Genome-wide prioritization of disease genes and identification of disease-disease associations from an integrated human functional linkage network

Background

Recent studies have shown that the regulatory effect of microRNAs can be investigated by examining expression changes of their target genes. Given this, it is useful to define an overall metric of regulatory effect for a specific microRNA and see how this changes across different conditions.

Results

Here, we define a regulatory effect score (RE-score) to measure the inhibitory effect of a microRNA in a sample, essentially the average difference in expression of its targets versus non-targets. Then we compare the RE-scores of various microRNAs between two breast cancer subtypes: estrogen receptor positive (ER⁺) and negative (ER^-). We applied this approach to five microarray breast cancer datasets and found that the expression of target genes of most microRNAs was more repressed in ER^- than ER⁺; that is, microRNAs appear to have higher RE-scores in ER^- breast cancer. These results are robust to the microRNA target prediction method. To interpret these findings, we analyzed the level of microRNA expression in previous studies and found that higher microRNA expression was not always accompanied by higher inhibitory effects. However, several key microRNA processing genes, especially Ago2 and Dicer, were differentially expressed between ER^- and ER⁺ breast cancer, which may explain the different regulatory effects of microRNAs in these two breast cancer subtypes.

Conclusions

The RE-score is a promising indicator to measure microRNAs' inhibitory effects. Most microRNAs exhibit higher RE-scores in ER^- than in ER⁺ samples, suggesting that they have stronger inhibitory effects in ER^- breast cancers.     

Genome-wide detection of tissue-specific alternative splicing in the human transcriptome

We have developed an automated method for discovering tissue-specific regulation of alternative splicing through a genome-wide analysis of expressed sequence tags (ESTs). Using this approach, we have identified 667 tissue-specific alternative splice forms of human genes. We validated our muscle-specific and brain-specific splice forms for known genes. A high fraction (8/10) were reported to have a matching tissue specificity by independent studies in the published literature. The number of tissue-specific alternative splice forms is highest in brain, while eye-retina, muscle, skin, testis and lymph have the greatest enrichment of tissue-specific splicing. Overall, 10-30% of human alternatively spliced genes in our data show evidence of tissue-specific splice forms. Seventy-eight percent of our tissue-specific alternative splices appear to be novel discoveries. We present bioinformatics analysis of several tissue-specific splice forms, including automated protein isoform sequence and domain prediction, showing how our data can provide valuable insights into gene function in different tissues. For example, we have discovered a novel kidney-specific alternative splice form of the WNK1 gene, which appears to specifically disrupt its N-terminal kinase domain and may play a role in PHAII hypertension. Our database greatly expands knowledge of tissue-specific alternative splicing and provides a comprehensive dataset for investigating its functional roles and regulation in different human tissues.     

Analysis of a Charcot-Marie-Tooth disease mutation reveals an essential internal ribosome entry site element in the connexin-32 gene

A mutation located in the 5'-untranslated region (5'-UTR) of the nerve-specific connexin-32 mRNA, previously found in a family with Charcot-Marie-Tooth disease (CMTX), was analyzed for its effect on the expression of a reporter gene (luciferase) in transgenic mice and in transfected cells. Whereas both mutant and wild-type genes appeared to be transcribed and spliced efficiently, no luciferase was detected from the mutant in either system, suggesting that the mutation affects translation of the mRNA. When the 5'-UTR of nerve-specific connexin-32 mRNA was inserted between the two genes of a bicistronic vector and transfected into various cell lines, expression of the second gene was significantly increased. Because the mutant did not facilitate translation of the second gene in the bicistronic mRNA system, this result suggested that the CMTX mutation abolished function of an internal ribosome entry site (IRES) in the 5'-UTR of the wild-type connexin-32 mRNA. The CMTX phenotype of the mutant 5'-UTR further suggested that the wild-type IRES was essential for the translation of the connexin-32 mRNA in nerve cells. In addition, other sequence elements of the connexin-32 IRES were characterized by mutation analysis. A mutation in either of the first two elements investigated showed loss of IRES function, whereas mutation of a third element showed gain of function.     

Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos

The development of efficient non-viral methodologies for genome-wide insertional mutagenesis and gene tagging in mammalian cells is highly desirable for functional genomic analysis. Here we describe transposon mediated mutagenesis (TRAMM), using naked DNA vectors based on the Drosophila hydei transposable element Minos. By simple transfections of plasmid Minos vectors in HeLa cells, we have achieved high frequency generation of cell lines, each containing one or more stable chromosomal integrations. The Minos-derived vectors insert in different locations in the mammalian genome. Genome-wide mutagenesis in HeLa cells was demonstrated by using a Minos transposon containing a lacZ–neo gene-trap fusion to generate a HeLa cell library of at least 10⁵ transposon insertions in active genes. Multiple gene traps for six out of 12 active genes were detected in this library. Possible applications of Minos-based TRAMM in functional genomics are discussed.     

