubiquitous expression of R-Ras2/TC21 in many cells including untransformed NIH3T3 cells, are consistent with the possibility that R-Ras2/TC21 may be one of the factors that render normal cells insensitive to the growth inhibitory action of FT inhibitors.

Chromosome 17 allelic loss and NF1-GRD mutations do not play a significant role as molecular mechanisms leading to melanoma tumorigenesis.

Allelic loss in human cutaneous melanoma has been detected on chromosomes 1p, 6q, 9p, 10q, and 11q. Chromosome 17 contains important tumor suppressor genes such as p53, NM23, and neurofibromatosis type 1 (NF1), which have been implicated in melanoma tumorigenesis. The role of p53 has already been studied by a number of laboratories, showing contrasting results. In the present study, two restriction fragment length polymorphism (RFLP) probes for the NM23 and NF1 genes, together with five other RFLP and four variable number of tandem repeat chromosome 17 probes, were investigated at the loss of heterozygosity (LOH) level in a Southern blot-based assay. The NF1 gene was also tested for LOH by a polymerase chain reaction (PCR)-based approach in two different experiments, using a dinucleotide repeat polymorphic probe at locus D17S250 (17q11.2-q12), and an Alu probe intragenic to the NF1 gene (17q11.2). A PCR single-strand conformation polymorphism assay was included in the study for mutation detection at the NF1-GTPase-activating protein-related domain (GRD). A total of 68 melanocytic tumors were analyzed. LOH was detected in 9 of 87 informative cases (10%). LEW301 (17p11.2-pcen) presented the highest LOH frequency (22%). NM23 showed LOH in 17% of the informative cases, while NF1 did not show either LOH in the Southern blot- and PCR-based experiments or mutations at the NF1-GRD. These results are in concordance with those of previous smaller studies, but when compared with higher LOH frequencies obtained from other chromosomes, these findings indicate that the LOH values found in our study can most likely be attributed to background effect. Thus, chromosome 17 LOH is likely to play and unimportant role as a genetic event in melanoma tumorigenesis. Nevertheless, NF1 merits further study, since homozygous deletions have been detected at this locus in melanoma cell lines.

Comparative study of p53 gene and protein alterations in human astrocytic tumors.

The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, III and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.

Evidence for a differential postnatal development of proenkephalin B (= prodynorphin)-derived opioid peptides in the rat hypothalamus.

[he concentrations of immunoreactive (ir-) peptides derived from the opioid peptide precursors proenkephalin A (Met-enkephalin), proenkephalin B [dynorphin (DYN)-(1-17), dynorphin-(1-8), dynorphin B, alpha-neoendorphin (alpha-NEO-E), beta-NEO-E] and proopiomelanocortin [beta-endorphin (beta-END)], and of the neurosecretory hormones vasopressin and oxytocin increased between approximately 10-fold and 50-fold from birth to adulthood in the rate hypothalamus. Gel filtration and HPLC analysis of proenkephalin B-derived opioid peptides revealed that in 3-day-old rats the predominant portion of ir-dynorphin-(1-17) and a substantial part of ir-dynorphin B consisted of a high (6000) mol wt species, a common precursor peptide for DYN-(1-17) and DYN B. In adults rats, however, authentic DYN-(1-17) and DYN B were found to be the major ir-forms. The mol wt patterns of ir-DYN-(1-8), ir-alpha-NEO-E and ir-beta-NEO-E did not differ between 3-day-old and adult rats and reflected predominantly the respective authentic opioid peptides. Taking into consideration the developmental changes in the mol wt pattern of ir-DYN-(1-17), authentic DYN-(1-17) was 5 times lower in concentration than DYN-(1-8) in 3-day-old rats, whereas in adults these opioid peptides occurred in equimolar concentrations. These findings suggest that the posttranslational processing of the precursor proenkephalin B changes in the course of postnatal development. Ir-beta-END in the hypothalamus of newborn and adult rats consisted exclusively of beta-END-sized peptides which were not (unlike those in the intermediate pituitary lobe) alpha-N-acetylated. Thus, in the hypothalamus, the enzymatic processing of the opioid peptide precursor proopiomelanocortin to beta-END seems to be fully active at birth, in contrast to that of proenkephalin B.

