ANO7 ancestral diversity
A total of 809 germline variants were reported within the ANO7 gene region for 166 genomes, with as expected (ref. 17,30), greater numbers observed for Africans over Europeans (median 125 vs 110). Exhibiting 45 unique amino acid sequences of ANO7 containing germline missenses, with median pairwise genetic distance 0.003 (range, 0.001 to 0.005). The number of African-specific sequences were more than twice that of the Europeans (29 vs 12), with African patients exhibiting three times as many individually unique African-specific sequences as Europeans (21 vs 7). Phylogenetic analysis divided the sequences into eight groups (Fig. 1) including three African-specific (Groups B, D and F) and one European-specific (Group A).
ANO7 germline predicted deleterious variants (PDVs)
Of the 13 germline variants annotated as PDVs (Table 1, Figure S1), nine were African-specific, including p.Leu734Pro and p.Asp789Leu novel to this study, while p.Ala744Gly has previously been reported in a single East Asian. Known PCa variants included the European-exclusive PDV p.Ala759Thr (ref. 13,31,32) and the recently described African-related p.Ser914* (ref. 12). Ancestrally-shared PDVs p.Ile740Leu and p.Ser914* showed a higher prevalence in African patients (logistic regression models, p.Ile740Leu: Europeans vs Africans; odds ratio/OR = 0.04, 95% confident interval/CI = 0.005-0.30, P-value = 0.02; p.Ser914*: OR = 0.01, 95% CI = 0.002–0.09, P-value = 2.03e-05).
Common germline ANO7 variants associated with African-ancestral PCa risk
Of the 17 ANO7 variants represented within our 473 genotyped southern African PCa cases and 307 cancer-free controls, 12 variants (9 PDVs) overlapped with our sequencing data (Table 1, Figure S2). While PDV p.Ile740Leu was associated with PCa risk (Wilcoxon test, FDR = 0.03), the European-derived PCa risk variant p.Ala759Thr was rare in our study with no associated risk.
Intercorrelation of germline ANO7 variants
We investigated the correlation between germline ANO7 PDVs or SVs, with known PCa risk variants (Spearman’s test, FDRs = 0 to 2.842E-03, Table 2). Correlations identified exclusively in African patients involved four germline SVs while the correlation specific to European patients involve a PCa risk variant rs62187431, the latter also correlated with two germline SVs exclusively in the African patients (Table S2). Ancestrally shared PDV correlated pairs included; p.Ile740Leu and p.Ser914*, cooccurring in 29 Africans and a single European, and p.Asp789Leu and p.Asp789Val, cooccurring in five African patients and which together truncates the Anoctamin 7 protein by 100 amino acids. As the less frequent variant only occurred in a subject when the more frequent variant was present, most were defined as inclusive correlated (IC) pairs (Figure S3). The IC pair p.Ala759Thr and rs62187431 whose linkage disequilibrium (LD) was also reported in Asian patients(ref. 15) were in the same haplotype block with strong LD in Europeans patients.
ANO7 germline PDVs linked with early-onset PCa
Correlating PCa measurements and ANO7 variants using linear regression analyses with adjustments for the number of small germline variants and PCa risk levels, we found African patients to present six years earlier at diagnosis (-6.42 years, 95% CI=-10.68 to -2.16, P-value = 0.003) if carrying three or more selected germline variants (PDVs and/or SVs), regardless of zygosity (Figure S4). Of these 15 African patients, 14 presented with at least two germline PDVs and one with two germline SVs, suggesting an accumulation of inherited PCa risk from selected ANO7 variants. While none of our European patients presented with greater than two selected variants, it should be noted that ANO7 rs77559646 has previously been reported to be associated with early-onset PCa in a Finnish study (ref. 11).
ANO7 as a putative cancer driver
Identified somatic variants, 23 small variants and two inter-chromosomal fusion SVs, were biased towards high-risk tumours (Gleason score \(\ge\)4+3) derived from patients of predominantly African ancestry (8 out of 9). An African missense p.Phe79Leu (rs1217170132) was annotated with a deleterious impact on an alternative transcript (ENST00000451047), which has not been reported in population data or previous studies. Additionally, and novel to this study, we found ANO7 to act as a fusion partner for oncogenic genes G3BP Stress Granule Assembly Factor 1 (G3BP1) at 5q33.1 (ref. 33–35) and PTPRF interacting protein alpha 4 (PPFIA4) at 1q32.1 (ref. 36–38)
Impact of ANO7 PDVs on protein structure
Of the 13 PDVs identified in WGS (Table 1), 11 clustered in the Ca2+-activated chloride channel (Figure S1), while a single PDV was located in an anoctamin dimer region. Two clustering of PDVs in proximity with distances < 13Å were predicted in the tertiary structure (Fig. 2, Figure S5), with one clustering, p.Ile740Leu and p.Ala744Gly, located adjacent to putative Ca2+-binding sites originally identified in Anoctamin 1 protein (ref. 39).
Two potential ion conduction pores were identified in the transmembrane domains (TMDs) and pass through putative Ca2+-binding sites (Fig. 3a). The two pores were close among helices α5–8 at the end connecting to the cytoplasm and apart at the other end where Pore 1 was circled by helices α5–9 (Figure S6), while Pore 2 was tilted with helices α4–6 surrounded (Fig. 3b), similar placement of ion conduction of Anoctamin 1 protein (ref. 39). Their properties were similar that were hydrophobic and less ionisable in the central part near the Ca2+-binding sites and with radii larger than 1Å within TMDs (bottlenecks, Pores 1 = 1.6Å, Pores 2 = 1.3Å). The result of pore identification changed with presence of PDVs, which may hinder the movement of ions and affect the interaction between Ca2+ and Anoctamin 7 protein. For a protein with p.Ala470Val and p.Ile740Leu, Pore 1 was not identified, while Pore 2 was identified as narrower with 0.4Å bottleneck radius and was less hydrophilic and ionisable at the centre of the pore above the bottleneck (Fig. 3c). The pore alteration could be the consequence of positional changes of residues 673–693 (Figure S7), which were also observed when other PDVs were present (Figure S8).