A novel somatic mutation in GNAQ in a capillary malformation provides insight into molecular pathogenesis.

Sturge-Weber syndrome (SWS) is a sporadic, congenital, neuro-cutaneous disorder characterized by a mosaic, capillary malformation. SWS and non-syndromic capillary malformations are both caused by a somatic activating mutation in GNAQ encoding the G protein subunit alpha-q protein. The missense mutation R183Q is the sole GNAQ mutation identified thus far in 90% of SWS-associated or isolated capillary malformations. In this study, we sequenced skin biopsies of capillary malformations from 9 patients. We identified the R183Q mutation in nearly all samples, but one sample exhibited a Q209R mutation. This new mutation occurs at the same residue as the constitutively-activating Q209L mutation, commonly seen in tumors. However, Q209R is a rare variant in this gene. To compare the effect of the Q209R mutation on downstream signaling, we performed reporter assays with a GNAQ-responsive reporter co-transfected with either GNAQ WT, R183Q, Q209L, Q209R, or C9X (representing a null allele). Q209L showed the highest reporter activation, with R183Q and Q209R showing significantly lower activation. To determine whether these mutations had similar or different downstream consequences we performed RNA-seq analysis in microvascular endothelial cells (HMEC-1) electroporated with the same GNAQ variants. The R183 and Q209 missense variants caused extensive dysregulation of a broad range of transcripts compared to the WT or null allele, confirming that these are all activating mutations. However, the missense variants exhibited very few differentially expressed genes (DEGs) when compared to each other. These data suggest that these activating GNAQ mutations differ in magnitude of activation but have similar downstream effects.

Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene hypothesis.

Individuals with neurofibromatosis type 1 (NF1) have an increased risk of developing benign and malignant tumours. The NF1 gene is thought to be a tumour suppressor gene, yet no direct proof at the molecular level exists to support this hypothesis. Here we describe a neurofibrosarcoma from a patient with NF1 with loss of heterozygosity for all chromosome 17 polymorphisms tested. On the remaining chromosome 17 homologue, a 200 kilobase (kb) tumour specific deletion of NF1 was demonstrated. This is the first example of a homozygous inactivation of NF1 at the molecular level in a malignant tumour from an NF1 patient and the results strongly support the tumour suppressor gene hypothesis for this disease.

Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2.

Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair.

Randomized, double-blind, placebo-controlled, crossover trial of oral doxycycline for epistaxis in hereditary hemorrhagic telangiectasia.

BACKGROUND: Vascular malformations in hereditary hemorrhagic telangiectasia (HHT) lead to chronic recurrent bleeding, hemorrhage, stroke, heart failure, and liver disease. There is great interest in identifying novel therapies for epistaxis in HHT given its associated morbidity and impact on quality of life. We aimed to measure the effectiveness of oral doxycycline for the treatment of epistaxis and explore mechanisms of action on angiogenic, inflammatory and pathway markers in HHT using a randomized controlled trial. METHODS: 13 HHT patients with epistaxis were recruited from the Toronto HHT Center at St. Michael's Hospital. Recruitment was stopped early due to COVID-19-related limitations. The study duration was 24 months. Patients were randomly assigned to the treatment-first or placebo-first study arm. We compared the change in weekly epistaxis duration and frequency, biomarkers, blood measurements, and intravenous iron infusion and blood transfusion requirements between treatment and placebo. RESULTS: There was no significant difference in the change in weekly epistaxis duration (p = 0.136) or frequency (p = 0.261) between treatment and placebo. There was no significant difference in the levels of MMP-9, VEGF, ANG-2, IL-6 or ENG with treatment. Hemoglobin levels were significantly higher (p = 0.0499) during treatment. Ferritin levels were not significantly different between treatment and placebo. There was no significant difference in RBC transfusions between treatment periods (p = 0.299). CONCLUSION: Overall, our study did not demonstrate effectiveness of doxycycline as a treatment for epistaxis in patients with HHT, though the study was underpowered. Secondary analyses provided new observations which may help guide future trials in HHT. Trial Registration ClinicalTrials.gov, NCT03397004. Registered 11 January 2018 - Prospectively registered, https://clinicaltrials.gov/ct2/show/NCT03397004.

