Dumbbell-Shaped DNA Analytes Amplified by Polymerase Chain Reaction for Robust Single-Nucleotide Polymorphism Genotyping by Affinity Capillary Electrophoresis.

Anal Chem. 2013 May 9;

Authors: Shibata H, Ogawa A, Kanayama N, Takarada T, Maeda M

Abstract
A sample preparation method was developed for single-nucleotide polymorphism (SNP) genotyping based on hybridization between a single-stranded DNA (ssDNA) analyte and an allele-specific oligonucleotide (ASO) probe. When the SNP site is located in the stable secondary structure, the folding of this analyte imposes kinetic penalties on the hybridization with the ASO probe. To address this issue, the sequence of the ssDNA analyte was converted from the original one so that the analyte exhibited a clear dumbbell-shaped structure composed of two stem-loop moieties and an unfolded probe-binding site. The as-prepared analyte was structurally favorable for hybridization with the ASO probe, irrespective of the original sequence and secondary structure of the analyte. The sequence conversion was easily achieved by polymerase chain reaction using forward and reverse primers having an additional sequence at the 5'-terminus. These ssDNA analytes were subjected to affinity capillary electrophoresis using a diblock copolymer probe composed of an ASO segment and a poly(ethylene glycol) segment. The 70-base dumbbell-shaped analytes with a single-base difference were clearly separated within 12 min, although the original ones exhibited almost no separation due to the undesired folding of the probe-binding site. This sample preparation method should open up a wide range of applications for the ASO probes in genetic analysis.

PMID: 23659631 [PubMed - as supplied by publisher]

Apolipoprotein m (APOM) levels and APOM rs805297 G/T polymorphism are associated with increased risk of rheumatoid arthritis.

Joint Bone Spine. 2013 May 6;

Authors: Huang Y, Liu Y, Jiang L, Sun R, Zhang H, Liu R, Xu N

Abstract
OBJECTIVE: To examine whether the apolipoprotein M (APOM) rs805297 G/T polymorphism is associated with risk of rheumatoid arthritis (RA) in a Chinese population. METHODS: We studied APOM rs805297 G/T gene polymorphism in 520 RA patients, and 520 controls in a Chinese population. Genotyping was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The blood plasma concentration of APOM was measured by an enzyme-linked immunosorbent assay in 84 RA patients and 84 controls. RESULTS: When the APOM rs805297 G/T GG homozygote genotype was used as the reference group, the TT or GT/TT genotype was associated with an increased risk for RA (TT vs. GG, adjusted odds ratio 1.76, 95% CI 1.11-2.77, P=0.016; GT + TT vs. GG, adjusted odds ratio 1.30, 95% CI 1.02-1.67, P=0.037). The average concentration of APOM in plasma was significantly higher in RA patients compared to controls. Stratification analysis found a significantly increased risk for RA associated with the APOM rs805297 TT genotype among male patients, C-reactive protein (CRP)-positive patients, anticitrullinated protein/peptide antibodies (ACPA) - positive patients, rheumatoid factor (RF) - positive patients, patients with higher levels of the erythrocyte sedimentation rate (ESR), patients with higher DAS28 score and patients with higher functional class compared to the APOM rs805297 GG genotype. CONCLUSION: These findings suggest that the functional single-nucleotide polymorphism APOM rs805297 G/T variant allele was associated with RA risk.

PMID: 23660425 [PubMed - as supplied by publisher]

Gene-based single nucleotide polymorphism discovery in bovine muscle using next-generation transcriptomic sequencing.

BMC Genomics. 2013 May 7;14(1):307

Authors: Djari A, Esquerré D, Weiss B, Martins F, Meersseman C, Boussaha M, Klopp C, Rocha D

Abstract
BACKGROUND: Genetic information based on molecular markers has increasingly being used in cattle breeding improvement programmes, as a mean to improve conventionally phenotypic selection. Advances in molecular genetics have led to the identification of several genetic markers associated with genes affecting economic traits. Until recently, the identification of the causative genetic variants involved in the phenotypes of interest has remained a difficult task. The advent of novel sequencing technologies now offers a new opportunity for the identification of such variants. Despite sequencing costs plummeting, sequencing whole-genomes or large targeted regions is still too expensive for most laboratories. A transcriptomic-based sequencing approach offers a cheaper alternative to identify a large number of polymorphisms and possibly to discover causative variants. In the present study, we performed a gene-based single nucleotide polymorphism (SNP) discovery analysis in bovine Longissimus thoraci, using RNA-Seq. To our knowledge, this represents the first study done in bovine muscle. RESULTS: Messenger RNAs from Longissimus thoraci from three Limousin bull calves were subjected to high-throughput sequencing. Approximately 36--46 million paired-end reads were obtained per library. A total of 19,752 transcripts were identified and 34,376 different SNPs were detected. Fifty-five percent of the SNPs were found in coding regions and ~22% resulted in an amino acid change. Applying a very stringent SNP quality threshold, we detected 8,407 different high-confidence SNPs, 18% of which are non synonymous coding SNPs. To analyse the accuracy of RNA-Seq technology for SNP detection, 48 SNPs were selected for validation by genotyping. No discrepancies were observed when using the highest SNP probability threshold. To test the usefulness of the identified SNPs, the 48 selected SNPs were assessed by genotyping 93 bovine samples, representing mostly the nine major breeds used in France. Principal component analysis indicates a clear separation between the nine populations. CONCLUSIONS: The RNA-Seq data and the collection of newly discovered coding SNPs improve the genomic resources available for cattle, especially for beef breeds. The large amount of variation present in genes expressed in Limousin Longissimus thoracis, especially the large number of non synonymous coding SNPs, may prove useful to study the mechanisms underlying the genetic variability of meat quality traits.

PMID: 23651547 [PubMed - as supplied by publisher]

Allele-specific programming of Npy and epigenetic effects of physical activity in a genetic model of depression.

Transl Psychiatry. 2013;3:e255

Authors: Melas PA, Lennartsson A, Vakifahmetoglu-Norberg H, Wei Y, Aberg E, Werme M, Rogdaki M, Mannervik M, Wegener G, Brené S, Mathé AA, Lavebratt C

Abstract
Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA-protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP's T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy's epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

PMID: 23652932 [PubMed - in process]