Massively parallel sequencing of customised forensically informative SNP panels on the MiSeq.

Forensic DNA-based intelligence, or forensic DNA phenotyping, utilises SNPs to infer the biogeographical ancestry and externally visible characteristics of the donor of evidential material. SNaPshot® is a commonly employed forensic SNP genotyping technique, which is limited to multiplexes of 30-40 SNPs in a single reaction and prone to PCR contamination. Massively parallel sequencing has the ability to genotype hundreds of SNPs in multiple samples simultaneously by employing an oligonucleotide sample barcoding strategy. This study of the Illumina MiSeq massively parallel sequencing platform analysed 136 unique SNPs in 48 samples from SNaPshot PCR amplicons generated by five established forensic DNA phenotyping assays comprising the SNPforID 52-plex, SNPforID 34-plex, Eurasiaplex, Pacifiplex and IrisPlex. Approximately 3 GB of sequence data were generated from two MiSeq flow cells and profiles were obtained from just 0.25 ng of DNA. Compared with SNaPshot, an average 98% genotyping concordance was achieved. Our customised approach was successful in attaining SNP profiles from extremely degraded, inhibited, and compromised casework samples. Heterozygote imbalance and sequence coverage in negative controls highlight the need to establish baseline sequence coverage thresholds and refine allele frequency thresholds. This study demonstrates the potential of the MiSeq for forensic SNP analysis.

Identification of common genetic variation that modulates alternative splicing.

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

Variations within oxygen-regulated gene expression in humans.

The effects of hypoxia on gene transcription are mainly mediated by a transcription factor complex termed hypoxia-inducible factor (HIF). Genetic manipulation of animals and studies of humans with rare hereditary disease have shown that modifying the HIF pathway affects systems-level physiological responses to hypoxia. It is, however, an open question whether variations in systems-level responses to hypoxia between individuals could arise from variations within the HIF system. This study sought to determine whether variations in the responsiveness of the HIF system at the cellular level could be detected between normal individuals. Peripheral blood lymphocytes (PBL) were isolated on three separate occasions from each of 10 healthy volunteers. After exposure of PBL to eight different oxygen tensions ranging from 20% to 0.1%, the expression levels of four HIF-regulated transcripts involved in different biological pathways were measured. The profile of expression of all four transcripts in PBL was related to oxygen tension in a curvilinear manner. Double logarithmic transformation of these data resulted in a linear relationship that allowed the response to be parameterized through a gradient and intercept. Analysis of variance (ANOVA) on these parameters showed that the level of between-subject variation in the gradients of the responses that was common across all four HIF-regulated transcripts was significant (P = 0.008). We conclude that statistically significant variation within the cellular response to hypoxia can be detected between normal humans. The common nature of the variability across all four HIF-regulated genes suggests that the source of this variation resides within the HIF system itself.

Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry in genomics research.

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS-based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS-based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quantification, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies.

P2RX7: A bipolar and unipolar disorder candidate susceptibility gene?

The chromosomal region 12q24 has been previously implicated by linkage studies of both bipolar disorder and unipolar mood disorder and we have reported two pedigrees segregating both bipolar disorder and Darier's disease that show linkage across this region. The gene P2RX7 is located in this chromosomal region and has been recently reported as a susceptibility gene for bipolar disorder and unipolar depression. The non-synonymous SNP rs2230912 (resulting in amino-acid polymorphism Q460R) showed the strongest association and has been postulated to be pathogenically relevant. We have investigated this gene in a large UK case-control sample (bipolar I disorder N = 687, unipolar recurrent major depression N = 1,036, controls N = 1,204). Neither rs2230912 nor any of 8 other SNPs genotyped across P2RX7 was found to be associated with mood disorder in general, nor specifically with bipolar or unipolar disorder. Further, sequencing of our two chromosome 12-linked bipolar-Darier families showed no evidence of rare variants at P2RX7 that could explain the linkage. Our data do not provide support for rs2230912 or the other polymorphisms studied within the P2RX7 locus, being involved in susceptibility to mood disorders.

Microarray expression technology: from start to finish.

