Advances in genetic and epigenetic analyses of gliomas: a neuropathological perspective

Gliomas, the most common malignant primary brain tumors, are universally fatal once they progress from low-grade into high-grade neoplasms. In recent years, we have accumulated unprecedented data about the genetic and epigenetic abnormalities in gliomas; yet, our appreciation of how these deadly tumors arise is still rudimentary. One of the major deterrents in understanding gliomagenesis is the remarkably complex and heterogeneous molecular composition of gliomas, as well as their ability to change phenotypically as they progress and recur. In the past decade, several monumental studies have begun to define better glioma heterogeneity. Four distinct molecular subgroups have emerged: proneural, classical, mesenchymal, and neural; which have unique gene expression signatures and prognostic significance. Of these, gliomas of the proneural subtype, which encompass most grade II/III diffuse gliomas and secondary glioblastomas and often carry isocitrate dehydrogenase (IDH) mutations, have emerged as a distinct tumor subclass with a notably superior prognosis. Important molecular markers with prognostic relevance, such as mutant IDH1/2, have already been incorporated into clinical neuropathological practice. The recent molecular discoveries in gliomas have also emphasized the intimate link between epigenetics and genetics in gliomagenesis. Several of the novel genetic mutations described are responsible for distinct epigenetic remodeling in gliomas, the mechanisms of which are currently being elucidated. Importantly, these epigenetic and genomic alterations represent new and exciting drug targets for future therapeutic interventions in our continuous fight with this fatal malignancy.     

KDM1 is a novel therapeutic target for the treatment of gliomas

Glioma development is a multistep process, involving alterations in genetic and epigenetic mechanisms. Understanding the mechanisms and enzymes that promote epigenetic changes in gliomas are urgently needed to identify novel therapeutic targets. We examined the role of histone demethylase KDM1 in glioma progression. KDM1 was overexpressed in gliomas and its expression positively correlated with histological malignancy. Knockdown of KDM1 expression or its pharmacological inhibition using pargyline or NCL-1 significantly reduced the proliferation of glioma cells. Inhibition of KDM1 promoted up regulation of the p53 target genes p21 and PUMA. Patient-derived primary GBM cells expressed high levels of KDM1 and pharmacological inhibition of KDM1 decreased their proliferation. Further, KDM1 inhibition reduced the expression of stemness markers CD133 and nestin in GBM cells. Mouse xenograft assays revealed that inhibition of KDM1 significantly reduced glioma xenograft tumor growth. Inhibition of KDM1 increased levels of H3K4-me2 and H3K9-Ac histone modifications, reduced H3K9-me2 modification and promoted expression of p53 target genes (p21 and PUMA), leading to apoptosis of glioma xenograft tumors. Our results suggest that KDM1 is overexpressed in gliomas and could be a potential therapeutic target for the treatment of gliomas.     

Methylation profiling identifies 2 groups of gliomas according to their tumorigenesis

Extensive genomic and gene expression studies have been performed in gliomas, but the epigenetic alterations that characterize different subtypes of gliomas remain largely unknown. Here, we analyzed the methylation patterns of 807 genes (1536 CpGs) in a series of 33 low-grade gliomas (LGGs), 36 glioblastomas (GBMs), 8 paired initial and recurrent gliomas, and 9 controls. This analysis was performed with Illumina's Golden Gate Bead methylation arrays and was correlated with clinical, histological, genomic, gene expression, and genotyping data, including IDH1 mutations. Unsupervised hierarchical clustering resulted in 2 groups of gliomas: a group corresponding to de novo GBMs and a group consisting of LGGs, recurrent anaplastic gliomas, and secondary GBMs. When compared with de novo GBMs and controls, this latter group was characterized by a very high frequency of IDH1 mutations and by a hypermethylated profile similar to the recently described glioma CpG island methylator phenotype. MGMT methylation was more frequent in this group. Among the LGG cluster, 1p19q codeleted LGG displayed a distinct methylation profile. A study of paired initial and recurrent gliomas demonstrated that methylation profiles were remarkably stable across glioma evolution, even during anaplastic transformation, suggesting that epigenetic alterations occur early during gliomagenesis. Using the Cancer Genome Atlas data set, we demonstrated that GBM samples that had an LGG-like hypermethylated profile had a high rate of IDH1 mutations and a better outcome. Finally, we identified several hypermethylated and downregulated genes that may be associated with LGG and GBM oncogenesis, LGG oncogenesis, 1p19q codeleted LGG oncogenesis, and GBM oncogenesis.     

