Limits...
Study of transcriptional effects in Cis at the IFIH1 locus.

Zouk H, Marchand L, Polychronakos C - PLoS ONE (2010)

Bottom Line: We investigated the effect of the T1D-associated variation on mRNA transcript expression of these genes.Using single-nucleotide primer extension, we found no difference between mRNA transcripts in 9 LCLs, 6 pancreas and 13 thymus samples, suggesting that GCA and FAP are not involved.The mechanism of the association of the nsSNP with T1D remains to be determined, but does not involve mRNA modulation.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Genetics Laboratory, McGill University Health Center, Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT

Background: The Thr allele at the non-synonymous single-nucleotide polymorphism (nsSNP) Thr946Ala in the IFIH1 gene confers risk for Type 1 diabetes (T1D). The SNP is embedded in a 236 kb linkage disequilibrium (LD) block that includes four genes: IFIH1, GCA, FAP and KCNH7. The absence of common nsSNPs in the other genes makes the IFIH1 SNP the strongest functional candidate, but it could be merely a marker of association, due to LD with a variant regulating expression levels of IFIH1 or neighboring genes.

Methodology/principal findings: We investigated the effect of the T1D-associated variation on mRNA transcript expression of these genes. Heterozygous mRNA from lymphoblastoid cell lines (LCLs), pancreas and thymus was examined by allelic expression imbalance, to detect effects in cis on mRNA expression. Using single-nucleotide primer extension, we found no difference between mRNA transcripts in 9 LCLs, 6 pancreas and 13 thymus samples, suggesting that GCA and FAP are not involved. On the other hand, KCNH7 was not expressed at a detectable level in all tissues examined. Moreover, the association of the Thr946Ala SNP with T1D is not due to modulation of IFIH1 expression in organs involved in the disease, pointing to the IFIH1 nsSNP as the causal variant.

Conclusions/significance: The mechanism of the association of the nsSNP with T1D remains to be determined, but does not involve mRNA modulation. It becomes necessary to study differential function of the IFIH1 protein alleles at Thr946Ala to confirm that it is responsible for the disease association.

Show MeSH

Related in: MedlinePlus

Allelic ratio distribution at the IFIH1 locus.9 Lymphoblastoid cell lines (LCL), 6 pancreas (Panc.) and 13 thymus (Thym.) tissue from individuals heterozygous for the selected marker SNPs for each gene were used to assess allelic imbalance at the IFIH1 locus. Relative allelic abundance in individual samples has been normalized to the mean genomic DNA ratio (equal to1) and normalized sample RNA ratios were compared to those of normalized genomic DNA for each gene in each tissue. The average means ± SEM are summarized in table 2, along with the statistical analysis. Our power to detect a difference of 40% in the means of DNA and LCL RNA was >99%.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2903489&req=5

pone-0011564-g001: Allelic ratio distribution at the IFIH1 locus.9 Lymphoblastoid cell lines (LCL), 6 pancreas (Panc.) and 13 thymus (Thym.) tissue from individuals heterozygous for the selected marker SNPs for each gene were used to assess allelic imbalance at the IFIH1 locus. Relative allelic abundance in individual samples has been normalized to the mean genomic DNA ratio (equal to1) and normalized sample RNA ratios were compared to those of normalized genomic DNA for each gene in each tissue. The average means ± SEM are summarized in table 2, along with the statistical analysis. Our power to detect a difference of 40% in the means of DNA and LCL RNA was >99%.

Mentions: The calculated allelic ratios for each SNP representing each gene in the IFIH1 locus and their distribution in the different tissues that were assayed are shown in Table 2 and Figure 1 respectively. The allelic ratio distribution of IFIH1, GCA, and FAP cDNA do not significantly differ from that of their corresponding DNA. After correction by the genomic DNA allele proportion, the average ratio (mean ± SEM) of the major allele (T) over the minor allele (C) of the rs1990760 SNP in IFIH1 in LCLs is 1.0029±0.0106, p = 0.8477 (Table 2), indicating the absence of an AI effect due to a common genetic variation at the IFIH1 gene in LCLs. The same is observed in pancreas and thymus. No evidence of significant transcriptional effect was seen in any of the other genes in all the assayed tissues and cells (Table 2).Our approach had a 99% power to detect a transcriptional effect of rs1990760 on IFIH1 in LCLs, of the magnitude reported by Liu et al. [14] (40% allelic difference at α = 0.01). We also have 99% power to detect a 25% difference in expression between the two IFIH1alleles. Since all RNA samples were DNAse-treated prior to RT-PCR and did not generate detectable PCR product in the absence of an RT step, it is highly improbable that our results were influenced in some way by genomic DNA contamination. It has been recently suggested [31] that differential secondary structure of RNA alleles may interfere with quantitative comparisons through a differential effect on the efficiency of reverse transcription, creating spurious allelic imbalance or conceivably masking true imbalance (if it happens to be exactly equal and in the opposite direction). To deal with this, minimization of secondary structure by fragmenting the RNA prior to reverse transcription (RT) was recommended. To see whether this may be a problem in the specific case of IFIH1, we compared allelic ratios obtained with or without fragmentation of the RNA prior to RT. In twelve independent comparisons, the mean allelic ratio (normalized for the average DNA ratio) was 0.95±0.07 (SEM) for unfragmented vs. 1.00±0.05 for fragmented RNA (p = 0.56, 99% power to detect a 40% effect at α = 0.01, 86.7% power to detect a 25% effect at α = 0.05). Oligo-dT priming of the RT, suggested as an alternative, also gave nearly-identical results (0.98±0.05 [SEM]). We, therefore, concluded that interference by secondary structure was not an issue in allelic IFIH1 measurements.