Genome-wide linkage analysis of an autosomal recessive hypotrichosis identifies a novel P2RY5 mutation

While there have been significant advances in understanding the genetic etiology of human hair loss over the previous decade, there remain a number of hereditary disorders for which a causative gene has yet to be identified. We studied a large, consanguineous Brazilian family that presented with woolly hair at birth that progressed to severe hypotrichosis by the age of 5, in which 6 of the 14 offspring were affected. After exclusion of known candidate genes, a genome-wide scan was performed to identify the disease locus. Autozygosity mapping revealed a highly significant region of extended homozygosity (lod score of 10.41) that contained a haplotype with a linkage lod score of 3.28. Results of these two methods defined a 9-Mb region on chromosome 13q14.11-q14.2. The interval contains the P2RY5 gene, in which we recently identified pathogenic mutations in several families of Pakistani origin affected with autosomal recessive woolly and sparse hair. After the exclusion of several other candidate genes, we sequenced the P2RY5 gene and identified a homozygous mutation (C278Y) in all affected individuals in this family. Our findings show that mutations in P2RY5 display variable expressivity, underlying both hypotrichosis and woolly hair, and underscore the essential role of P2RY5 in the tissue integrity and maintenance of the hair follicle.     

Interaction of TFIID in the minor groove of the TATA element.

TFIID binding in the minor groove of DNA at the TATA element was demonstrated by methylation interference and hydroxyl radical footprinting assays, and by binding studies with thymine analog substituted oligonucleotides. These results provide an explanation for TFIID-dependent DNA bending at the TATA element. TFIID binding shows phosphate contacts with the same residues that were found to be essential for TFIID interactions by methylation and thymine-specific modification interference assays. Based on previous studies implicating residues conserved between the direct repeats in DNA binding, as well as models of prokaryotic DNA binding proteins, these results also suggest a model in which the direct repeats of TFIID form two basic antiparallel beta ribbon arms that could contact DNA through the minor groove.     

Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.     

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.     

Genome-wide detection of allelic gene expression in hepatocellular carcinoma cells using a human exome SNP chip

Allelic variations in gene expression influence many biological responses and cause phenotypic variations in humans. In this study, Illumina Human Exome BeadChips containing more than 240,000 single nucleotide polymorphisms (SNPs) were used to identify changes in allelic gene expression in hepatocellular carcinoma cells following lipopolysaccharide (LPS) stimulation. We found 17 monoallelically expressed genes, 58 allelic imbalanced genes, and 7 genes showing allele substitution. In addition, we also detected 33 differentially expressed genes following LPS treatment in vitro using these human exome SNP chips. However, alterations in allelic gene expression following LPS treatment were detected in only three genes (MLXIPL, TNC, and MX2), which were observed in one cell line sample only, indicating that changes in allelic gene expression following LPS stimulation of liver cells are rare events. Among a total of 75 genes showing allelic expression in hepatocellular carcinoma cells, either monoallelic or imbalanced, 43 genes (57.33%) had expression quantitative trait loci (eQTL) data, indicating that high-density exome SNP chips are useful and reliable for studying allelic gene expression. Furthermore, most genes showing allelic expression were regulated by cis-acting mechanisms and were also significantly associated with several human diseases. Overall, our study provides a better understanding of allele-specific gene expression in hepatocellular carcinoma cells with and without LPS stimulation and potential clues for the cause of human disease due to alterations in allelic gene expression.     

Genome-wide detection of genetic loci triggering uneven descending of gametes from a natural hybrid pine

Marker transmission ratio distortion (TRD) revealed in genetic mapping studies on distant crosses can be used to infer the genetic basis relating to reproductive barriers between species. Unlike measuring the degree of TRD by the overall number of segregation distorted markers in the affected genome regions, mapping the segregation distorting loci (SDL) provides reliable statistic parameters that help to confine the target genomic regions for further characterization at molecular level. Using the linkage map constructed for a natural hybrid of Pinus hwangshanensis and Pinus massoniana, we perform SDL analyses and align the established map to the loblolly pine consensus map. Altogether, six SDLs with relatively strong LOD supports are detected on four linkage groups of the established map. Since gametes inheriting different alternate chromatid blocks from the SDL affecting genome regions have uneven chance to descend to the offspring, the corresponding genome regions are supposed to play more significant roles in rendering the reproductive isolations between P. hwangshanensis and P. massoniana. This paper presents a case study on a crucial step for uncovering the hidden genetic factors that trigger the uneven descending of gametes in a natural hybrid pine.     

Genome-wide SNP association-based localization of a dwarfism gene in Friesian dwarf horses

The recent completion of the horse genome and commercial availability of an equine SNP genotyping array has facilitated the mapping of disease genes. We report putative localization of the gene responsible for dwarfism, a trait in Friesian horses that is thought to have a recessive mode of inheritance, to a 2-MB region of chromosome 14 using just 10 affected animals and 10 controls. We successfully genotyped 34,429 SNPs that were tested for association with dwarfism using chi-square tests. The most significant SNP in our study, BIEC2-239376 (P(2df)=4.54 × 10(-5), P(rec)=7.74 × 10(-6)), is located close to a gene implicated in human dwarfism. Fine-mapping and resequencing analyses did not aid in further localization of the causative variant, and replication of our findings in independent sample sets will be necessary to confirm these results.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

Genome-wide identification of genes likely to be involved in human genetic disease

Sequence analysis of the group of proteins known to be associated with hereditary diseases allows the detection of key distinctive features shared within this group. The disease proteins are characterized by greater length of their amino acid sequence, a broader phylogenetic extent, and specific conservation and paralogy profiles compared with all human proteins. This unique property pattern provides insights into the global nature of hereditary diseases and moreover can be used to predict novel disease genes. We have developed a computational method that allows the detection of genes likely to be involved in hereditary disease in the human genome. The probability score assignments for the human genome are accessible at http://maine.ebi. ac.uk:8000/services/dgp.