Postnatal development of beta-endorphin-related peptides in rat anterior and intermediate pituitary lobes: evidence for contrasting development of proopiomelanocortin processing.

The concentration of immunoreactive (ir) beta-endorphin (beta-END) in the neurointermediate pituitary lobe was 15-fold higher in adult than in newborn rats; in contrast, that of ir-beta-END in the anterior lobe was twice as high in newborn as in adult animals. Ir-beta-END in the neurointermediate lobe of newborn rats consisted exclusively of beta-END-sized peptides, indicating that at birth rats are capable of processing the opioid peptide precursor proopiomelanocortin (POMC) to beta-END. Moreover, beta-END-related peptides in the neurointermediate lobe of newborn rats were found to be predominantly alpha-N-acetylated and, therefore, inactivated with respect to their opiate-like properties. Further analysis of these alpha-N-acetylated forms on high performance liquid chromatography indicated that newborn rats predominantly contained alpha-N-acetyl-(Ac-)beta-END-(1-31), whereas the major forms in adult rats were Ac-beta-END-(1-27) and -(1-26). Thus, the C-terminal processing of Ac-beta-END-(1-31) to -(1-27) and -(1-26) may not yet be fully active at birth, in contrast to the processing of POMC to beta-END. In the anterior lobe of newborn rats, however, the ratio of beta-lipotropin/beta-END resembled that of adults, and more than 80% of beta-END-sized ir-material was found to consist of nonacetylated (and therefore opiate-active) beta-END-(1-31), as in adults, suggesting that the enzymatic system responsible for processing of POMC to beta-lipotropin and beta-END is already mature at birth. The high concentrations of beta-END in the anterior lobe of newborn rats suggest a possible role of this opioid peptide in perinatal development and/or parturition.

Proenkephalin B (prodynorphin)-derived opioid peptides: evidence for a differential processing in lobes of the pituitary.

The distribution of peptides derived from the novel opioid peptide precursor proenkephalin B (prodynorphin) was studied in lobes of the pituitary with antibodies against alpha-neoendorphin (alpha-neo-E) beta-neoE, dynorphin (DYN)-(1-17), DYN-(1-8), and DYN B in combination with gel filtration and high pressure liquid chromatography. In the posterior pituitary, all five opioid peptides occurred in high and about equimolar concentrations, whereas putative precursor peptides were found in only minor quantities. In contrast, in the anterior pituitary immunoreactive (ir-) DYN-(1-17) and ir-DYN B consisted exclusively of a common precursor species with a mol wt of about 6000. Six thousand-dalton DYN may be comprised of the C-terminal portion of proenkephalin B, with the sequence of DYN-(1-17) at its N-terminus. Moreover, the major portions of ir-alpha-neo-E and ir-beta-neoE in the anterior pituitary were found to be of an apparent mol wt of 8000. These findings indicate a differential processing of the opioid peptide precursor proenkephalin B in the two lobes of the pituitary. The anterior pituitary seems to process proenkephalin B predominantly into high mol wt forms of neo-E and DYNs, whereas in the posterior pituitary proenkephalin B undergoes further proteolytic processing to the smaller opioid peptides alpha-neo-E, beta-neo-E, DYN-(1-17), DYN-(1-8), and DYN B. Thus, processing differences may enable the selective liberation of different (opioid) peptides with distinct biological properties from one precursor within different tissues.

Chronic haloperidol treatment increases the level of in vitro translatable messenger ribonucleic acid coding for the beta-endorphin/adrenocorticotropin precursor proopiomelanocortin in the pars intermedia of the rat pituitary.