Gliomas display a microRNA expression profile reminiscent of neural precursor cells

Gliomas express many genes that play a role in neural precursor cells (NPCs), but no direct comparison between glioma and stem cell (SC) gene expression profiles has been performed. To investigate the similarities and differences between gliomas and SCs, we compared the microRNA (miRNA) expression signatures of glial tumors, embryonic SCs (ESCs), NPCs, and normal adult brains from both human and mouse tissues. We demonstrated that both human gliomas (regardless of their grade) and methylcholanthrene-induced mouse glioma shared an miRNA expression profile that is reminiscent of NPCs. About half of the miRNAs expressed in the shared profile clustered in seven genomic regions susceptible to genetic/epigenetic alterations in various cancers. These clusters comprised the miR17 family, mir183-182, and the SC-specific clusters mir367-302 and mir371-373, which are upregulated in gliomas, ESCs, and NPCs. The bipartite cluster of 7 + 46 miRNAs on chromosome 14q32.31, which might represent the largest tumor suppressor miRNA cluster, was downregulated in the shared expression profile. This study provides the first evidence for association between these clusters and gliomas. Despite the broad similarity in the miRNA expression profiles, 15 miRNAs showed disparate expression between SC and gliomas. Ten miRNAs belong to the 2 SC-specific clusters and the remaining (mir135b, mir141, mir205, mir200C, and mir301a) have been previously shown to associate with malignancies. Our finding showed that all gliomas displayed NPC-like miRNA signatures, which may have implications for studies of glioma origins. Furthermore, careful study of the 15 miRNAs that differ in expression between SCs and gliomas, particularly those 5 that are not SC-specific, may enhance our understanding of gliomagenesis.     

Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.     

Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish

Background

Ongoing characterization of glioma has revealed that Akt signaling plays a crucial role in gliomagenesis. In mouse models, however, Akt alone was not sufficient to induce glioma.

Methods

We established transgenic zebrafish that overexpressed dominant-active (DA) human Akt1 or Rac1^G12V (DARac1) at ptf1a domain and investigated transgenic phenotypes and mechanisms leading to gliomagenesis.

Results

Transgene expressions were spatiotemporally restricted without any developmental abnormality of embryos and persisted at cerebellum and medulla in adult zebrafish. DAAkt1 alone induced glioma (with visible bumps at the head), with incidences of 36.6% and 49% at 6 and 9 months, respectively. Histologically, gliomas showed various histologic grades, increased proliferation, and frequent invasion into the fourth ventricle. Preferential location of small tumors at periventricular area and coexpression of Her4 suggested that tumors originated from Ptf1a- and Her4-positive progenitor cells at ventricular zone. Gliomagenesis was principally mediated by activation of survival pathway through upregulation of survivin genes. Although DARac1 alone was incapable of gliomagenesis, when coexpressed with DAAkt1, gliomagenesis was accelerated, showing higher tumor incidences (62.0% and 73.3% at 6 and 9 months, respectively), advanced histologic grade, invasiveness, and shortened survival. DARac1 upregulated survivin2, cyclin D1, β-catenin, and snail1a but downregulated E-cadherin, indicating that DARac1 promotes gliomagenesis by enhancing proliferation, survival, and epithelial-to-mesenchymal transition. On pharmacologic tests, only Akt1/2 inhibitor effectively suppressed gliomagenesis, inhibited cellular proliferation, and induced apoptosis in established gliomas.

