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An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform.

Parikh I, Medway C, Younkin S, Fardo DW, Estus S - Mol Neurodegener (2014)

Bottom Line: To evaluate whether this polymorphism was associated with AD, we genotyped 3269 individuals and found that rs588076 was modestly associated with AD.However, when both the primary AD SNP rs3851179 was added to the logistic regression model, only rs3851179 was significantly associated with AD.Conditional on rs3851179, rs588076 was not associated with AD risk, suggesting that D18-19 PICALM is not critical in AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Physiology, Sanders-Brown Center on Aging, University of Kentucky, 800 S, Limestone St,, Lexington, KY 40536, USA. Steve.estus@uky.edu.

ABSTRACT

Background: Although genome wide studies have associated single nucleotide polymorphisms (SNP)s near PICALM with Alzheimer's disease (AD), the mechanism underlying this association is unclear. PICALM is involved in clathrin-mediated endocytosis and modulates Aß clearance in vitro. Comparing allelic expression provides the means to detect cis-acting regulatory polymorphisms. Thus, we evaluated whether PICALM showed allele expression imbalance (AEI) and whether this imbalance was associated with the AD-associated polymorphism, rs3851179.

Results: We measured PICALM allelic expression in 42 human brain samples by using next-generation sequencing. Overall, PICALM demonstrated equal allelic expression with no detectable influence by rs3851179. A single sample demonstrated robust global PICALM allelic expression imbalance (AEI), i.e., each of the measured isoforms showed AEI. Moreover, the PICALM isoform lacking exons 18 and 19 (D18-19 PICALM) showed significant AEI in a subset of individuals. Sequencing these individuals and subsequent genotyping revealed that rs588076, located in PICALM intron 17, was robustly associated with this imbalance in D18-19 PICALM allelic expression (p = 9.54 x 10-5). This polymorphism has been associated previously with systolic blood pressure response to calcium channel blocking agents. To evaluate whether this polymorphism was associated with AD, we genotyped 3269 individuals and found that rs588076 was modestly associated with AD. However, when both the primary AD SNP rs3851179 was added to the logistic regression model, only rs3851179 was significantly associated with AD.

Conclusions: PICALM expression shows no evidence of AEI associated with rs3851179. Robust global AEI was detected in one sample, suggesting the existence of a rare SNP that strongly modulates PICALM expression. AEI was detected for the D18-19 PICALM isoform, and rs588076 was associated with this AEI pattern. Conditional on rs3851179, rs588076 was not associated with AD risk, suggesting that D18-19 PICALM is not critical in AD. In summary, this analysis of PICALM allelic expression provides novel insights into the genetics of PICALM expression and AD risk.

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Linearity of allelic expression assay. Different proportions of rs76719109 T and G homozygous cDNA were mixed to test the linearity of the AEI assay. The T:G ratios were 1:4, 1:2, 1:1, 2:1, and 4:1. An overall linear relationship was found (r2 = 0.99). The slope was 0.999, i.e., the assay detected the T and G alleles with equal efficiency. The graphs are plotted log2 to avoid compression at the lower ratios and thereby better visualize the data[22].
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Figure 2: Linearity of allelic expression assay. Different proportions of rs76719109 T and G homozygous cDNA were mixed to test the linearity of the AEI assay. The T:G ratios were 1:4, 1:2, 1:1, 2:1, and 4:1. An overall linear relationship was found (r2 = 0.99). The slope was 0.999, i.e., the assay detected the T and G alleles with equal efficiency. The graphs are plotted log2 to avoid compression at the lower ratios and thereby better visualize the data[22].

Mentions: To detect the presence of regulatory cis-acting SNPs in human brain samples, we measured allelic ratios in cDNA from reverse transcribed mRNA. Heterozygosity for an exonic "reporter" SNP provides the means to compare the expression of one allele with another allele within an individual. Our criteria for reporter SNPs for AEI analysis is that the SNPs are present in exons and have a minor allele frequency (MAF) greater than 15%, which allows for sufficient sample numbers for analysis. Only two PICALM SNPs satisfied these criteria, rs76719109 and rs592297 (Figure 1). Rs76719109 has a MAF of 0.44 and resides within exon 17; PCR amplification from exon 17–20 allowed us to measure AEI for total PICALM as well as PICALM splice variants lacking exon 18 or exons 18–19 (Figure 1a). Rs592297 has a MAF of 0.20 and resides in exon 5. PCR amplification from exon 5–6 produced a single PCR product for cDNA (Figure 1b). The AEI assay was validated in two ways. First, we tested the linearity of the assay by generating a cDNA standard curve consisting of five different rs76719109 T:G ratios (Figure 2). Our input T:G ratios ranged from 1:4 to 4:1. We found a robust linear relationship between input and observed T:G ratios. Second, we applied our experimental approach to genomic DNA (gDNA), which represented a positive control with an expected "allelic" ratio of 1.0. Rs76719109 and rs592297 showed gDNA ratios of 1.01 ± 0.03 (mean ± SD, n = 35) and 0.96 ± 0.05 (mean ± SD, n = 19), respectively (Figure 3). Hence, this AEI assay appears robust for detecting and quantifying variations in allelic expression.


An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform.