Study of transcriptional effects in Cis at the IFIH1 locus.

Zouk H, Marchand L, Polychronakos C - PLoS ONE (2010)

Allelic ratio distribution at the IFIH1 locus.9 Lymphoblastoid cell lines (LCL), 6 pancreas (Panc.) and 13 thymus (Thym.) tissue from individuals heterozygous for the selected marker SNPs for each gene were used to assess allelic imbalance at the IFIH1 locus. Relative allelic abundance in individual samples has been normalized to the mean genomic DNA ratio (equal to1) and normalized sample RNA ratios were compared to those of normalized genomic DNA for each gene in each tissue. The average means ± SEM are summarized in table 2, along with the statistical analysis. Our power to detect a difference of 40% in the means of DNA and LCL RNA was >99%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903489&req=5

pone-0011564-g001: Allelic ratio distribution at the IFIH1 locus.9 Lymphoblastoid cell lines (LCL), 6 pancreas (Panc.) and 13 thymus (Thym.) tissue from individuals heterozygous for the selected marker SNPs for each gene were used to assess allelic imbalance at the IFIH1 locus. Relative allelic abundance in individual samples has been normalized to the mean genomic DNA ratio (equal to1) and normalized sample RNA ratios were compared to those of normalized genomic DNA for each gene in each tissue. The average means ± SEM are summarized in table 2, along with the statistical analysis. Our power to detect a difference of 40% in the means of DNA and LCL RNA was >99%.
Mentions: The calculated allelic ratios for each SNP representing each gene in the IFIH1 locus and their distribution in the different tissues that were assayed are shown in Table 2 and Figure 1 respectively. The allelic ratio distribution of IFIH1, GCA, and FAP cDNA do not significantly differ from that of their corresponding DNA. After correction by the genomic DNA allele proportion, the average ratio (mean ± SEM) of the major allele (T) over the minor allele (C) of the rs1990760 SNP in IFIH1 in LCLs is 1.0029±0.0106, p = 0.8477 (Table 2), indicating the absence of an AI effect due to a common genetic variation at the IFIH1 gene in LCLs. The same is observed in pancreas and thymus. No evidence of significant transcriptional effect was seen in any of the other genes in all the assayed tissues and cells (Table 2).Our approach had a 99% power to detect a transcriptional effect of rs1990760 on IFIH1 in LCLs, of the magnitude reported by Liu et al. [14] (40% allelic difference at α = 0.01). We also have 99% power to detect a 25% difference in expression between the two IFIH1alleles. Since all RNA samples were DNAse-treated prior to RT-PCR and did not generate detectable PCR product in the absence of an RT step, it is highly improbable that our results were influenced in some way by genomic DNA contamination. It has been recently suggested [31] that differential secondary structure of RNA alleles may interfere with quantitative comparisons through a differential effect on the efficiency of reverse transcription, creating spurious allelic imbalance or conceivably masking true imbalance (if it happens to be exactly equal and in the opposite direction). To deal with this, minimization of secondary structure by fragmenting the RNA prior to reverse transcription (RT) was recommended. To see whether this may be a problem in the specific case of IFIH1, we compared allelic ratios obtained with or without fragmentation of the RNA prior to RT. In twelve independent comparisons, the mean allelic ratio (normalized for the average DNA ratio) was 0.95±0.07 (SEM) for unfragmented vs. 1.00±0.05 for fragmented RNA (p = 0.56, 99% power to detect a 40% effect at α = 0.01, 86.7% power to detect a 25% effect at α = 0.05). Oligo-dT priming of the RT, suggested as an alternative, also gave nearly-identical results (0.98±0.05 [SEM]). We, therefore, concluded that interference by secondary structure was not an issue in allelic IFIH1 measurements.

Bottom Line: We investigated the effect of the T1D-associated variation on mRNA transcript expression of these genes.Using single-nucleotide primer extension, we found no difference between mRNA transcripts in 9 LCLs, 6 pancreas and 13 thymus samples, suggesting that GCA and FAP are not involved.The mechanism of the association of the nsSNP with T1D remains to be determined, but does not involve mRNA modulation.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Genetics Laboratory, McGill University Health Center, Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT

Background: The Thr allele at the non-synonymous single-nucleotide polymorphism (nsSNP) Thr946Ala in the IFIH1 gene confers risk for Type 1 diabetes (T1D). The SNP is embedded in a 236 kb linkage disequilibrium (LD) block that includes four genes: IFIH1, GCA, FAP and KCNH7. The absence of common nsSNPs in the other genes makes the IFIH1 SNP the strongest functional candidate, but it could be merely a marker of association, due to LD with a variant regulating expression levels of IFIH1 or neighboring genes.

Methodology/principal findings: We investigated the effect of the T1D-associated variation on mRNA transcript expression of these genes. Heterozygous mRNA from lymphoblastoid cell lines (LCLs), pancreas and thymus was examined by allelic expression imbalance, to detect effects in cis on mRNA expression. Using single-nucleotide primer extension, we found no difference between mRNA transcripts in 9 LCLs, 6 pancreas and 13 thymus samples, suggesting that GCA and FAP are not involved. On the other hand, KCNH7 was not expressed at a detectable level in all tissues examined. Moreover, the association of the Thr946Ala SNP with T1D is not due to modulation of IFIH1 expression in organs involved in the disease, pointing to the IFIH1 nsSNP as the causal variant.

Conclusions/significance: The mechanism of the association of the nsSNP with T1D remains to be determined, but does not involve mRNA modulation. It becomes necessary to study differential function of the IFIH1 protein alleles at Thr946Ala to confirm that it is responsible for the disease association.

Show MeSH
Related in: MedlinePlus