Minh Nguyen

A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig

Most traits and disorders have a multifactorial background indicating that they are controlled by environmental factors as well as an unknown number of quantitative trait loci (QTLs). The identification of mutations underlying QTLs is a challenge because each locus explains only a fraction of the phenotypic variation. A paternally expressed QTL affecting muscle growth, fat deposition and size of the heart in pigs maps to the IGF2 (insulin-like growth factor 2) region. Here we show that this QTL is caused by a nucleotide substitution in intron 3 of IGF2. The mutation occurs in an evolutionarily conserved CpG island that is hypomethylated in skeletal muscle. The mutation abrogates in vitro interaction with a nuclear factor, probably a repressor, and pigs inheriting the mutation from their sire have a threefold increase in IGF2 messenger RNA expression in postnatal muscle. Our study establishes a causal relationship between a single-base-pair substitution in a non-coding region and a QTL effect. The result supports the long-held view that regulatory mutations are important for controlling phenotypic variation.

Comparative sequence analysis of the INS-IGF2-H19 gene cluster in pigs.

IGF2 is the major candidate gene for a paternally expressed Quantitative Trait Locus (QTL) in the pig primarily affecting muscle development. Here we report two sequence contigs together comprising almost 90 kb containing the INS-IGF2 and H19 genes. A comparative sequence analysis of the pig, human, and mouse genomic sequences was conducted to identify the exon/intron organization, all promoters, and other evolutionarily conserved elements. RT-PCR analysis showed that IGF2 transcripts originated from four different promoters and included various combinations of seven untranslated exons together with three coding exons, in agreement with previous findings in other mammals. The observed sequence similarity in intronic and intragenic regions among the three species is remarkable and is most likely explained by the complicated regulation of imprinting and expression of these genes. The general trend was, as expected, a higher sequence similarity between human and pig than between these species and the mouse, but a few exceptions to this rule were noted. This genomic region exhibits several striking features, including a very high GC content, many CpG islands, and a low amount of interspersed repeats. The high GC and CpG content were more pronounced in the pig than in the two other species. The results will facilitate the further characterization of this important QTL in the pig.

Differentiation Among Potyviruses Infecting Sweet Potato Based on Genus- and Virus-Specific Reverse Transcription Polymerase Chain Reaction

Knowledge of virus diseases affecting sweet potato has been complicated due to the frequent occurrence of mixed infections and difficulties in isolating and purifying sweet potato viruses. A combined assay of reverse transcription and polymerase chain reaction (PCR) utilizing degenerate genus-specific primers POT1 and POT2 was applied to 18 sweet potato clones from China. The primers were designed to amplify the variable 5 terminal region of the potyvirus coat protein gene. Molecular analysis of the amplified fragments identified the Chinese strains of sweet potato feathery mottle virus (SPFMV-CH), sweet potato latent virus (SPLV-CH), and sweet potato virus G (SPVG-CH). Among the detected potyviruses, a distantly related strain of SPFMV-CH, tentatively named SPFMV-CH2, was identified in sweet potatoes from China. On the basis of sequence identity, SPFMV-CH2 was closely related to the common (-C) strain of that virus. Identification of a closely related strain of SPVG-CH in one sweet potato clone from China further illustrated the usefulness of broad-spectrum PCR for detecting uncharacterized viruses. The acquisition of sequence information permitted the design of virus-specific primers for detecting and differentiating SPFMV, SPLV, and SPVG.

Chromosomal patterns of gene expression from microarray data: methodology, validation and clinical relevance in gliomas

BackgroundExpression microarrays represent a powerful technique for the simultaneous investigation of thousands of genes. The evidence that genes are not randomly distributed in the genome and that their coordinated expression depends on their position on chromosomes has highlighted the need for mathematical approaches to exploit this dependency for the analysis of expression data-sets.ResultsWe have devised a novel mathematical technique (CHROMOWAVE) based on the Haar wavelet transform and applied it to a dataset obtained with the Affymetrix® HG-U133_Plus_2 array in 27 gliomas. CHROMOWAVE generated multi-chromosomal pattern featuring low expression in chromosomes 1p, 4, 9q, 13, 18, and 19q. This pattern was not only statistically robust but also clinically relevant as it was predictive of favourable outcome. This finding was replicated on a data-set independently acquired by another laboratory. FISH analysis indicated that monosomy 1p and 19q was a frequent feature of tumours displaying the CHROMOWAVE pattern but that allelic loss on chromosomes 4, 9q, 13 and 18 was much less common.ConclusionThe ability to detect expression changes of spatially related genes and to map their position on chromosomes makes CHROMOWAVE a valuable screening method for the identification and display of regional gene expression changes of clinical relevance. In this study, FISH data showed that monosomy was frequently associated with diffuse low gene expression on chromosome 1p and 19q but not on chromosomes 4, 9q, 13 and 18. Comparative genomic hybridisation, allelic polymorphism analysis and methylation studies are in progress in order to identify the various mechanisms involved in this multi-chromosomal expression pattern.

I-Kappa-B Kinase-epsilon activates nuclear factor-kappa B and STAT5B and supports glioblastoma growth but amlexanox shows little therapeutic potential in these tumors

Aim: This study aims to analyze the role of I-kappa-B kinase (IKK)-epsilon in glioblastoma (GBM). Methods: A series of in vitro, in vivo, microarray, and immunohistochemical assessments were performed to evaluate the biological effects of IKK-epsilon on cell signaling, radiation sensitivity, and patient survival in GBM condition. Results: IKK-epsilon was strongly expressed in 75% of 195 primary GBM samples but did not correlate with patient survival. No correlation was established between the copy number, messenger RNA (mRNA) expression, and protein expression in 38 fresh tumor samples, nor between IKK-epsilon mRNA expression and survival in 543 GBM of the TCGA repository. In vitro, IKK-epsilon contributed to the growth and migration of glioma cells, independent of their EGFRVIII status. IKK-epsilon activated nuclear factor (NF)-κB and STAT5B in vitro, confirming the observed correlation surgical GBM samples. IKK-epsilon silencing did not alter the sensitivity of GBM cells to ionizing radiation. Amlexanox, inhibitor of IKK-epsilon and TBK1, poorly (IC50 > 100 μM) decreased cell growth and increased NF-κB activity in GBM cells, in vitro, notably due to TBK1 inhibition. In vivo, oral amlexanox failed to inhibit the growth of intracerebral U87 GBM xenografts in nude mice. Conclusion: The results confirm a moderate pro-oncogenic role of IKK-epsilon in GBM, but question the potential of amlexanox as a therapeutic drug.