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Pair of Studies Examine Low-frequency Variants Linked to Cholesterol, Lipid Levels

NEW YORK (GenomeWeb News) – Two studies appearing in the American Journal of Human Genetics today uncovered a handful of low-frequency variants, including novel variants in ANGPTL8, PNPLA5, and PCSK7, among others, that seem to contribute to cholesterol or lipid levels in the blood.

Lipid levels, especially LDL-C levels, are risk factors for coronary artery disease. According to the US Centers for Disease Control and Prevention, heart disease is the most common cause of death in the US, as some 600,000 people die a year from the condition.

Previous studies have uncovered a few rare mutations that lead to Mendelian lipid-related diseases, and genome-wide association studies have found some common variants linked to lipid levels, but researchers in the US, Netherlands, and elsewhere led by Cristen Willer at the University of Michigan noted that the role of rare and low-frequency variants in population-wide cholesterol levels is relatively unknown.

"We evaluated the hypothesis that rare or low-frequency variants, which are not well covered by GWASs and not easily imputed, are also associated with LDL-C [low-density lipoprotein cholesterol]," Willer and her colleagues said in their AJHG paper.

To find low frequency variants linked to cholesterol levels, the researchers, as part of the NHLBI Grand Opportunity Exome Sequencing Project, performed exome sequencing on more than 2,000 people culled from seven population-based cohorts. That group was enriched for individuals with either very high or very low fasting LDL-C levels as determined by the Friedewald formula.

They performed exome sequencing using either the Roche Nimblegen SeqCap EZ or Aglient SureSelect Human All Exon 50Mb capture approaches followed by paired-end sequencing on either the Illumina GAII or HiSeq machines, and generated a 127x mean depth of targeted regions.

From this, the researchers identified variants in three genes, PCSK9, LDLR, and APOB, all of which were previously associated with LDL-C.

By folding in exome data from an additional 1,302 individuals, also enriched for those extremes of LDL-C levels, the researchers teased out variants in a gene — PNPLA5 — that had not been linked to LDL-C before. The researchers confirmed the association using the Go-T2D dataset.

The genetic architecture, effect direction, and effect size varied by variant. For instance, the researchers noted single-copy nonsense variants in APOB as well as low-frequency missense mutations in PCSK9. Meanwhile, loss of function variants led to larger effect sizes than did missense mutation. And rare variants in PCSK9 and APOB variants were linked to low LDL-C, while PNPLA5 and LDL9 variants were associated with high LDL-C levels.

In addition, the researchers noted that variants in LDL9 and PCSK9 could be found in both European and African Americans, while APOB variants were mostly found in European Americans and PNPLA5 variants were mostly found in African Americans.

"[We] present clear evidence that uncommon and rare variants contribute to variation of LDL-C levels in the general population," the study authors wrote.

Meanwhile, in their AJHG paper, researchers led by Adrienne Cupples from the Boston University School of Medicine and the National Heart, Lung, and Blood Institute Framingham Heart Study noted that a recent study found that certain low-frequency variants in the PCSK9 gene were linked to lower plasma levels of LDL-C and a lower risk of coronary heart disease.

"These observations have raised the question of whether the PCSK9 example is a paradigm for complex diseases like [coronary heart disease] or an exception," Cupples and her colleagues said.

To identify new low-frequency nonsynonymous and splice site variants linked to lipid levels, Cupples and her colleagues genotyped more than 56,500 individuals from 13 studies on the Illumina Human Exome array and analyzed those participants' fasting LDL-C, HDL-C, and triglyceride levels. The researchers estimated that the array captured 78 percent of nonsynonymous and splice site variation with less than one in 1,000 allele frequency in European Americans, and 71 percent of nonsynonymous and splice site variation in African Americans.

From this, the investigators uncovered four novel variants linked to HDL-C or triglyceride levels, two of which — in COL18A1 and PCSK7 — were found in African-American participants and two of which — in ANGPTL8 and PAFAH1B2— were found in European-American participants. Unlike the other group, they did not find any variants linked to LDL-C levels.

Each of these novel variants, the researchers noted, had large effects on lipid levels. For instance, mice overexpressing PCSK7 in the liver have lower HDL-C and higher triglyceride levels as compared to controls.

Cupples and her colleagues also investigated whether these variants are associated with cardiovascular disease. They tested those novel variants alongside PCSK9, as a positive control, in more than 63,000 European-American individuals from 15 studies and nearly 14,000 African-American individuals from seven studies, of whom roughly 20 percent had coronary heart disease.

While the investigators confirmed the link between PCSK9 and heart disease, they did not find an association between the novel variants at ANGPTL8, COL18A1, PAFAH1B, or PCSK7 and heart disease risk. Cupples and colleagues noted that those variants were all linked to HDL-C, which may not be a coronary heart disease risk factor, and that could, in part, explain the lack of association between the variants and disease. They also pointed out the mechanism behind how the genes affect blood lipoprotein is not fully understood.

Still, the researchers uncovered four low-frequency variants that affect plasma HDL-C and/or triglyceride levels, though not CHD risk. "These results suggest that the example of PCSK9 with low-frequency alleles that affect both plasma lipids and CHD is likely to be an exception rather than a paradigm," they said.