Nonfluorescent Denaturing HPLC–Based Primer-Extension Method for Allele-Specific Expression: Application to Analysis of Mismatch Repair Genes

Aceto, Gitana María; Lellis, Laura De; Catalano, Teresa; Veschi, Serena; Radice, Paolo; Iorio, Angelo Di; Mariani-Costantini, Renato; Cama, Alessandro; Curia, Maria Cristina

doi:10.1373/clinchem.2009.126300

Cited by 4 publications

(12 citation statements)

References 23 publications

(27 reference statements)

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“…The mean ratio of peak heights corresponding to the two alleles was 0.88 (SD 0.04) and the overall coefficient of variation (CV) was 4.99%. Peak ratios deviating from the expected 1:1 using templates with equimolar allelic representation, such as gDNA, are commonly observed and potential explanations have been previously discussed (26). The mean ratio obtained with gDNA was employed to normalize the data generated in primer extension experiments conducted using cDNA templates.…”

Section: Methodsmentioning

confidence: 88%

“…DNA and RNA were extracted as previously described (26). Synthesis of cDNA was performed using DNase I-treated RNA, random hexamers and the Superscript-II Reverse Transcriptase kit according to manufacturer specifications (Invitrogen, Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…ASE analyses were carried out using a previously described method based on denaturing high performance liquid chromatography (DHPLC) (26). Primer sequences are provided in Supplementary Table 1.…”

Section: Methodsmentioning

confidence: 99%

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Increased Variance in Germline Allele-Specific Expression of APC Associates With Colorectal Cancer

et al. 2012

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Background and Aims Germline variation in allele-specific expression (ASE) is associated with highly penetrant familial cancers, but its role in common sporadic cancers is unclear. ASE of the adenomatous polyposis coli (APC) gene plays a role in familial adenomatous polyposis coli. We hypothesized that moderate ASE variation in APC contributes to common forms of colorectal cancer (CRC). Methods Denaturing high performance liquid chromatography (DHPLC) was employed for germline APC ASE analysis in CRC cases (n=53) and controls (n=68). Means, medians, and variances of ASE were compared. Mutation analysis and SNP genotyping were performed. Results ASE distributions differed significantly between groups; cases had a significantly larger variance than controls (p = 0.0004). Importantly, CRC risk increased proportionally with the degree of deviation from the mean. Individuals with ASE deviating more than 1 SD from the mean had an odds ratio (OR) of 3.97 (1.71, 9.24 95% CI; p = 0.001); those deviating more than 1.645 SDs had an OR of 13.46 (1.76, 609.40 95% CI; p = 0.005). In support of these findings, sequence analysis revealed that a patient with marked ASE, who was negative for CRC family history, carried a nonsense APC mutation (p.Arg216X). Furthermore, APC genotyping showed that multiple SNPs were associated with ASE values and/or ASE variance in cases, but not in controls. Thus, cis variants may explain at least some of the ASE results. Conclusion Our results indicate that imbalanced germline ASE of APC is more frequent in CRC patients than controls, and represents an indicator of risk for common forms of CRC.

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Section: Methodsmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

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Increased Variance in Germline Allele-Specific Expression of APC Associates With Colorectal Cancer

et al. 2012

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“…ASE analyses were performed by dHPLC-based single nucleotide primer extension (SNuPE) [ 26 ]. This technique allows to measure the relative allele expression comparing the heights of the peaks corresponding to the two alleles obtained by PCR-amplified gDNA and cDNA templates.…”

Section: Methodsmentioning

confidence: 99%

Correlation between mutations and mRNA expression of APC and MUTYH genes: new insight into hereditary colorectal polyposis predisposition