Predictors of mortality in patients with hereditary hemorrhagic telangiectasia.

BACKGROUND: Retrospective questionnaire and healthcare administrative data suggest reduced life expectancy in untreated hereditary hemorrhagic telangiectasia (HHT). Prospective data suggests similar mortality, to the general population, in Denmark's centre-treated HHT patients. However, clinical phenotypes vary widely in HHT, likely affecting mortality. We aimed to measure predictors of mortality among centre-treated HHT patients. HHT patients were recruited at 14 HHT centres of the Brain Vascular Malformation Consortium (BVMC) since 2010 and followed annually. Vital status, organ vascular malformations (VMs) and clinical symptoms data were collected at baseline and during follow-up (N = 1286). We tested whether organ VMs, HHT symptoms and HHT genes were associated with increased mortality using Cox regression analysis, adjusting for patient age, sex, and smoking status. RESULTS: 59 deaths occurred over average follow-up time of 3.4 years (max 8.6 years). A history of anemia was associated with increased mortality (HR = 2.93, 95% CI 1.37-6.26, p = 0.006), as were gastro-intestinal (GI) bleeding (HR = 2.63, 95% CI 1.46-4.74, p = 0.001), and symptomatic liver VMs (HR = 2.10, 95% CI 1.15-3.84, p = 0.015). Brain VMs and pulmonary arteriovenous malformations (AVMs) were not associated with mortality (p > 0.05). Patients with SMAD4 mutation had significantly higher mortality (HR = 18.36, 95% CI 5.60-60.20, p < 0.001) compared to patients with ACVRL1 or ENG mutation, but this estimate is imprecise given the rarity of SMAD4 patients (n = 33, 4 deaths). CONCLUSIONS: Chronic GI bleeding, anemia and symptomatic liver VMs are associated with increased mortality in HHT patients, independent of age, and in keeping with the limited treatment options for these aspects of HHT. Conversely, mortality does not appear to be associated with pulmonary AVMs or brain VMs, for which patients are routinely screened and treated preventatively at HHT Centres. This demonstrates the need for development of new therapies to treat chronic anemia, GI bleeding, and symptomatic liver VMs in order to reduce mortality among HHT patients.

Utility of modified Rankin Scale for brain vascular malformations in hereditary hemorrhagic telangiectasia.

BACKGROUND: Approximately 10% of hereditary hemorrhagic telangiectasia (HHT) patients harbour brain vascular malformations (VMs). Intracranial hemorrhage (ICH) from brain VMs can lead to death or morbidity, while treatment options for brain VMs also have associated morbidity. The modified Rankin Scale (mRS) may provide an approach to identifying HHT-brain VM patients with poor outcomes, and their predictors. We aimed to measure the relationship between mRS score and brain VM, brain VM number, as well as other aspects of HHT, at enrollment and during prospective follow-up. METHODS: 1637 HHT patients (342 with brain VMs) were recruited from 14 HHT centres of the Brain Vascular Malformation Consortium since 2010 and followed prospectively (mean = 3.4 years). We tested whether the presence of brain VM, other HHT organ involvement, and HHT mutation genotype were associated with worse mRS scores at baseline and during follow-up, using linear mixed models, adjusting for age, sex, and year of visit. RESULTS: Presence of brain VMs was not associated with worse mRS score at baseline and there was no significant worsening of mRS with prospective follow-up in these patients; 92% had baseline mRS of 0-2. HHT-related gastrointestinal (GI) bleeding was associated with worse mRS scores at baseline (0.37, 95% CI 0.26-0.47, p < 0.001), as were history of anemia (0.35, 95% CI 0.27-0.43, p < 0.001) and liver VMs (0.19, 95% CI 0.09-0.30, p < 0.001). Presence of pulmonary arteriovenous malformations (AVMs) was not associated with worse mRS scores at baseline. mRS score was not associated with either HHT genotype (Endoglin vs ACVRL1). Only GI bleeding was associated with a significantly worsening mRS during prospective follow-up (0.64, 95% CI 0.21-1.08, p = 0.004). CONCLUSION: Most HHT-brain VM patients had good functional capacity (mRS scores 0-2) at baseline that did not change significantly over 3.4 mean years of follow-up, suggesting that mRS may not be useful for predicting or measuring outcomes in these patients. However, HHT patients with GI bleeding, anemia history or liver VMs had worse mRS scores, suggesting significant impact of these manifestations on functional capacity. Our study demonstrates the insensitivity of the mRS as an outcomes measure in HHT brain VM patients and reinforces the continued need to develop outcomes measures, and their predictors, in this group.

Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients.

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the neural crest. No reliable cellular phenotypic marker has been identified, which has hampered direct efforts to identify the gene. The chromosome location of the NF1 gene has been previously mapped genetically to 17q11.2, and data from two NF1 patients with balanced translocations in this region have further narrowed the candidate interval. The use of chromosome jumping and yeast artificial chromosome technology has now led to the identification of a large (approximately 13 kilobases) ubiquitously expressed transcript (denoted NF1LT) from this region that is definitely interrupted by one and most likely by both translocations. Previously identified candidate genes, which failed to show abnormalities in NF1 patients, are apparently located within introns of NF1LT, on the antisense strand. A new mutation patient with NF1 has been identified with a de novo 0.5-kilobase insertion in the NF1LT gene. These observations, together with the high spontaneous mutation rate of NF1 (which is consistent with a large locus), suggest that NF1LT represents the elusive NF1 gene.

ZPLD1 gene is disrupted in a patient with balanced translocation that exhibits cerebral cavernous malformations.

The past few years have seen rapid advances in our understanding of the genetics and molecular biology of cerebral cavernous malformations (CCM) with the identification of the CCM1, CCM2, and CCM3 genes. Recently, we have recruited a patient with an X/3 balanced translocation that exhibits CCM. By fluorescent in situ hybridization analysis, sequence analysis tools and database mining procedures, we refined the critical region to an interval of 200-kb and identified the interrupted ZPLD1 gene. We detected that the mRNA expression level of ZPLD1 gene is consistently decreased 2.5-fold versus control (P=0.0006) with allelic loss of gene expression suggesting that this protein may be part of the complex signaling pathway implicated in CCM formation.

Genetic considerations relevant to intracranial hemorrhage and brain arteriovenous malformations.

Brain arteriovenous malformations (AVMs) cause intracranial hemorrhage (ICH), especially in young adults. Molecular characterization of lesional tissue provides evidence for involvement of both angiogenic and inflammatory pathways, but the pathogenesis remains obscure and medical therapy is lacking. Abnormal expression patterns have been observed for proteins related to angiogenesis (e.g., vascular endothelial growth factor, angiopoietin-2, matrix metalloproteinase-9), and inflammation (e.g., interleukin-6 [IL-6] and myeloperoxidase). Macrophage and neutrophil invasion have also been observed in the absence of prior ICH. Candidate gene association studies have identified a number of germline variants associated with clinical ICH course and AVM susceptibility. A single nucleotide polymorphism (SNP) in activin receptor-like kinase-1 (ALK-1) is associated with AVM susceptibility, and SNPs in IL-6, tumor necrosis factor-alpha (TNF-alpha), and apolipoprotein-E (APOE) are associated with AVM rupture. These observations suggest that even without a complete understanding of the determinants of AVM development, the recent discoveries of downstream derangements in vascular function and integrity may offer potential targets for therapy development. Further, biomarkers can now be established for assessing ICH risk. These data will generate hypotheses that can be tested mechanistically in model systems, including surrogate phenotypes, such as vascular dysplasia and/or models recapitulating the clinical syndrome of recurrent spontaneous ICH.