Conclusions

The zebrafish model reinforces the pivotal role of Akt signaling in gliomagenesis and suggests Rac1 as an important protein involved in progression.     

miR-19a and miR-19b Overexpression in Gliomas

Astrocytic gliomas are the most common type of human primary brain tumors with poor prognosis. MicroRNAs(miRs) are frequently deregulated in gliomas and play an oncogenic or tumor suppressor role. In our previous study we found that miR-19a and miR-19b were up-regulated in malignant glioma cell lines by microRNA array. For further validation of this finding, the expression of miR-19a and miR-19b was detected by qRT-PCR and in situ hybridization(ISH) in 8 malignant glioma cell lines, 43 freshly resected glioma samples and 75 archival paraffin embedded glioma specimens with different grades of malignancy in the present study. The results demonstrate that miR-19a and miR-19b are overexpressed in glioma cell lines and astrocytic glioma tissues, and their expression level is positively correlated with tumor grades. Additionally, the tumor suppressor gene PTEN is identified as the target of miR-19a and miR-19b by Luciferase assay. It is speculated that miR-19a and miR-19b may have an oncogenic role in gliomagenesis at least partially via the negative regulation of PTEN and the molecular mechanism of gliomagenesis in which miR 19a and miR-19b involved should be investigated further.     

Clinical characterization and immunosuppressive regulation of CD161 (KLRB1) in glioma through 916 samples

BackgroundGlioblastoma is a paradigm of cancer‐associated immunosuppression, limiting the effects of immunotherapeutic strategies. Thus, identifying the molecular mechanisms underlying immune surveillance evasion is critical. Recently, the preferential expression of inhibitory natural killer (NK) cell receptor CD161 on glioma‐infiltrating cytotoxic T cells was identified. Focusing on the molecularly annotated, large‐scale clinical samples from different ethnic origins, the data presented here provide evidence of this immune modulator''s essential roles in brain tumor biology.MethodsRetrospective RNA‐seq data analysis was conducted in a cohort of 313 patients with glioma in the Chinese Glioma Genome Atlas (CGGA) database and 603 patients in The Cancer Genome Atlas (TCGA) database. In addition, single‐cell sequencing data from seven surgical specimens of glioblastoma patients and a model in which patient‐derived glioma stem cells were cocultured with peripheral lymphocytes, were used to analyze the molecular evolution process during gliomagenesis.ResultsCD161 was enriched in high‐grade gliomas and isocitrate dehydrogenase (IDH)‐wildtype glioma. CD161 acted as a potential biomarker for the mesenchymal subtype of glioma and an independent prognostic factor for the overall survival (OS) of patients with glioma. In addition, CD161 played an essential role in inhibiting the cytotoxicity of T cells in glioma patients. During the process of gliomagenesis, the expression of CD161 on different lymphocytes dynamically evolved.ConclusionThe expression of CD161 was closely related to the pathology and molecular pathology of glioma. Meanwhile, CD161 promoted the progression and evolution of gliomas through its unique effect on T cell dysfunction. Thus, CD161 is a promising novel target for immunotherapeutic strategies in glioma treatment.     

