Profile of genetic variations in severely calcified carotid plaques by whole-exome sequencing

Abstract

Background: The precise mechanisms of carotid calcification and its clinical significance have not been established.

Methods: We classified ten plaques from carotid endarterectomy patients into high- and low-calcified plaques based on the Agatston calcium scores. We performed whole-exome sequencing for genetic profiles with single nucleotide variations (SNVs), insertions, and deletions. Bioinformatic data mining was then conducted to disclose specific gene variations to either high- or low-calcified carotid plaques.

Results: In the carotid plaques, G:C>A:T/C:G>T:A transitions as SNVs, insT after C/insC after A as insertions, and delA after G/delT after C as deletions were most frequently observed, but no significant difference was observed between the high- and low-calcified plaque groups in their proportion of base-pair substitution types. In the bioinformatic analysis, SNVs of ATP binding cassette subfamily C member 6 (ADCC6) were more commonly found in high-calcified plaques and SNVs of KLKB1 were more commonly found in low-calcified plaques compared to the other group. No new genetic variants related to calcification or atherosclerosis among those not registered in dbSNP was detected.

Conclusion: Our findings clarified the features of base-pair substitutions in carotid plaques, showing no relation to calcification. However, genetic variants in ADCC6 relating to vascular calcification for high-calcified plaques, and in KLKB1 encoding kallikrein associated with vascular regulation of atherosclerosis for low-calcified plaques were more specifically extracted. These results contribute to a better understanding of the genetic basis of molecular activity and calcium formation in carotid plaques.

Keywords: Calcification; Carotid endarterectomy; Carotid plaque; Single nucleotide variation; Whole exome sequencing.

Figure 1:

The proportions of the identified principal variants in the targeted exome region. Synonymous and missense variants represented the most common types of variants in all specimens. Similar proportions of variants were observed in the high- and low- calcified plaque groups. High: high-calcified plaques, Low: low- calcified plaques.

Figure 2:

(a) The percentages of single nucleotide changes in individual plaques demonstrated no remarkable difference in each specimen for base-pair substitution. (b) The percentages of insertions in individual plaques demonstrated no remarkable difference in each specimen for base-pair substitution. (c) The percentages of deletions in individual plaques demonstrated no remarkable difference in each specimen for base-pair substitution. H1–5: high-calcified plaques, L1–5: low-calcified plaques.

Figure 3:

(a) Details of the alterations of single nucleotides. Regarding SNVs, G:C>A:T and C:G>T:A transitions were the most frequent followed by T:A>A:T transversion and A:T>G:C transition. No significant differences in nucleotide changes between the high- and low- calcified plaque groups were apparent for the SNVs, except for a slight difference in A:T>G:C transversion. (b) Details of the alterations of single nucleotides. The most frequent insertions were insT after C and insC after A, followed by insA after T, insT after A, insG after A, and insT after G. No significant differences in nucleotide changes between the high- and low-calcified plaque groups were apparent for the ins. (c) Details of the alterations of single nucleotides. Among the deletions, delA after G and delT after C were the most common followed by delA after T and delA after C. No significant differences in nucleotide changes between the high- and low-calcified plaque groups were apparent for the deals. High: high-calcified plaques, low: low-calcified plaques.