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

SMAD4 mutations found in unselected HHT patients.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disease exhibiting multifocal vascular telangiectases and arteriovenous malformations. The majority of cases are caused by mutations in either the endoglin (ENG) or activin receptor-like kinase 1 (ALK1, ACVRL1) genes; both members of the transforming growth factor (TGF)-beta pathway. Mutations in SMAD4, another TGF-beta pathway member, are seen in patients with the combined syndrome of juvenile polyposis (JP) and HHT (JP-HHT). METHODS: We sought to determine if HHT patients without any apparent history of JP, who were undergoing routine diagnostic testing, would have mutations in SMAD4. We tested 30 unrelated HHT patients, all of whom had been referred for DNA based testing for HHT and were found to be negative for mutations in ENG and ALK1. RESULTS: Three of these people harboured mutations in SMAD4, a rate of 10% (3/30). The SMAD4 mutations were similar to those found in other patients with the JP-HHT syndrome. CONCLUSIONS: The identification of SMAD4 mutations in HHT patients without prior diagnosis of JP has significant and immediate clinical implications, as these people are likely to be at risk of having JP-HHT with the associated increased risk of gastrointestinal cancer. We propose that routine DNA based testing for HHT should include SMAD4 for samples in which mutations in neither ENG nor ALK1 are identified. HHT patients with SMAD4 mutations should be screened for colonic and gastric polyps associated with JP.

Clinical manifestations in a large hereditary hemorrhagic telangiectasia (HHT) type 2 kindred.

HHT type 2 (HHT 2) is a multi-system vascular dysplasia caused by a mutation in the ALK-1 gene, but the phenotype has not been well defined. We report on 51 members of an HHT 2 kindred with an ALK-1 gene mutation shown to be associated with the disorder. This ALK-1 mutation was detected in 38 kindred members who were evaluated systematically for associated vascular abnormalities. Pulmonary arteriovenous malformations (AVMs) were found in 6% of those screened, cerebral AVM in 7%, hepatic AVM in 17%, and spinal AVM in 3%. We discuss these and other findings in the 38 affected kindred members, as well as findings in the 13 kindred members in whom the mutation was not detected. This study shows that pulmonary, cerebral, spinal, and hepatic AVMs can all occur in HHT 2. It also adds to the evidence suggesting that pulmonary AVMs are more common in HHT 1 than in HHT 2. We identify a higher prevalence of hepatic AVMs than previously reported in either HHT 1 or 2. This may be specific to the mutation in this kindred, but probably reflects the lack of routine screening for this manifestation. Even in this family in which all affected individuals have the same mutation, the clinical manifestations of HHT and their severity varied tremendously. Intrafamilial variation in expression of HHT is clearly significant, emphasizing the difficulty in establishing the diagnosis in individuals and in sub-typing families when DNA testing is not available.

Genetic mapping of a novel familial form of infantile hemangioma.

Infantile hemangiomas are the most common tumor of infancy, occurring with an incidence of up to 10% of all births. They are benign but highly proliferative lesions involving aberrant localized growth of capillary endothelium. Although most hemangiomas occur sporadically and as single lesions, or in conjunction with pleiotropic genetic syndromes, we have previously identified six kindreds where hemangiomas appear to segregate as an autosomal dominant trait with high penetrance. Four such families contain affected individuals in three or more generations. In the current study, blood samples from five of these families were collected and used in a whole genome linkage search at 10-cM resolution. We established evidence for linkage to 5q in three families, and evidence for locus heterogeneity. The three 5q-linked families were further genotyped to generate haplotype information and narrow the candidate interval. Based on recombination breakpoint analysis, the interval exists between markers D5S2490 and D5S408, spanning 55 cM, and corresponding to 5q31-33. Using information from affected and unaffected individuals, the interval spans 38 cM between markers D5S1469 and D5S211. Three candidate genes involved with blood vessel growth map to this region: fibroblast growth factor receptor-4 (FGFR4), platelet-derived growth factor receptor-beta (PDG-FRB), and fms-related tyrosine kinase-4 (FLT4). The genes and gene products associated with familial hemangiomas may be involved somatically in the more common sporadic cases.

