A Noncoding Variant Near PPP1R3B Promotes Liver Glycogen Storage and MetS, but Protects Against Myocardial Infarction

Abstract

Context: Glycogen storage diseases are rare. Increased glycogen in the liver results in increased attenuation.

Objective: Investigate the association and function of a noncoding region associated with liver attenuation but not histologic nonalcoholic fatty liver disease.

Design: Genetics of Obesity-associated Liver Disease Consortium.

Setting: Population-based.

Main outcome: Computed tomography measured liver attenuation.

Results: Carriers of rs4841132-A (frequency 2%-19%) do not show increased hepatic steatosis; they have increased liver attenuation indicative of increased glycogen deposition. rs4841132 falls in a noncoding RNA LOC157273 ~190 kb upstream of PPP1R3B. We demonstrate that rs4841132-A increases PPP1R3B through a cis genetic effect. Using CRISPR/Cas9 we engineered a 105-bp deletion including rs4841132-A in human hepatocarcinoma cells that increases PPP1R3B, decreases LOC157273, and increases glycogen perfectly mirroring the human disease. Overexpression of PPP1R3B or knockdown of LOC157273 increased glycogen but did not result in decreased LOC157273 or increased PPP1R3B, respectively, suggesting that the effects may not all occur via affecting RNA levels. Based on electronic health record (EHR) data, rs4841132-A associates with all components of the metabolic syndrome (MetS). However, rs4841132-A associated with decreased low-density lipoprotein (LDL) cholesterol and risk for myocardial infarction (MI). A metabolic signature for rs4841132-A includes increased glycine, lactate, triglycerides, and decreased acetoacetate and beta-hydroxybutyrate.

Conclusions: These results show that rs4841132-A promotes a hepatic glycogen storage disease by increasing PPP1R3B and decreasing LOC157273. rs4841132-A promotes glycogen accumulation and development of MetS but lowers LDL cholesterol and risk for MI. These results suggest that elevated hepatic glycogen is one cause of MetS that does not invariably promote MI.

Keywords: GWAS; NALFD; genetics; glycogen; metabolic syndrome; triglyceride.

Figure 1.

Association of rs4841132 with liver attenuation across studies and ancestries. (A) Effect of index variants on liver attenuation by study (European [EUR]; African American [AFA]; Hispanic [HIS]; Chinese [CHN]) and (B) by ancestry. Effect allele frequency (EAF) is listed with the corresponding P value (P), effect size on the inverse-normally transformed scale (Beta), and 95% confidence interval in effect size (95% CI) for association with liver attenuation adjusted for age, age², sex, alcoholic drinks per week, and population substructure. Values greater than 0 indicate an increase in the amount of liver attenuation. For the pooled analyses, heterogeneity P values are also reported. Effect sizes and confidence intervals are summarized in the Forest plots at right. The solid vertical line represents beta = 0.

Figure 2.

Allele-specific expression shows that rs4841132-A increases PPP1R3B expression in cis. (A) Theoretical ratio of PPP1R3B RNA transcripts with respect to rs4841132 genotype and (B) PPP1R3B allelic balance is closer to 1 in rs4841132-G/G individuals than in rs4841132-A/G individuals suggesting a cis effect of rs4841132-A. Box plots: dark line indicates the median value, a box indicating the 25th and 75th percentiles, whiskers extending from the median for twice the interquartile range, and individual points representing measurements outside that interval.

Figure 3.

CRISPR/Cas9 105 bp deletion of rs4841132 region increases glycogen, PPP1R3B RNA, and decreases LOC157273 RNA in HuH-7 cells. (A) Fold-change in glycogen in μg/mg protein. (B) Quantitative PCR of PPP1R3B and LOC157273 normalized to wild-type (WT) control (black). Control, no stimulation of cells (circles); hetero: heterozygously deleted rs4841132 region (blue); homo, homozygously deleted rs4841132 region (red); insulin, cells treated with 30 μM insulin (squares); OA, cells treated with 200 μM oleic acid (triangles). *P < 0.05; **P < 0.01.

Figure 4.

LOC157273 overexpression and knockdown decreases and increases glycogen and has variable effects on PPP1R3B RNA levels in Huh-7 cells. Overexpression of vector only or vector with LOC157273. (A) Fold change in glycogen in μg/mg protein and (B) quantitative PCR of PPP1R3B and LOC157273 normalized to vector control. Antisense oligonucleotide knockdown of LOC157273. (C) Fold-change in glycogen in μg/mg protein and (D) quantitative PCR of PPP1R3B and LOC157273 normalized to scrambled control. ASO, cells exposed to LOC157273 antisense oligonucleotides; control, no stimulation of cells (circles); insulin, cells treated with 30 μM insulin (squares); LOC, cells expressing LOC157273; OA; cells treated with 200uM oleic acid (triangles); scrambled: cells exposed to scrambled oligonucleotide; vector, cells expressing vector. *P < 0.05; **P < 0.01.

Figure 5.

Overexpression and knockout of PPP1R3B increases and decreases glycogen and has variable effects on LOC157273 RNA levels in HuH-7 cells. Overexpression of vector or vector with PPP1R3B. (A) Fold-change in glycogen in μg/mg protein and (B) QPCR of PPP1R3B and LOC157273 normalized to vector control. Knockout of PPP1R3B with CRISPR-Cas9 engineered insertion causing protein early termination and nonsense mediated decay. (C) Fold-change in glycogen in μg/mg protein and (D) quantitative PCR of PPP1R3B and LOC157273 normalized to HuH-7 control. Control, no stimulation of cells; KO, knock-out; OE, overexpression; vector, cells expressing vector. P value between experimental (red) and control (black) conditions are noted, with * representing P < 0.05.

Figure 6.

Pleiotropic effects of rs4841132. Each row summarizes the effect of rs4841132-A on a trait using an additive model. Trait names (Traits) are grouped by system. Sources include: GWAS summary statistics (public), the Michigan Genomics Initiative (MGI), or the UK Biobank (UKBB). For each trait, effect sizes (beta) are in common units with a P value (pval) and corresponding z score from the largest analysis in source manuscripts. The z scores are graphically summarized to the right; a red bar indicates rs4841132-A associates with an increase, whereas a blue bar indicates a decrease. GWAS, genome-wide association study.

Figure 7.

Physiological model of the metabolic effects of rs4841132-A. Purple arrow and inhibition sign note putative increased and inhibitory effects of increased PPP1R3B on the enzyme activity of glycogen synthase and glycogen phosphorylase, respectively. Green arrows represent possible net flow of metabolites to account for traits, diseases, and metabolite levels seen in the population. Traits, diseases, or metabolites that are increased or decreased in the population are noted in red and blue, respectively.