To fully understand the allelic variation that underlies common diseases, complete genome sequencing for many individuals with and without disease is required. This is still not technically feasible. However, recently it has become possible to carry out partial surveys of the genome by genotyping large numbers of common SNPs in genome-wide association studies. Here, we outline the main factors - including models of the allelic architecture of common diseases, sample size, map density and sample-collection biases - that need to be taken into account in order to optimize the cost efficiency of identifying genuine disease-susceptibility loci.

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Genome-wide association studies: theoretical and practical concerns

Alternative splicing modulates the expression of many oncogene and tumor-suppressor isoforms. We have tested whether some alternative splicing factors are involved in cancer. We found that the splicing factor SF2/ASF is upregulated in various human tumors, in part due to amplification of its gene, SFRS1. Moreover, slight overexpression of SF2/ASF is sufficient to transform immortal rodent fibroblasts, which form sarcomas in nude mice. We further show that SF2/ASF controls alternative splicing of the tumor suppressor BIN1 and the kinases MNK2 and S6K1. The resulting BIN1 isoforms lack tumor-suppressor activity; an isoform of MNK2 promotes MAP kinase–independent eIF4E phosphorylation; and an unusual oncogenic isoform of S6K1 recapitulates the transforming activity of SF2/ASF. Knockdown of either SF2/ASF or isoform-2 of S6K1 is sufficient to reverse transformation caused by the overexpression of SF2/ASF in vitro and in vivo. Thus, SF2/ASF can act as an oncoprotein and is a potential target for cancer therapy.

The gene encoding the splicing factor SF2/ASF is a proto-oncogene

The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration.

genetic factors underlying common disease are largely unknown. Discovery of disease-causing genes will transform our knowledge of the genetic contribution to human disease, lead to new genetic screens, and underpin research into new cures and improved lifestyles. The se-quencing of the human genome has catalyzed efforts to search for disease genes by the strategy of associating sequence variants with measurable phenotypes. In particular , the Human Genome Project and follow-on efforts to characterize genetic variation have resulted in the discovery of millions of single-nucleotide polymorphisms (SNPs) (Patil et al. 2001; Sachidanandam et al. 2001; Reich et al. 2003). This represents a significant fraction of common genetic variation in the human genome and creates an unprecedented opportunity to associate genes with phenotypes via large-scale SNP genotyping studies. To make use of this information, efficient and accurate SNP genotyping technologies are needed. However, most methods were designed to analyze only one or a few SNPs per assay, and are costly to scale up (Kwok 2001; Syvanen 2001). To help enable genome-wide association studies and other large-scale genetic analysis projects, we have developed an integrated SNP genotyping system that combines a highly multiplexed assay with an accurate readout technology based on random arrays of DNA-coated beads (Michael et al. 1998; Oliphant et al. 2002; Gunderson et al. 2004). Our aim was to reduce costs and increase productivity by ~2 orders of magnitude. We chose a multiplexed approach because it is more easily scalable and is intrinsically cost-efficient (Wang et al. 1998). Although existing multiplexed approaches lacked the combination of accuracy, robustness, scalability, and cost-effectiveness needed for truly large-scale endeavors, we hypothesized that some of these limitations could be overcome by designing an assay specifically for multiplexing. To increase throughput and decrease costs by ~2 orders of magnitude, it was necessary to eliminate bottlenecks throughout the genotyping process. It was also desirable to minimize sources of variability and human error in order to ensure data quality and reproducibility. We therefore took a systems-level view to technology design, development , and integration. Although the focus of this paper is on a novel, highly multiplexed genotyping assay, the GoldenGate ™ assay, four other key technologies that were developed in parallel, as part of the complete BeadLab system (Oliphant et al. 2002), are briefly described below. BEADARRAY™ PLATFORM We developed an array technology based on random assembly of beads in micro-wells located at the end of an …

Highly parallel SNP genotyping.

A new field of genetic analysis of global gene expression has emerged in recent years, driven by the realization that traditional techniques of linkage and association analysis can be applied to thousands of transcript levels measured by microarrays. Genetic dissection of transcript abundance has shed light on the architecture of quantitative traits, provided a new approach for connecting DNA sequence variation with phenotypic variation, and improved our understanding of transcriptional regulation and regulatory variation.

Genetics of global gene expression.

Variations in gene sequence and expression underlie much of human variability. Despite the known biological roles of differential allelic gene expression resulting from X-chromosome inactivation and genomic imprinting, a large-scale analysis of allelic gene expression in human is lacking. We examined allele-specific gene expression of 1063 transcribed single-nucleotide polymorphisms (SNPs) by using Affymetrix HuSNP oligo arrays. Among the 602 genes that were heterozygous and expressed in kidney or liver tissues from seven individuals, 326 (54%) showed preferential expression of one allele in at least one individual, and 170 of those showed greater than fourfold difference between the two alleles. The allelic variation has been confirmed by real-time quantitative PCR experiments. Some of these 170 genes are known to be imprinted, such as SNRPN, IPW, HTR2A, and PEG3. Most of the differentially expressed genes are not in known imprinting domains but instead are distributed throughout the genome. Our studies demonstrate that variation of gene expression between alleles is common, and this variation may contribute to human variability.

Allelic Variation in Gene Expression Is Common in the Human Genome

A genome-wide computational screen was performed to identify tumor-associated alternative RNA splicing isoforms. A BLAST algorithm was used to compare 11,014 genes from RefSeq with 3,471,822 human expressed sequence tag sequences. The screen identified 26,258 alternative splicing isoforms of which 845 were significantly associated with human cancer, and 54 were specifically associated with liver cancer. Furthermore, canonical GT-AG splice junctions were used significantly less frequently in the alternative splicing isoforms in tumors. Reverse transcription-PCR experiments confirmed association of the alternative splicing isoforms with tumors. These results suggest that alternative splicing may have potential as a diagnostic marker for cancer.

https://cancerres.aacrjournals.org/content/canres/63/3/655.full.pdf

Computational Analysis and Experimental Validation of Tumor-associated Alternative RNA Splicing in Human Cancer

We mapped a tumor suppressor gene locus to an 800-kb interval on human chromosome 13q12.11 for esophageal squamous cell carcinoma (ESCC). Two genes, ML-1 and RNF6, are located within this 800-kb interval. We analyzed both genes for the presence of mutations in 24 ESCC primary tumors and 16 tumor cell lines by directly sequencing the PCR products that were amplified from each exon. No mutation was detected in ML-1. In contrast, three somatic mutations in the RNF6 gene were detected in the ESCC primary tumors, and one mutation was also found in a tumor cell line. Identification of multiple somatic mutations in RNF6 suggests that RNF6 is a potential tumor suppressor gene involved in the pathogenesis of ESCC.

H. Shuen Lo

Papers

Allelic Variation in Gene Expression Is Common in the Human Genome

Computational Analysis and Experimental Validation of Tumor-associated Alternative RNA Splicing in Human Cancer

Identification of somatic mutations of the RNF6 gene in human esophageal squamous cell carcinoma.