The recent introduction of new microarray expression technologies and the further development of established platforms ensure that the researcher is presented with a range of options for performing an experiment. Whilst this has opened up the possibilities for future applications, such as exon-specific arrays, increased sample throughput and 'chromatin immunoprecipitation (ChIP) on chip' experiments, the initial decision processes and experiment planning are made more difficult. This review will give an overview of the various technologies that are available to perform a microarray expression experiment, from the initial planning stages through to the final data analysis. Both practical aspects and data analysis options will be considered. The relative advantages and disadvantages will be discussed with insights provided for future directions of the technology.

Localization of bipolar susceptibility locus by molecular genetic analysis of the chromosome 12q23-q24 region in two pedigrees with bipolar disorder and Darier's disease.

OBJECTIVE: The authors previously reported two families (pedigrees 324 and 5501) in which Darier's disease-a rare, autosomal dominant skin disease-and bipolar disorder cosegregate. In each of these families there is complete cosegregation of mood disorder with a segment of chromosome 12q23-q24, consistent with the existence of a highly penetrant dominant variant. Here molecular genetic analyses aimed at localizing and identifying the susceptibility gene in this region are reported. METHOD: In the two families, the authors undertook 1) linkage and haplotype studies using 45 highly polymorphic molecular genetic markers in order to delineate the region of interest and 2) direct analysis of genes within this region. RESULTS: Linkage and haplotype information from the most severely affected individuals defined a region of interest that spanned two neighboring regions of 19 megabases (Mb) (D12S362-D12S1646) and 7 Mb (D12S1718-D12S837). Information from all individuals refined the region of interest to 6.5 Mb (D12S127-D12S1646). Systematic study of the coding and flanking intronic regions of 25 known genes within this latter region failed to identify any highly penetrant autosomal dominant disease-conferring mutations in these pedigrees. CONCLUSIONS: This linkage and haplotype analysis, together with data from several other linkage studies, provides compelling evidence for the existence in the 12q23-q24 region of one or more genes involved in the pathogenesis of bipolar disorder. Further molecular genetic analysis of this region is required to identify the gene(s).

Mutational analysis of two positional candidate susceptibility genes for bipolar disorder on chromosome 12q23-q24: phenylalanine hydroxylase and human LIM-homeobox LHX5.

OBJECTIVES: In the search for chromosome 12 genes potentially involved in the pathogenesis of bipolar disorder we will screen Phenylalanine hydroxylase and human LIM-homeobox LHX5 genes for sequence variants, both of which have been suggested as candidate genes. The genes lie on chromosome 12q23-24, near the Darier's disease gene, ATP2A2. We have previously reported two families in which the pattern of segregation of illness is consistent with genetic linkage between this chromosomal region and a putative highly penetrant autosomal dominant major affective disorder locus (pedigree 324, maximum LOD=2.1; pedigree 5501, maximum LOD=3.6). METHODS: We screened the coding and intronic flanking regions of the phenylalanine hydroxylase and LHX5 genes for sequence variation by denaturing high-performance liquid chromatography in individuals from the pedigrees. RESULTS: In total, nine single nucleotide polymorphisms and one 6 base pair deletion were identified. CONCLUSION: Our studies allowed us to conclude that none of these variants act as a highly penetrant autosomal dominant susceptibility locus for mood disorder in our families.

Affymetrix GeneChip system: moving from research to the clinic.

Affymetrix Inc.'s GeneChip technology has become the industry standard in microarray-based research. Due to the high density of content per array that can be achieved today (an industry leading 6.5 million features) the GeneChips can be used for high-throughput mutation detection, single nucleotide polymorphism genotyping, expression profiling and detection of chromosomal aberrations. This in turn opens the way for clinical applications in genetics, cytogenetics, pharmacogenetics, oncology and pathogen recognition. Establishing standards is a central issue in improving data quality and, in combination with automatic, easy-to-interpret reports, will form the basis of the clinical applicability. Future pricing policies and the resolving of ethical considerations will also dictate the technology's full translation from research into clinical laboratories.

Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1alpha, HIF-2alpha, and other pathways.

Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.

Development and evaluation of real competitive PCR for high-throughput quantitative applications.

Real competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility, and high-throughput potential. We describe further development and evaluation of this methodology as a tool for measuring nucleic acid abundance within a cell. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thereby increasing throughput and ease of reaction setup while decreasing optimization time. In addition, we have developed a software package, TITAN, to automatically analyze the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. It is also demonstrated that the technique is capable of detecting submicroscopic chromosomal DNA deletions.