Rare and potentially pathogenic variants in hydroxycarboxylic acid receptor genes identified in breast cancer cases

Abstract

Background: Three genes clustered together on chromosome 12 comprise a group of hydroxycarboxylic acid receptors (HCARs): HCAR1, HCAR2, and HCAR3. These paralogous genes encode different G-protein coupled receptors responsible for detecting glycolytic metabolites and controlling fatty acid oxidation. Though better known for regulating lipid metabolism in adipocytes, more recently, HCARs have been functionally associated with breast cancer proliferation/survival; HCAR2 has been described as a tumor suppressor and HCAR1 and HCAR3 as oncogenes. Thus, we sought to identify germline variants in HCAR1, HCAR2, and HCAR3 that could potentially be associated with breast cancer risk.

Methods: Two different cohorts of breast cancer cases were investigated, the Alabama Hereditary Cancer Cohort and The Cancer Genome Atlas, which were analyzed through nested PCRs/Sanger sequencing and whole-exome sequencing, respectively. All datasets were screened for rare, non-synonymous coding variants.

Results: Variants were identified in both breast cancer cohorts, some of which appeared to be associated with breast cancer BC risk, including HCAR1 c.58C > G (p.P20A), HCAR2 c.424C > T (p.R142W), HCAR2 c.517_518delinsAC (p.G173T), HCAR2 c.1036A > G (p.M346V), HCAR2 c.1086_1090del (p.P363Nfs*26), HCAR3 c.560G > A (p.R187Q), and HCAR3 c.1117delC (p.Q373Kfs*82). Additionally, HCAR2 c.515C > T (p.S172L), a previously identified loss-of-function variant, was identified.

Conclusions: Due to the important role of HCARs in breast cancer, it is vital to understand how these genetic variants play a role in breast cancer risk and proliferation and their consequences on treatment strategies. Additional studies will be needed to validate these findings. Nevertheless, the identification of these potentially pathogenic variants supports the need to investigate their functional consequences.

Keywords: And protein elongation; Breast cancer; G-protein coupled receptor; Genetic variants; Hydroxycarboxylic acid receptor.

Fig. 1

Rare missense variants in HCAR1 and HCAR3 detected in the AHCC. A Sanger sequencing comparisons of wildtype and mutant for HCAR1 variants p.P20A and p.L241F, and HCAR3 variant p.R187Q. B WebLogo amino acid conservation. The y-axis shows relative sequence conservation and the height of symbols in the stack show relative frequency for that position (HCAR1 was aligned with the following orthologues: Homo sapiens (Q9BXC0), Mus musculus (Q8C131), Canis lupus familiaris (B9UM26), Pan troglodytes (G2HJ56), Pongo pygmaeus (A0A4Y1JWL9), and Pan paniscus (A0A2R8ZEB9); HCAR3 was aligned with: Homo sapiens (P49019), Xenopus laevis (A0A1L8I0Z3), Pan troglodytes (H2RAM9), Pongo abelii (H2NJ01), Pan paniscus (A0A4Y1JWN7), and Pongo pygmaeus (A0A4Y1JWR4))

Fig. 2

Topological illustration of HCAR1, HCAR2 and HCAR3 highlighting missense variants identified in the AHCC, TCGA, and previous functional studies. A HCAR1 (Q9BXC0); red: HCAR1 p.P20A detected in a BC case from the AHCC; orange: HCAR1 missense variants detected in TCGA BC cases, including p.Y74C, p.D112N, p.S172L, p.H257R; light blue/silver: p.L241F detected in both TCGA and AHCC BC cases; yellow: HCAR1 transmembrane domain variants, p.R99A, p.Y233A, p.R240A, and p.T267A, reported by Liu et al. to diminish the response to L-lactate [33]; green: HCAR1 residue R71 and motif C165-E166-S167-F168 identified by Kuei et al. as being critical for protein function [31]; blue: HCAR1 extracellular Cys residues, C6, 7, 88, 157, 165 (green) and 252, reported by Kuei et al. to abolish receptor activity when substituted with alanine or serine [31]; purple: HCAR1 variants p.A110V, p.S172L (covered by orange since it overlaps with variant detected in TCGA), and p.D253H identified by Doyle et al. as loss-of-function variants [32]. B HCAR2 (Q8TDS4); orange: HCAR2 missense variants detected in TCGA BC cases, including TCGA variants p.L11P, p.R142W, p.M167L, p.P168L, p.G173T, p.R311H, p.M346V, and p.G350S; yellow: N86/W91, R111, S178, F276, and Y284 were reported by Tunaru et al. as critical for binding of nicotinic acid [29]; green: Yasuda et al. identified an atypical motif N17-C18-C19 that is crucial for surface trafficking. They also identified C100, C177, C183, and C266, in the extracellular regions, which are important for HCAR2 activation [30]; blue: Li et al. identified a sequence of residues from 329–343 that plays a crucial role in keeping HCAR2 in an inactive conformation [28]. C HCAR3 (P49019); red: HCAR3 p.R187Q identified in a BC case in the AHCC; orange: HCAR3 missense variants detected in TCGA BC cases, including p.R3W, p.A27V, and p.R216W

Fig. 3

HCAR3 frameshift mutation, c.1117delC (p.Q373Kfs*82). A Sanger sequencing comparisons of wildtype and mutant, illustrating the deletion of the cytosine. B Mutalyzer comparison of the HCAR3 wildtype and mutant proteins, highlighting the different C-termini in red. C Phyre2 protein prediction software displaying secondary structure differences between wildtype and mutant. D I-TASSER protein prediction comparisons of the wildtype and mutant protein (each in two different views) to show the C-terminus extension and difference in tertiary structures; PyMOL was used to display the molecular graphics

Fig. 4

HCAR2 frameshift mutation, c. 1086_1090del (p.P363Nfs*26). A IGV screenshot (with sequences in the reverse complement) and HCAR2/3 sequence alignment displaying the locations of the mutation (yellow) and an additional HCAR2/3 sequence difference (red). B Mutalyzer comparison of the HCAR2 wildtype, HCAR2 mutant, and HCAR3 wildtype proteins, highlighting the different C-termini in red. C I-TASSER protein prediction comparisons of the HCAR2 wildtype, HCAR2 mutant, and HCAR3 wildtype proteins