Evidence for loss of heterozygosity of 5q in sporadic haemangiomas: are somatic mutations involved in haemangioma formation?

BACKGROUND/AIMS: Haemangiomas are common benign tumours of infancy that consist of rapidly proliferating endothelial cells. A locus for an autosomal dominant predisposition to haemangioma has been identified recently on chromosome 5q. This study aimed to investigate loss of heterozygosity on chromosomes 5 and 9 in haemangiomas. METHODS: Sporadic proliferative phase haemangiomas were microdissected. Polymerase chain reaction amplification and analysis of microsatellite markers on chromosomes 5 and 9 was carried out. RESULTS: There was a significant loss of heterozygosity for markers on chromosome 5q in haemangioma tissue, when compared with either markers from chromosome 5p (p < 0.05) or markers from chromosome 9 (p < 0.05). CONCLUSIONS: These results suggest that haemangioma formation might be associated with somatic mutational events, and provides evidence that a locus on 5q is involved in the formation of sporadic haemangiomas.

Chromosome breakpoint at 17q11.2 and insertion of DNA from three different chromosomes in a glioblastoma with exceptional glial fibrillary acidic protein expression.

A glioblastoma that retained glial fibrillary acidic protein (GFAP) in culture has a break in the long arm of chromosome 17 at band 17q11.2. DNA inserted at this breakpoint came from chromosome bands 3p21, 3q23, 16q11.2, and 22q11.2. These chromosome fragments were inserted in band 17q11.2 proximal to the neurofibromatosis-1 (NF-1) gene and neu (HER2; erbB2) oncogene loci. The glioblastoma also contained a reciprocal translocation between 16p12 and 20p12. These structural abnormalities, previously undescribed in gliomas, were demonstrated by high-resolution chromosome banding, microdissection, and fluorescence in situ hybridization (FISH). Numerical changes typical of glioblastoma were present: gain of chromosome 7 and losses of chromosomes 10, 13, and 22. The complex chromosome origin of DNA inserted in this glioma chromosome is described. The association of two infrequent events in this single glioblastoma line, this complex insertion and retention of GFAP expression, is not likely to be a chance occurrence. It raises the possibility of an association between the two events.

Familial segregation of hemangiomas and vascular malformations as an autosomal dominant trait.

BACKGROUND: The pathogenesis of infantile hemangiomas is not yet understood. Growth factors and hormonal and mechanical influences have been thought to affect the focal abnormal growth of endothelial cells in these lesions. However, these influences may represent secondary responses to an underlying primary molecular event leading to the development of hemangiomas. OBSERVATIONS: We report the rare familial occurrence of hemangiomas and/or vascular malformations in 6 kindreds, suggesting autosomal dominant inheritance. In these families, multiple generations (2-4) were affected by hemangiomas or vascular malformations. In contrast to the generally accepted female-male ratio of 3:1 to 4:1 associated with sporadic hemangiomas, the families with hemangiomas in our study demonstrated a 2:1 ratio. Additionally, vascular malformations and hemangiomas were present in different members of the same family. The vascular lesions appeared to be transmitted in an autosomal dominant fashion with moderate to high penetrance. CONCLUSIONS: We have identified 6 families demonstrating autosomal dominant segregation of childhood hemangiomas. Additionally, family members with vascular malformations were identified in these kindreds. Physicians caring for children with hemangiomas and vascular malformations should include in their medical histories inquiries about vascular lesions in other family members, even when obvious lesions are not present in the parents. The identification of the mutation(s) underlying vascular lesions will provide insight into the pathogenesis of these familial hemangiomas and, potentially, common sporadic hemangiomas. In addition, such research would shed light on the regulation of angiogenic processes during development.

Abnormal pattern of Tie-2 and vascular endothelial growth factor receptor expression in human cerebral arteriovenous malformations.

