Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas ( TCGA) pilot project aims to assess the value of large- scale multi- dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas - the most common type of primary adult brain cancer - and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol- 3- OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

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Comprehensive genomic characterization defines human glioblastoma genes and core pathways

Point mutations in the coding regions of genes are commonly assumed to exert their effects by altering single amino acids in the encoded proteins. However, there is increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing. Nonsense, missense and even translationally silent mutations can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency. As the splicing mechanisms that depend on exonic signals are elucidated, new therapeutic approaches to treating certain genetic diseases can begin to be explored.

/pdf/listening-to-silence-and-understanding-nonsense-exonic-41age9auze.pdf

Listening to silence and understanding nonsense: exonic mutations that affect splicing

The germline mutation rate in human males, especially older males, is generally much higher than in females, mainly because in males there are many more germ-cell divisions. However, there are some exceptions and many variations. Base substitutions, insertion-deletions, repeat expansions and chromosomal changes each follow different rules. Evidence from evolutionary sequence data indicates that the overall rate of deleterious mutation may be high enough to have a large effect on human well-being. But there are ways in which the impact of deleterious mutations can be mitigated.

The origins, patterns and implications of human spontaneous mutation

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is a cancer predisposition syndrome, and somatic NF1 mutations are also common in tumours. The neurofibromin protein (encoded by NF1) is a RAS GTPase-activating protein that activates RAS–MAPK signalling when lost. This Review discusses NF1 disease, neurofibromin signalling pathways and recent developments in NF1 therapeutics.

A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.

https://www.cell.com/ajhg/pdf/S0002-9297(07)64009-9.pdf

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain

Christian Ehlers

Papers

Minor Lesion Mutational Spectrum of the Entire NF1 Gene Does Not Explain Its High Mutability but Points to a Functional Domain Upstream of the GAP-Related Domain