Chronic treatment of rats with haloperidol (1.5 mg/kg, once daily) over a period of 7--21 days resulted in a 80--100% increase in the tissue levels of immunoreactive beta-endorphin and in the in vitro release of immunoreactive beta-endorphin from the neurointermediate pituitary. Incorporation of [3H]phenylalanine into isolated neurointermediate pituitaries of haloperidol-treated rats revealed an increase in the amount of label incorporated into the beta-endorphin/ACTH precursor proopiomelanocortin (POMC) to a similar extent (about 80%) but had essentially no effect on the conversion of the precursor into beta-lipotropin and beta-endorphin. Extraction of messenger (m) RNA from neurointermediate pituitaries followed by cell-free translation in a reticulocyte system showed an increase in the total level of translatable mRNA (about 25%). The content of translatable mRNA coding for POMC, however, was increased by 100-150%. Time-course studies revealed a parallelism between the effect of haloperidol on the level of in vitro translatable mRNA coding for POMC and the ability of the drug to increase the concentrations of beta-endorphin in the neurointermediate pituitary. A complete reversal of the effects of haloperidol was seen 2 weeks after discontinuation of the drug. These findings suggest that the chronic blockade of dopaminergic receptors by haloperidol causes a reversible increase in the beta-endorphin biosynthesis in the rat intermediate pituitary at the pretranslational level by markedly increasing the level of translatable mRNA coding for POMC.

Progress toward the isolation and characterization of the genes causing neurofibromatosis.

Neurofibromatosis 1 and neurofibromatosis 2 are clinically distinct autosomal dominant disorders that affect an estimated 1.5 million individuals throughout the world. The genetic defect in each disorder has been mapped to different chromosomes, NF1 to chromosome 17 and NF2 to chromosome 22. Progress towards the cloning of the NF1 gene has proceeded rapidly. The NF1 locus was bracketed using genetic linkage analysis on NF1 affected pedigrees. Physical mapping methods were then used to precisely map the translocation breakpoints in each of two NF1 affected individuals who harbored constitutional chromosomal translocations in the putative NF1 region of chromosome 17. The region of DNA located between the two translocations has been cloned in cosmids and yeast artificial chromosomes and a number of RNA coding sequences have been identified. The identification of the NF1 gene will depend on finding mutations in the DNA of affected individuals. In the case of NF2, progress seems to have been less rapid, in part due to the lower availability of NF2 affected pedigrees. The genetic defect has been mapped to the long arm of chromosome 22 by studies of chromosomal loss in the tumours associated with this disease. Subsequent genetic mapping has confirmed this location. Flanking DNA markers for the NF2 locus have been identified. The region of DNA between these markers is in the order of 5-10 Mb. The identification of chromosomal aberrations in patients with NF2 that involve chromosome 22 will play an important role in the identification of the NF2 gene in much the same way as they have in NF1.

Subsets of glioblastoma multiforme defined by molecular genetic analysis.

Glioblastoma multiforme is a clinically and histologically heterogeneous lesion; however, to date, it has not been possible to subdivide glioblastomas on a clinical, histopathological or biological basis. Previous studies have demonstrated that loss of portions of chromosomes 10 and 17 and amplification of the epidermal growth factor receptor (EGFR) gene are the most frequent genetic alterations in glioblastoma. We therefore examined 74 glioblastomas from 67 patients for loss of heterozygosity on chromosomes 10 and 17, and for amplification of the epidermal growth factor receptor gene, to determine whether glioblastomas can be subtyped on a genetic basis. Using Southern blot analysis we were able to detect different patterns of genomic alterations. Eighteen of 67 informative patients were characterized by a loss of heterozygosity on the short arm of chromosome 17 in the tumor tissue. Forty-five of 64 informative patients showed a loss of heterozygosity on chromosome 10. Amplification of the epidermal growth factor receptor gene was noted in 25 of 67 patients and was restricted to those glioblastomas that had lost portions of chromosome 10. Epidermal growth factor receptor gene amplification occurred significantly more often in patients without chromosome 17p loss than in patients with chromosome 17p loss (p = 0.01). In addition, those glioblastomas with a loss of chromosome 17p occurred in patients significantly younger than those with glioblastomas characterized by EGFR gene amplification (p = 0.001). These data emphasize the genetic heterogeneity of glioblastoma and suggest the division of glioblastoma into genetic subsets.