OBJECTIVE: Human cerebral arteriovenous malformations (AVMs) are speculated to result from abnormal angiogenesis. Vascular endothelial growth factor receptors (VEGF-Rs) and Tie-2 play critical roles in vasculogenesis and angiogenesis. We hypothesized that the abnormal vascular phenotype of AVMs may be associated with abnormal expression of VEGF-Rs and Tie-2. METHODS: We measured the expression of Tie-2, VEGF-R1, and VEGF-R2 in AVMs and normal brain tissue, using immunoblotting. To assess active vascular remodeling, we also measured endothelial nitric oxide synthase expression. CD31 expression was used to control for endothelial cell mass for Tie-2, VEGF-Rs, and endothelial nitric oxide synthase. Immunoblotting data were presented as relative expression, using normal brain tissue values as 100%. RESULTS: CD31 was expressed to similar degrees in AVMs and normal brain tissue (99+/-29% versus 100+/-20%, mean +/- standard error, P = 0.98). Tie-2 expression was markedly decreased in all AVMs, compared with normal brain tissue (16+/-9% versus 100+/-37%, P = 0.04). VEGF-R1 expression was decreased in four of five AVMs, but the difference between the mean values was not significant (35+/-8% versus 100+/-42%, P = 0.14). VEGF-R2 expression was decreased in all AVMs, compared with normal brain tissue (28+/-6% versus 100+/-29%, P = 0.03). There was no difference in endothelial nitric oxide synthase expression between AVMs and normal brain tissue (106+/-42% versus 100+/-25%, P = 0.91). CONCLUSION: AVM vessels exhibited abnormal expression of Tie-2 and VEGF-Rs, both of which may contribute to the pathogenesis of AVMs.

Analysis of yeast artificial chromosome clones.

The technology of cloning large fragments of DNA in yeast as yeast artificial chromosomes (YACs) (1), combined with that of electro-phoretic separation of large fragments by pulsed-field gel electrophoresis (PFGE), has revolutionized research in molecular genetics. In addition to the large insert size, the YAC cloning system offers relatively unbiased genome representation compared to the conventional bacterial cloning systems based on plasmids or bacteriophages (2,3). The enormous impact of YAC cloning and PFGE techniques on physical mapping is being realized with the building of multimegabase contigs (contiguous cloned stretches of DNA). Some examples include mapping and cloning of a large gene like cystic fibrosis (CF) (4), of a chromosomal band like that of 18q21.3 (5), of a substantial portion of an entire human chromosome, Xq24-28 (3), and even an entire organism, C. eI.egum (2). Similarly, the use of YAC clones in the identification of disease-related genes like neurofibromatosis (NFl) is well documented (6). PFGE techniques play a major and critical role in the construction (see Chapter 16) and characterization (later in this chapter) of YAC clones.

Somatic mutations in the neurofibromatosis 1 gene in gliomas and primitive neuroectodermal tumours.

The increased frequency of glioma among neurofibromatosis 1 (NF1) patients suggests a general involvement of the NF1 gene in glioma tumourigenesis. Using the methodology of conventional Southern blotting with a complete panel of overlapping partial cDNAs covering the whole NF1 gene, we screened 31 gliomas of several different subtypes and 3 primitive neuroectodermal tumours (PNETs) from non-NF1 patients for aberrant restriction patterns in their tumour DNA samples. Clear evidence for somatic mutation events at the NF1 gene locus was found in 1 astrocytoma, 2 glioblastomas, 1 ependymoma and 1 PNET with an astrocytic component. These results suggest that the NF1 gene is important in suppressing tumours of neuroectodermal origin.

Sequencing and analysis of genomic fragments from the NF1 locus.

The sequence of five non-contiguous genomic fragments encompassing 14.4 kilobases from the NF1 locus have been determined by fluorescence-based automated DNA sequence analysis. These fragments included one kilobase of the NF1 coding region, which resulted in the identification of the intron/exon boundaries of five exons. Based on these sequences, five new NF1 exon-PCR assays have been developed, that could be useful for detecting new NF1 mutations. The genomic sequences were analyzed for the presence of Alu repetitive elements and their classification is described. This analysis may provide some insight into the characterization of genetic rearrangements resulting in disruption of the NF1 gene.