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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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Evaluation of PICALM isoform AEI with respect to rs3851179. The indicated PICALM isoforms were analyzed for AEI as a function of rs3851179 (a-c) or rs588076 (d). Each allelic ratio was normalized to the sample’s gDNA ratio. a) The full length PICALM isoform contained exons 18–19 and showed equal allelic ratios, with a non-significant trend towards an increase in the G allele. b)D18-PICALM showed equal allelic ratios and c)D18-19 PICALM showed significant unequal allelic ratios in 9 samples (*p < 0.05), d) The D18-19 PICALM AEI was associated with rs588076 heterozygosity. This pattern of significant AEI was not associated with sex, age or AD status (p > 0.05).
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Figure 5: Evaluation of PICALM isoform AEI with respect to rs3851179. The indicated PICALM isoforms were analyzed for AEI as a function of rs3851179 (a-c) or rs588076 (d). Each allelic ratio was normalized to the sample’s gDNA ratio. a) The full length PICALM isoform contained exons 18–19 and showed equal allelic ratios, with a non-significant trend towards an increase in the G allele. b)D18-PICALM showed equal allelic ratios and c)D18-19 PICALM showed significant unequal allelic ratios in 9 samples (*p < 0.05), d) The D18-19 PICALM AEI was associated with rs588076 heterozygosity. This pattern of significant AEI was not associated with sex, age or AD status (p > 0.05).

Mentions: Having considered overall PICALM allelic expression, we proceeded to apply this AEI analysis to PICALM isoforms. The exon 17 to exon 20 PCR amplicons captured three different PICALM isoforms because exons 18 and 19 are variably spliced. These isoforms were termed full length PICALM (contains exons 18–19), D18-PICALM (lacked exon 18) or D18-19 PICALM (lacked both exons 18 and 19). We analyzed each of these isoforms for the presence of AEI as a function of rs3851179 heterozygosity. For the full length PICALM and D18-PICALM isoforms, significant AEI was observed only in the AD40 sample. To evaluate whether a subtle difference in allelic expression may be present, we compared the allelic expression of the full length PICALM isoform between the rs3851179 homozygous and heterozygous groups by using a t-test. However, no difference was observed as the average T:G ratio in the rs3851179 homozygous and heterozygous groups was 0.93 ± 0.07 (n = 17) and 0.93 ± 0.06 (n = 18), respectively (Figure 5a, p = 0.81). Likewise, for the D18-PICALM isoform, the rs3851179 homozygous and heterozygous groups showed mean T:G ratios of 0.95 ± 0.09 (n = 17) and 1.01 ± 0.12 (n = 18), respectively (Figure 5b, p = value = 0.13). When we evaluated the D18-19 PICALM isoform, significant AEI was detected in multiple samples (Figure 5c). We analyzed these results in two ways. First, we compared the frequency of samples with significant AEI between the rs3851179 homozygous and heterozygous individuals by using a Fisher’s exact test; a significant difference between groups was not detected (Figure 5c, p = 0.44). Second, we compared the mean T:G ratio between rs3851179 homozygous and heterozygous individuals by using a t-test. However, the rs3851179 homozygous and heterozygous individuals showed similar values that did not achieve significance, i.e., 1.19 ± 0.16 and 1.11 ± 0.22, respectively (p = 0.08). Hence, rs3851179 heterozygosity was not associated with AEI for these PICALM isoforms.


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)

Evaluation of PICALM isoform AEI with respect to rs3851179. The indicated PICALM isoforms were analyzed for AEI as a function of rs3851179 (a-c) or rs588076 (d). Each allelic ratio was normalized to the sample’s gDNA ratio. a) The full length PICALM isoform contained exons 18–19 and showed equal allelic ratios, with a non-significant trend towards an increase in the G allele. b)D18-PICALM showed equal allelic ratios and c)D18-19 PICALM showed significant unequal allelic ratios in 9 samples (*p < 0.05), d) The D18-19 PICALM AEI was associated with rs588076 heterozygosity. This pattern of significant AEI was not associated with sex, age or AD status (p > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Evaluation of PICALM isoform AEI with respect to rs3851179. The indicated PICALM isoforms were analyzed for AEI as a function of rs3851179 (a-c) or rs588076 (d). Each allelic ratio was normalized to the sample’s gDNA ratio. a) The full length PICALM isoform contained exons 18–19 and showed equal allelic ratios, with a non-significant trend towards an increase in the G allele. b)D18-PICALM showed equal allelic ratios and c)D18-19 PICALM showed significant unequal allelic ratios in 9 samples (*p < 0.05), d) The D18-19 PICALM AEI was associated with rs588076 heterozygosity. This pattern of significant AEI was not associated with sex, age or AD status (p > 0.05).
Mentions: Having considered overall PICALM allelic expression, we proceeded to apply this AEI analysis to PICALM isoforms. The exon 17 to exon 20 PCR amplicons captured three different PICALM isoforms because exons 18 and 19 are variably spliced. These isoforms were termed full length PICALM (contains exons 18–19), D18-PICALM (lacked exon 18) or D18-19 PICALM (lacked both exons 18 and 19). We analyzed each of these isoforms for the presence of AEI as a function of rs3851179 heterozygosity. For the full length PICALM and D18-PICALM isoforms, significant AEI was observed only in the AD40 sample. To evaluate whether a subtle difference in allelic expression may be present, we compared the allelic expression of the full length PICALM isoform between the rs3851179 homozygous and heterozygous groups by using a t-test. However, no difference was observed as the average T:G ratio in the rs3851179 homozygous and heterozygous groups was 0.93 ± 0.07 (n = 17) and 0.93 ± 0.06 (n = 18), respectively (Figure 5a, p = 0.81). Likewise, for the D18-PICALM isoform, the rs3851179 homozygous and heterozygous groups showed mean T:G ratios of 0.95 ± 0.09 (n = 17) and 1.01 ± 0.12 (n = 18), respectively (Figure 5b, p = value = 0.13). When we evaluated the D18-19 PICALM isoform, significant AEI was detected in multiple samples (Figure 5c). We analyzed these results in two ways. First, we compared the frequency of samples with significant AEI between the rs3851179 homozygous and heterozygous individuals by using a Fisher’s exact test; a significant difference between groups was not detected (Figure 5c, p = 0.44). Second, we compared the mean T:G ratio between rs3851179 homozygous and heterozygous individuals by using a t-test. However, the rs3851179 homozygous and heterozygous individuals showed similar values that did not achieve significance, i.e., 1.19 ± 0.16 and 1.11 ± 0.22, respectively (p = 0.08). Hence, rs3851179 heterozygosity was not associated with AEI for these PICALM isoforms.

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