Shared epigenetic mechanisms in human and mouse gliomas inactivate expression of the growth suppressor SLC5A8

Tumors arise in part from the deleterious effects of genetic and epigenetic mechanisms on gene expression. In several mouse models of human tumors, the tumorigenic phenotype is reversible, suggesting that epigenetic mechanisms also contribute significantly to tumorigenesis in mice. It is not known whether these are the same epigenetic mechanisms in human and mouse tumors or whether they affect homologous genes. Using an integrated approach for genome-wide methylation and copy number analyses, we identified SLC5A8 on chromosome 12q23.1 that was affected frequently by aberrant methylation in human astrocytomas and oligodendrogliomas. SLC5A8 encodes a sodium monocarboxylate cotransporter that was highly expressed in normal brain but was significant down-regulated in primary gliomas. Bisulfite sequencing analysis showed that the CpG island was unmethylated in normal brain but frequently localized methylated in brain tumors, consistent with the tumor-specific loss of gene expression. In glioma cell lines, SLC5A8 expression was also suppressed but could be reactivated with a methylation inhibitor. Expression of exogenous SLC5A8 in LN229 and LN443 glioma cells inhibited colony formation, suggesting that it may function as a growth suppressor in normal brain cells. Remarkably, 9 of 10 murine oligodendroglial tumors (from p53+/- or ink4a/arf+/- animals transgenic for S100beta-v-erbB) showed a similar tumor-specific down-regulation of mSLC5A8, the highly conserved mouse homologue. Taken together, these data suggest that SLC5A8 functions as a growth suppressor gene in vitro and that it is silenced frequently by epigenetic mechanisms in primary gliomas. The shared epigenetic inactivation of mSLC5A8 in mouse gliomas indicates an additional degree of commonality in the origin and/or pathway to tumorigenesis between primary human tumors and these mouse models of gliomas.     

Innate neural stem cell heterogeneity determines the patterning of glioma formation in children

The concept that gliomas comprise a heterogeneous group of diseases distinguished by their developmental origin raises the intriguing possibility that neural stem cells (NSCs) from different germinal zones have differential capacities to respond to glioma-causing genetic changes. We demonstrate that lateral ventricle subventricular zone NSCs are molecularly and functionally distinct from those of the third ventricle. Consistent with a unique origin for pediatric low-grade glioma, third ventricle, but not lateral ventricle, NSCs hyperproliferate in response to mutations characteristic of childhood glioma. Finally, we demonstrate that pediatric optic gliomas in Nf1 genetically engineered mice arise from the third ventricle. Collectively, these observations establish the importance of innate brain region NSC heterogeneity in the patterning of gliomagenesis in children and adults.     

PDGF-B-driven gliomagenesis can occur in the absence of the proteoglycan NG2

Background  

In the last years, the transmembrane proteoglycan NG2 has gained interest as a therapeutic target for the treatment of diverse tumor types, including gliomas, because increases of its expression correlate with dismal prognosis. NG2 has been shown to function as a co-receptor for PDGF ligands whose aberrant expression is common in gliomas. We have recently generated a glioma model based on the overexpression of PDGF-B in neural progenitors and here we investigated the possible relevance of NG2 during PDGF-driven gliomagenesis.     

Constitutive Activation of Raf-1 Induces Glioma Formation in Mice

In human glioblastoma multiforme (GBM), RAS activity is upregulated in the majority of the tumors. Furthermore, the levels of phospho-mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), a downstream effector of RAS, are also increased. In mice, activated KRas cooperates with the loss of INK4a-ARF locus or with activated Akt to induce gliomas, confirming an important role for this pathway in glioma biology. However, to correctly target therapies against the RAS signaling pathway, it is necessary to identify the effectors that contribute to RAS-mediated gliomagenesis. In this study, we investigated the contribution of RAF signaling in glioma oncogenesis. We find that the levels of RAF-1 and BRAF proteins and RAF kinase activity are increased in human GBM samples. We confirm the importance of this finding by demonstrating a causal role for a constitutively active Raf-1 mutant in glioma formation in mice. Specifically, we find that activated Raf-1 cooperates with Arf loss or Akt activation to generate gliomas similar to activated KRas under the same conditions. Our study suggests that the oncogenic effect of KRas in glioma formation may be transduced at least in part through Raf signaling and that therapeutic targeting of this pathway may be beneficial in glioma treatment.     

