Feasibility of low-throughput next generation sequencing for germline DNA screening.

BACKGROUND: Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS). METHODS: Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1-12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases. RESULTS: The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤ 0.15, strand biases >-35, or genotype quality scores ≤ 80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤ 5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity). CONCLUSIONS: Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: a comprehensive profiling approach.

BACKGROUND: Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC. METHODS: The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations. RESULTS: A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy. CONCLUSIONS: High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

Five quantitative trait loci control radiation-induced adenoma multiplicity in Mom1R Apc Min/+ mice.

Ionising radiation is a carcinogen capable of inducing tumours, including colorectal cancer, in both humans and animals. By backcrossing a recombinant line of Apc(Min/+) mice to the inbred BALB/c mouse strain, which is unusually sensitive to radiation-induced tumour development, we obtained panels of 2Gy-irradiated and sham-irradiated N2 Apc(Min/+) mice for genotyping with a genome-wide panel of microsatellites at approximately 15 cM density and phenotyping by counting adenomas in the small intestine. Interval and composite interval mapping along with permutation testing identified five significant susceptibility quantitative trait loci (QTLs) responsible for radiation induced tumour multiplicity in the small intestine. These were defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (log of odds, LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p<0.00001, 6.2, p<0.00001) and two regions within chromosome 16 respectively (LOD 4.1, p = 4x10(-5), 4.8, p<0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p<0.005). Genes containing BALB/c specific non-synonymous polymorphisms were identified within Mrip regions and prediction programming used to locate potentially functional polymorphisms. Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in Apc(Min/+) mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Casc5, and Bub1), DNA repair (Recc1 and Prkdc) or inflammation (Duox2, Itgb2l and Cxcl5). Our study demonstrates use of in silico analysis in candidate gene identification as a way of reducing large-scale backcross breeding programmes.

Protein binding and metabolism influence the relative skin sensitization potential of cinnamic compounds.

Skin protein modification (haptenation) is thought to be a key step in the manifestation of sensitization to low molecular mass chemicals (<500 g/mol). For sensitizing chemicals that are not protein reactive, it is hypothesised that metabolic activation can convert such chemicals into protein reactive toxins within the skin. trans-Cinnamaldehyde, alpha-amyl cinnamaldehyde, and trans-cinnamic alcohol are known sensitizers with differing potencies in man, where the former two are protein reactive and the latter is not. Here, we have used immunochemical methods to investigate the extent of protein-cinnamaldehyde binding in rat and human skin homogenates that have been incubated (for either 5, 15, 30, or 60 min) at 37 degrees C with cinnamaldehyde, alpha-amyl cinnamaldehyde (at concentrations of between 1 and 40 mM), and cinnamic alcohol (at higher concentrations of 200 or 400 mM). Cinnamaldehyde specific antiserum was raised specially. A broad range (in terms of molecular mass) of protein-cinnamaldehyde adducts was detected (as formed in a time- and concentration-dependent manner) in skin treated with cinnamaldehyde and cinnamic alcohol but not with alpha-amyl cinnamaldehyde. Mechanistic observations have been related to relative skin sensitization potential, as determined using the local lymph node assay (LLNA) as a biological read-out. The work presented here suggests that there is a common hapten involved in cinnamaldehyde and cinnamic alcohol sensitization and that metabolic activation (to cinnamaldehyde) is involved in the latter. Conversely, there does not appear to be a common hapten for cinnamaldehyde and alpha-amyl cinnamaldehyde. Such mechanistic work on protein modification is important in understanding the early mechanisms of skin sensitization. Such knowledge can then be used in order that effective and appropriate in vitro/in silico tools for predicting sensitization potential, with a high confidence, can be developed.

Linkage disequilibrium in human populations.

Whereas the human linkage map appears on limited evidence to be constant over populations, maps of linkage disequilibrium (LD) vary among populations that differ in gene history. The greatest difference is between populations of sub-Saharan origin and populations remotely derived from Africa after a major bottleneck that reduced their heterozygosity and altered their Malecot parameters, increasing the intercept M that reflects association in founders and decreasing the exponential decline epsilon. Variation among populations within this ethnic dichotomy is much smaller. These observations validate use of a cosmopolitan LD map based on a sizeable sample representing a large population reliably typed for markers at high density. Then an LD map for a region or isolate within an ethnic group may be created by fitting the sample LD to the cosmopolitan map, estimating Malecot parameters simultaneously. The cosmopolitan map scaled by epsilon recovers 95% of the information that a local map at the same density gives and therefore more than the information in a low-resolution local map. Relative to a Eurasian cosmopolitan map the scaling factors are estimated to be 0.82 for isolates of European descent, 1.53 for Yorubans, and 1.74 for African Americans. These observations are consistent with a common bottleneck (perhaps but not necessarily speciation) approximately 173,500 years ago, if the bottleneck associated with migration out of Africa was 100,000 years ago. Eurasian populations (especially isolates with numerous cases) are efficient for genome scans, and populations of recent African origin (such as African Americans) are efficient for identification of causal polymorphisms within a candidate sequence.