Transcriptional Profiling of Human Familial Longevity Indicates a Role for ASF1A and IL7R

The Leiden Longevity Study consists of families that express extended survival across generations, decreased morbidity in middle-age, and beneficial metabolic profiles. To identify which pathways drive this complex phenotype of familial longevity and healthy aging, we performed a genome-wide gene expression study within this cohort to screen for mRNAs whose expression changes with age and associates with longevity. We first compared gene expression profiles from whole blood samples between 50 nonagenarians and 50 middle-aged controls, resulting in identification of 2,953 probes that associated with age. Next, we determined which of these probes associated with longevity by comparing the offspring of the nonagenarians (50 subjects) and the middle-aged controls. The expression of 360 probes was found to change differentially with age in members of the long-lived families. In a RT-qPCR replication experiment utilizing 312 controls, 332 offspring and 79 nonagenarians, we confirmed a nonagenarian specific expression profile for 21 genes out of 25 tested. Since only some of the offspring will have inherited the beneficial longevity profile from their long-lived parents, the contrast between offspring and controls is expected to be weak. Despite this dilution of the longevity effects, reduced expression levels of two genes, ASF1A and IL7R, involved in maintenance of chromatin structure and the immune system, associated with familial longevity already in middle-age. The size of this association increased when controls were compared to a subfraction of the offspring that had the highest probability to age healthily and become long-lived according to beneficial metabolic parameters. In conclusion, an “aging-signature” formed of 21 genes was identified, of which reduced expression of ASF1A and IL7R marked familial longevity already in middle-age. This indicates that expression changes of genes involved in metabolism, epigenetic control and immune function occur as a function of age, and some of these, like ASF1A and IL7R, represent early features of familial longevity and healthy ageing

Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms

The hippocampal expression profiles of wild-type mice and mice transgenic for δC-doublecortin-like kinase were compared with Solexa/Illumina deep sequencing technology and five different microarray platforms. With Illumina's digital gene expression assay, we obtained ∼2.4 million sequence tags per sample, their abundance spanning four orders of magnitude. Results were highly reproducible, even across laboratories. With a dedicated Bayesian model, we found differential expression of 3179 transcripts with an estimated false-discovery rate of 8.5%. This is a much higher figure than found for microarrays. The overlap in differentially expressed transcripts found with deep sequencing and microarrays was most significant for Affymetrix. The changes in expression observed by deep sequencing were larger than observed by microarrays or quantitative PCR. Relevant processes such as calmodulin-dependent protein kinase activity and vesicle transport along microtubules were found affected by deep sequencing but not by microarrays. While undetectable by microarrays, antisense transcription was found for 51% of all genes and alternative polyadenylation for 47%. We conclude that deep sequencing provides a major advance in robustness, comparability and richness of expression profiling data and is expected to boost collaborative, comparative and integrative genomics studies

Human Mutation REVIEW High-Resolution Melting Analysis (HRMA)-More Than Just Sequence Variant Screening For the Focus Section on HRMA Technology

ABSTRACT: Transition of the double-stranded DNA molecule to its two single strands, DNA denaturation or melting, has been used for many years to study DNA structure and composition. Recent technological advances have improved the potential of this technology, especially to detect variants in the DNA sequence. Sensitivity and specificity were increased significantly by the development of so-called saturating DNA dyes and by improvements in the instrumentation to measure the melting behavior (improved temperature precision combined with increased measurements per time unit and drop in temperature). Melt analysis using these new instruments has been designated high-resolution melting curve analysis (HRM or HRMA). Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity, HRMA is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we will briefly discuss the latest developments in HRMA and review in particular other applications that have thus far received less attention, including presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants and mosaicism), an alternative to gel-electrophoresis and clone characterization. Together, these diverse applications make HRMA a multipurpose technology and a standard tool that should be present in any laboratory studying nucleic acids

Loss of maternal EED results in postnatal overgrowth

Background: Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes. Results: In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed. Conclusions: This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofReviewedFacult

The complete genome sequence of the murine pathobiont Helicobacter typhlonius

BackgroundImmuno-compromised mice infected with Helicobacter typhlonius are used to model microbially inducted inflammatory bowel disease (IBD). The specific mechanism through which H. typhlonius induces and promotes IBD is not fully understood. Access to the genome sequence is essential to examine emergent properties of this organism, such as its pathogenicity. To this end, we present the complete genome sequence of H. typhlonius MIT 97-6810, obtained through single-molecule real-time sequencing.ResultsThe genome was assembled into a single circularized contig measuring 1.92 Mbp with an average GC content of 38.8%. In total 2,117 protein-encoding genes and 43 RNA genes were identified. Numerous pathogenic features were found, including a putative pathogenicity island containing components of type IV secretion system, virulence-associated proteins and cag pathogenicity island protein. We compared the genome of H. typhlonius to those of the murine pathobiont Helicobacter hepaticus and human pathobiont Helicobacter pylori. H. typhlonius resembles H. hepaticus most with 1,594 (75.3%) of its genes being orthologous to genes in H. hepaticus. Determination of the global methylation state revealed eight distinct recognition motifs for adenine and cytosine methylation. H. typhlonius shares four of its recognition motifs with H. pylori. ConclusionsThe complete genome sequence of H. typhlonius MIT 97-6810 enabled us to identify many pathogenic features suggesting that H. typhlonius can act as a pathogen. Follow-up studies are necessary to evaluate the true nature of its pathogenic capabilities. We found many methylated sites and a plethora of restriction-modification systems. The genome, together with the methylome, will provide an essential resource for future studies investigating gene regulation, host interaction and pathogenicity of H. typhlonius. In turn, this work can contribute to unraveling the role of Helicobacter in enteric disease

Keratosis Follicularis Spinulosa Decalvans Is Caused by Mutations in MBTPS2

Keratosis Follicularis Spinulosa Decalvans (KFSD) is a rare genetic disorder characterized by development of hyperkeratotic follicular papules on the scalp followed by progressive alopecia of the scalp, eyelashes, and eyebrows. Associated eye findings include photophobia in childhood and corneal dystrophy. Due to the genetic and clinical heterogeneity of similar disorders, a definitive diagnosis of KFSD is often challenging. Toward identification of the causative gene we reanalyzed a large Dutch KFSD family. SNP arrays (1 M) redefined the locus to a 2.9-Mb region at Xp22.12-Xp22.11. Screening of all 14 genes in the candidate region identified MBTPS2 as the candidate gene carrying a c.1523A>G (p.Asn508Ser) missense mutation. The variant was also identified in two unrelated X-linked KFSD families and cosegregated with KFSD in all families. In symptomatic female carriers, skewed X-inactivation of the normal allele matched with increased severity of symptoms. MBTPS2 is required for cleavage of sterol regulatory element-binding proteins (SREBPs). In vitro functional expression studies of the c. 1523A>G mutation showed that sterol responsiveness was reduced by half. Other missense mutations in MBTPS2 have recently been identified in patients with IFAP syndrome. We postulate that both phenotypes are in the spectrum of one genetic disorder with a partially overlapping phenotype. Hum Mutat 31:1125-1133, 2010. (C) 2010 Wiley-Liss, Inc

第53回物性若手夏の学校(2008年度)

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