Examination of Genetic Susceptibility in Radiation-Associated Meningioma.

Previous epidemiological studies have demonstrated elevated susceptibility to ionizing radiation in some families, thus suggesting the presence of genetic components that conferred increased rate of radiation-associated meningioma (RAM). In this study, we exome-sequenced and investigated the segregation pattern of rare deleterious variants in 11 RAM pedigrees. In addition, we performed a rare-variant association analysis in 92 unrelated familial cases of RAM that were ancestry-matched with 88 meningioma-free controls. In the pedigree analysis, we found that each family carried mostly a unique set of rare deleterious variants. A follow-up pathway analysis of the union of the genes that segregated within each of the 11 pedigrees identified a single statistically significant (q value = 7.90E-04) "ECM receptor interaction" set. In the case-control association analysis, we observed no statistically significant variants or genes after multiple testing correction; however, examination of ontological categories of the genes that associated with RAM at nominal P values <0.01 identified biologically relevant pathways such as DNA repair, cell cycle and apoptosis. These results suggest that it is unlikely that a small number of highly penetrant genes are involved in the pathogenesis of RAM. Substantially larger studies are needed to identify genetic risk variants and genes in RAM.

DNA analysis with multiplex microarray-enhanced PCR.

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3' end of a universal amplification primer. The complex primer pair is covalently tethered through its 5' end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 10(6)-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 10(3) genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.

The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas.

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

[Heterogeneity of the structural organization of the chromatin domain including the dihydrofolate reductase gene in Chinese hamster ovary cell culture]. / Geterogennost' strukturnoi organizatsii khromatina domena, vkliuchaiushchego gen digidrofolatreduktazy, v kul'ture kletok iaichnikov kitaiskogo khomiachka.

We investigated histone-DNA interactions in chromatin of amplified about 1000 fold dihydrofolate reductase (DHFR) domain in methotrexate resistant Chinese hamster ovary cell line CHOC 400. We explored chromatin structure of DHFR-gene and extended region lying 3'-downstream of the DHFR-gene, which includes two zones of DNA replication initiation, Ori-beta and Ori-gamma, and matrix attachment region (MAR). Using the method of hybridization with "protein images" we found that the yield of both core histones and histone H1 cross-linked with DNA in 5'-region of the DHFR-gene, in Ori-gamma and regions flanked from either side MAR is significantly decreased in comparison with 3'-transcribed region of the DHFR-gene, Ori-beta and MAR. It was shown previously that decreased yield of histones cross-linked with DNA is typical for 5'-region of actively transcribed genes. So, histone-DNA interactions in chromatin of the 5'-region of the house-keeping and moderately transcribed DHFR-gene, Ori-gamma and regions which flank MAR resemble those in 5'-regions of actively transcribed genes. Significant differences in amount of histones cross-linked with DNA in various parts of the DHFR-domain testify to high level of heterogeneity of chromatin structure in the domain.

Proximal and long-range alterations in chromatin structure surrounding the Chinese hamster dihydrofolate reductase promoter.

The chromatin structure of the dihydrofolate reductase (DHFR) gene was examined by DNA/protein cross-linking, chemical DNA methylation, and micrococcal nuclease digestion. The 5' promoter region of the gene displays two nucleosome-free zones (-550 to -300 and -150 to +100 bp relative to the ATG codon), each of which contains a number of micrococcal nuclease-hypersensitive sites. Regions upstream from the distal hypersensitive zone (-900 to -550 bp), downstream from the proximal hypersensitive zone (+100 to +400 bp), and between these two zones (-300 to -150 bp) appear either to be more than 80% histone-free or to contain histones whose globular domains have lost most of their contacts with DNA. Overall, a broad zone extending from -4300 to +4700 bp is altered relative to bulk chromatin, and within this region there are positioned nucleosomes and/or nucleosome-free zones in which the DNA appears to interact with a number of different non-histone proteins. By comparison, the chromatin in the 3' end of the gene (including the right end of the 5th intron and the 6th exon) contains randomly positioned nucleosomes, and its structure is intermediate between that of the 5' end of the gene and a downstream matrix attachment region that contains regularly organized chromatin. A 2.3 kb zone in the central part of the 5th intron reveals some features similar to the 5' end of the gene, suggesting a hitherto unrecognized functional role.

Attachment to the nuclear matrix mediates specific alterations in chromatin structure.

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-beta and ori-gamma) being somewhat preferred. We show here that the chromatin in the ori-beta and ori-gamma regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.