Parikh I, Medway C, Younkin S, Fardo DW, Estus S - Mol Neurodegener (2014)

Linearity of allelic expression assay. Different proportions of rs76719109 T and G homozygous cDNA were mixed to test the linearity of the AEI assay. The T:G ratios were 1:4, 1:2, 1:1, 2:1, and 4:1. An overall linear relationship was found (r2 = 0.99). The slope was 0.999, i.e., the assay detected the T and G alleles with equal efficiency. The graphs are plotted log2 to avoid compression at the lower ratios and thereby better visualize the data[22].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150683&req=5

Figure 2: Linearity of allelic expression assay. Different proportions of rs76719109 T and G homozygous cDNA were mixed to test the linearity of the AEI assay. The T:G ratios were 1:4, 1:2, 1:1, 2:1, and 4:1. An overall linear relationship was found (r2 = 0.99). The slope was 0.999, i.e., the assay detected the T and G alleles with equal efficiency. The graphs are plotted log2 to avoid compression at the lower ratios and thereby better visualize the data[22].
Mentions: To detect the presence of regulatory cis-acting SNPs in human brain samples, we measured allelic ratios in cDNA from reverse transcribed mRNA. Heterozygosity for an exonic "reporter" SNP provides the means to compare the expression of one allele with another allele within an individual. Our criteria for reporter SNPs for AEI analysis is that the SNPs are present in exons and have a minor allele frequency (MAF) greater than 15%, which allows for sufficient sample numbers for analysis. Only two PICALM SNPs satisfied these criteria, rs76719109 and rs592297 (Figure 1). Rs76719109 has a MAF of 0.44 and resides within exon 17; PCR amplification from exon 17–20 allowed us to measure AEI for total PICALM as well as PICALM splice variants lacking exon 18 or exons 18–19 (Figure 1a). Rs592297 has a MAF of 0.20 and resides in exon 5. PCR amplification from exon 5–6 produced a single PCR product for cDNA (Figure 1b). The AEI assay was validated in two ways. First, we tested the linearity of the assay by generating a cDNA standard curve consisting of five different rs76719109 T:G ratios (Figure 2). Our input T:G ratios ranged from 1:4 to 4:1. We found a robust linear relationship between input and observed T:G ratios. Second, we applied our experimental approach to genomic DNA (gDNA), which represented a positive control with an expected "allelic" ratio of 1.0. Rs76719109 and rs592297 showed gDNA ratios of 1.01 ± 0.03 (mean ± SD, n = 35) and 0.96 ± 0.05 (mean ± SD, n = 19), respectively (Figure 3). Hence, this AEI assay appears robust for detecting and quantifying variations in allelic expression.

Bottom Line: To evaluate whether this polymorphism was associated with AD, we genotyped 3269 individuals and found that rs588076 was modestly associated with AD.However, when both the primary AD SNP rs3851179 was added to the logistic regression model, only rs3851179 was significantly associated with AD.Conditional on rs3851179, rs588076 was not associated with AD risk, suggesting that D18-19 PICALM is not critical in AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Physiology, Sanders-Brown Center on Aging, University of Kentucky, 800 S, Limestone St,, Lexington, KY 40536, USA. Steve.estus@uky.edu.

ABSTRACT

Background: Although genome wide studies have associated single nucleotide polymorphisms (SNP)s near PICALM with Alzheimer's disease (AD), the mechanism underlying this association is unclear. PICALM is involved in clathrin-mediated endocytosis and modulates Aß clearance in vitro. Comparing allelic expression provides the means to detect cis-acting regulatory polymorphisms. Thus, we evaluated whether PICALM showed allele expression imbalance (AEI) and whether this imbalance was associated with the AD-associated polymorphism, rs3851179.

Results: We measured PICALM allelic expression in 42 human brain samples by using next-generation sequencing. Overall, PICALM demonstrated equal allelic expression with no detectable influence by rs3851179. A single sample demonstrated robust global PICALM allelic expression imbalance (AEI), i.e., each of the measured isoforms showed AEI. Moreover, the PICALM isoform lacking exons 18 and 19 (D18-19 PICALM) showed significant AEI in a subset of individuals. Sequencing these individuals and subsequent genotyping revealed that rs588076, located in PICALM intron 17, was robustly associated with this imbalance in D18-19 PICALM allelic expression (p = 9.54 x 10-5). This polymorphism has been associated previously with systolic blood pressure response to calcium channel blocking agents. To evaluate whether this polymorphism was associated with AD, we genotyped 3269 individuals and found that rs588076 was modestly associated with AD. However, when both the primary AD SNP rs3851179 was added to the logistic regression model, only rs3851179 was significantly associated with AD.

Conclusions: PICALM expression shows no evidence of AEI associated with rs3851179. Robust global AEI was detected in one sample, suggesting the existence of a rare SNP that strongly modulates PICALM expression. AEI was detected for the D18-19 PICALM isoform, and rs588076 was associated with this AEI pattern. Conditional on rs3851179, rs588076 was not associated with AD risk, suggesting that D18-19 PICALM is not critical in AD. In summary, this analysis of PICALM allelic expression provides novel insights into the genetics of PICALM expression and AD risk.

Show MeSH
Related in: MedlinePlus