Animal Models of Cell Cycle Dysregulation and the Pathogenesis of Gliomas

Mutations in gliomas, for the most part, fall into two main categories. The first category of mutations affects genes that produce proteins which activate signal transduction pathways downstream of tyrosine kinase receptors; the second category disrupts the pathways leading to cell cycle arrest. Cell cycle arrest pathways normally maintain cells in the G1 phase of the cell cycle, preventing inappropriate proliferation. The role of disregulation of these pathways in tumor formation is currently the focus of many investigations. Studies carried out with astrocytes and other cell types indicate that these pathways may also function in maintenance of appropriate chromosome number and differentiated phenotype, and in acquisition of senescence. Genetically defined mouse models of gliomagenesis have been helpful in increasing our understanding of how cell cycle arrest pathways cooperate with alterations in signal transduction pathways to provoke tumor formation in many cell types, including glial cells. Various strategies for experimental cell cycle arrest disruption show minimal or no formation of gliomas. In contrast, gliomas are generated with a number of strategies that enhance signal transduction downstream of tyrosine kinase receptors. Experimental disruption of the cell cycle arrest pathways is required for gliomagenesis in some of these models, but not in others. Furthermore in some cases, although not required for gliomagenesis, disruption of the cell cycle arrest pathways appears to enhance glioma formation. The results of these mouse model experiments imply a potentially complex role for cell cycle arrest disruption in human gliomagenesis.     

Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma

Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.     

Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain

Stem cell factor (SCF) is overexpressed by neurons following brain injury as well as by glioma cells; however, its role in gliomagenesis remains unclear. Here, we demonstrate that SCF directly activates brain microvascular endothelial cells (ECs) in vitro and induces a potent angiogenic response in vivo. Primary human gliomas express SCF in a grade-dependent manner and induce normal neurons to express SCF in brain regions infiltrated by glioma cells, areas that colocalize with prominent angiogenesis. Downregulation of SCF inhibits tumor-mediated angiogenesis and glioma growth in vivo, whereas overexpression of SCF is associated with shorter survival in patients with malignant gliomas. Thus, the SCF/c-Kit pathway plays an important role in tumor- and normal host cell-induced angiogenesis within the brain.     

Molecular pathogenesis of IDH mutations in gliomas

The isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) genes are mutated in 50-80% of astrocytomas, oligodendrogliomas or oligoastrocytomas of grades II and III, and secondary glioblastomas; they are, however, seldom mutated in primary glioblastomas and never in other types of glioma. Gliomas with IDH1/2 mutations always harbor either TP53 mutations or total 1p/19q loss. This suggests these two types of tumor may arise from common progenitor cells that have IDH1/2 mutations, subsequently evolving into each tumor type with the acquisition of TP53 mutations or total 1p/19q loss. Survival is significantly longer for patients with IDH-mutated gliomas than for those with IDH-wild type tumors. This observation indicates that IDH status defines biologically different subgroups among gliomas. The molecular pathogenesis of IDH1/2 mutations in the development of gliomas is unclear. The mutated IDH1/2 enzyme generates D-2-hydroxyglutarate. Several theories have been proposed, including: increased angiogenesis because of accumulation of HIF-1α; a glioma CpG island methylator phenotype (G-CIMP) induced by inhibition of TET2; and increased vulnerability to oxidative stress because of depletion of antioxidants. Elucidating the pathogenesis of IDH mutations will aid better understanding of the molecular mechanisms of gliomagenesis and may lead to the development of novel molecular classification and therapy.     