Aceto

Fantini

Iure

et al. 2015

J Exp Clin Cancer Res

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BackgroundTranscript dosage imbalance may influence the transcriptome. To gain insight into the role of altered gene expression in hereditary colorectal polyposis predisposition, in the present study we analyzed absolute and allele-specific expression (ASE) of adenomatous polyposis coli (APC) and mutY Homolog (MUTYH) genes.MethodsWe analyzed DNA and RNA extracted from peripheral blood mononuclear cells (PBMC) of 49 familial polyposis patients and 42 healthy blood donors selected according similar gender and age. Patients were studied for germline alterations in both genes using dHPLC, MLPA and automated sequencing. APC and MUTYH mRNA expression levels were investigated by quantitative Real-Time PCR (qRT-PCR) analysis using TaqMan assay and by ASE assays using dHPLC-based primer extension.ResultsTwenty out of 49 patients showed germline mutations: 14 in APC gene and six in MUTYH gene. Twenty-nine patients did not show mutations in both genes. Results from qRT-PCR indicated that gene expression of both APC and MUTYH was reduced in patients analyzed. In particular, a significant reduction in APC expression was observed in patients without APC germline mutation vs control group (P < 0.05) while APC expression in the mutation carrier patients, although lower compared to control individuals, did not show statistical significance. On the other hand a significant reduced MUTYH expression was detected in patients with MUTYH mutations vs control group (P < 0.05). Altered ASE of APC was detected in four out of eight APC mutation carriers. In particular one case showed a complete loss of one allele. Among APC mutation negative cases, 4 out of 13 showed a moderate ASE. ASE of MUTYH did not show any altered expression in the cases analyzed. Spearman’s Rho Test analysis showed a positive and significant correlation between APC and MUTYH genes both in cases and in controls (P = 0.020 and P < 0.001).ConclusionsAPC and MUTYH showed a reduced germline expression, not always corresponding to gene mutation. Expression of APC is decreased in mutation negative cases and this appears to be a promising indicator of FAP predisposition, while for MUTYH gene, mutation is associated to reduced mRNA expression. This study could improve the predictive genetic diagnosis of at-risk individuals belonging to families with reduced mRNA expression regardless of presence of mutation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0244-4) contains supplementary material, which is available to authorized users.

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“…Pair-end reads passing the filtering were aligned to the Ovis aries v4.0 reference genome using BWA software (v0.7.13, bio-bwa.sourceforge.net) [31,32].…”

Section: Snp Genotyping and Quality Controlmentioning

confidence: 99%

Allele-specific expression reveals the phenotypic differences between thin- and fat-tailed sheep

Shao¹,

He²,

Pan³

et al. 2020

Preprint

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Background: The thin-tailed sheep breeds from Europe and the fat-tailed sheep breeds from China exhibit distinct phenotypic differences in fat deposition and meat production traits. However, the molecular mechanisms underlying gene expression related to these phenotypic differences are not well understood. Allele-specific expression (ASE) refers to the significant imbalance of expression levels of two parental alleles. Characterization of such events in F1 hybrid offspring generated from these two groups of sheep breeds can minimize the external factors influencing gene expression and reveal the variants with a cis -regulatory effect on gene expression. The aim of the present study was to investigate the genetic factors that influence different fat-deposition and meat production traits between thin- and fat-tailed sheep.Results: Fifteen F1 hybrids were generated from crosses between Texel and Kazakh sheep as the representative phenotypes of thin- and fat-tailed breeds, respectively. Totally, 33 whole genomes from F1 individuals and their parents were sequenced with an average depth of ~17.21× coverage per sample. ASE analysis results from 70 RNA-seq samples of adipose and skeleton muscle tissues showed 128 ASE candidate genes were related to the function of fat deposition and meat production traits. A genome-wide scan of selective sweeps was also conducted between these two groups of sheep breeds in an effort to identify genomic regions related to fat deposition and meat production, respectively. We detected signatures of selection in ASE genes associated with fat deposition (e.g., PDGFD ) and meat production traits (e.g., LRCC2 ). Further analysis suggested that PDGFD and LRCC2 genes were speculated to be causative genes for fat deposition and meat production traits in sheep, respectively. Furthermore, AMPK signaling pathway was significantly enriched in ASE genes related to fatty acid biosynthesis in both adipose and skeleton muscle tissues, while PPAR signaling pathway was significantly enriched in ASE genes related to lipid metabolism in adipose tissue. Conclusions: Our finding illustrates that the expression of identified ASE genes could potentially lead to the differences in traits of fat deposition and meat production between thin- and fat-tailed sheep. Keywords: allele-specific expression, phenotypic difference, thin- and fat-tailed sheep, whole-genome sequencing, transcriptome

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Nonfluorescent Denaturing HPLC–Based Primer-Extension Method for Allele-Specific Expression: Application to Analysis of Mismatch Repair Genes

Cited by 4 publications

References 23 publications

Increased Variance in Germline Allele-Specific Expression of APC Associates With Colorectal Cancer

Increased Variance in Germline Allele-Specific Expression of APC Associates With Colorectal Cancer

Correlation between mutations and mRNA expression of APC and MUTYH genes: new insight into hereditary colorectal polyposis predisposition

Allele-specific expression reveals the phenotypic differences between thin- and fat-tailed sheep

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