Comprehensive analysis of microRNA expression profile in malignant glioma tissues

Malignant gliomas represent the most devastating group of brain tumors in adults, among which glioblastoma multiforme (GBM) exhibits the highest malignancy rate. Despite combined modality treatment, GBM recurs and is invariably fatal. A further insight into the molecular background of gliomagenesis is required to improve patient outcomes. The primary aim of this study was to gain broad information on the miRNA expression pattern in malignant gliomas, mainly GBM. We investigated the global miRNA profile of malignant glioma tissues with miRNA microarrays, deep sequencing and meta‐analysis. We selected miRNAs that were most frequently deregulated in glioblastoma tissues, as well as in peritumoral areas, in comparison with normal human brain. We identified candidate miRNAs associated with the progression from glioma grade III to glioma grade IV. The meta‐analysis of miRNA profiling studies in GBM tissues summarizes the past and recent advances in the investigation of the miRNA signature in GBM versus noncancerous human brain and provides a comprehensive overview. We propose a list of 35 miRNAs whose expression is most frequently deregulated in GBM patients and of 30 miRNA candidates recognized as novel GBM biomarkers.     

Human glioma growth is controlled by microRNA-10b

MicroRNA (miRNA) expression profiling studies revealed a number of miRNAs dysregulated in the malignant brain tumor glioblastoma. Molecular functions of these miRNAs in gliomagenesis are mainly unknown. We show that inhibition of miR-10b, a miRNA not expressed in human brain and strongly upregulated in both low-grade and high-grade gliomas, reduces glioma cell growth by cell-cycle arrest and apoptosis. These cellular responses are mediated by augmented expression of the direct targets of miR-10b, including BCL2L11/Bim, TFAP2C/AP-2γ, CDKN1A/p21, and CDKN2A/p16, which normally protect cells from uncontrolled growth. Analysis of The Cancer Genome Atlas expression data set reveals a strong positive correlation between numerous genes sustaining cellular growth and miR-10b levels in human glioblastomas, while proapoptotic genes anticorrelate with the expression of miR-10b. Furthermore, survival of glioblastoma patients expressing high levels of miR-10 family members is significantly reduced in comparison to patients with low miR-10 levels, indicating that miR-10 may contribute to glioma growth in vivo. Finally, inhibition of miR-10b in a mouse model of human glioma results in significant reduction of tumor growth. Altogether, our experiments validate an important role of miR-10b in gliomagenesis, reveal a novel mechanism of miR-10b-mediated regulation, and suggest the possibility of its future use as a therapeutic target in gliomas.     

DMBT1 polymorphisms: relationship to malignant glioma tumorigenesis

The deleted in malignant brain tumors 1 (DMBT1) gene on 10q25-26 is a candidate tumor suppressor gene in malignant gliomas, but its role is controversial, e.g., some DMBT1 homozygous deletions reflect unmasking of constitutional deletion polymorphisms by 10q loss. To clarify the role of DMBT1 in gliomagenesis, we investigated three reported deletion hot spots. Homozygous deletions at DMBT1 repeat 2-4 to 2-7 were found in 10 of 73 gliomas with 10q loss, but all 10 deletions reflected unmasking of constitutional hemizygous deletions. Alleles bearing deletion 2-4/2-7 were not selected significantly for by 10q loss, with retention of only 10 of 16 deleted alleles. No homozygous deletion was detected at locus 74k in the 5' upstream region of DMBT1, and four tightly linked polymorphisms were found around this region; chromosome 10q loss randomly affected alleles with or without the variant sequences around locus 74k. Moreover, no significant selection pressure was detected for the haplotype with both deletion 2-4/2-7 and 5' polymorphisms. There was no segregation of deletion 2-4/2-7 in glioma patients compared with unrelated individuals from reference families but a suggestion of a difference in the distribution of the 5' polymorphisms between the reference individuals and glioma patients. Constitutional polymorphisms at DMBT1 repeat 2-9/2-10 appeared common in patients with both benign brain tumors and gliomas. A homozygote for both the 2-4/2-7 deletion and the 5' polymorphisms had a glioma arise at a typical age and without an apparent family cancer predisposition. These data suggest that DMBT1 polymorphisms are not likely primary targets of 10q loss in malignant gliomas and do not support a major role for DMBT1 in gliomagenesis.