Whole-exome sequencing of Nigerian benign prostatic hyperplasia reveals increased alterations in apoptotic pathways

Abstract

Background: Through whole-exome sequencing of 60 formalin-fixed paraffin-embedded Nigerian (NGRn) benign prostatic hyperplasia (BPH) samples, we identified germline and somatic alterations in apoptotic pathways impacting BPH development and progression. Prostate enlargement is a common occurrence in male aging; however, this enlargement can lead to lower urinary tract symptoms that negatively impact quality of life. This impact is disproportionately present in men of African ancestry. BPH pathophysiology is poorly understood and studies examining non-European populations are lacking.

Methods: In this study, NGRn BPH, normal prostate, and prostate cancer (PCa) tumor samples were sequenced and compared to characterize genetic alterations in NGRn BPH.

Results: Two hundred and two nonbenign, ClinVar-annotated germline variants were present in NGRn BPH samples. Six genes [BRCA1 (92%), HSD3B1 (85%), TP53 (37%), PMS2 (23%), BARD1 (20%), and BRCA2 (17%)] were altered in at least 10% of samples; however, compared to NGRn normal and tumor, the frequency of alterations in BPH samples showed no significant differences at the gene or variant level. BRCA2_rs11571831 and TP53_rs1042522 germline alterations had a statistically significant co-occurrence interaction in BPH samples. In at least two BPH samples, 173 genes harbored somatic variants known to be clinically actionable. Three genes (COL18A1, KIF16B, and LRP1) showed a statistically significant (p < 0.05) higher frequency in BPH. NGRn BPH also had five gene pairs (PKD1/KIAA0100, PKHD1/PKD1, DNAH9/LRP1B, NWD1/DCHS2, and TCERG1/LMTK2) with statistically significant co-occurring interactions. Two hundred and seventy-nine genes contained novel somatic variants in NGRn BPH. Three genes (CABP1, FKBP1C, and RP11-595B24.2) had a statistically significant (p < 0.05) higher alteration frequency in NGRn BPH and three were significantly higher in NGRn tumor (CACNA1A, DMKN, and CACNA2D2). Pairwise Fisher's exact tests showed 14 gene pairs with statistically significant (p < 0.05) interactions and four interactions approaching significance (p < 0.10). Mutational patterns in NGRn BPH were similar to COSMIC (Catalog of Somatic Mutations in Cancer) signatures associated with aging and dysfunctional DNA damage repair.

Conclusions: NGRn BPH contained significant germline alteration interactions (BRCA2_rs11571831 and TP53_rs1042522) and increased somatic alteration frequencies (LMTK2, LRP1, COL18A1, CABP1, and FKBP1C) that impact apoptosis. Normal prostate development is maintained by balancing apoptotic and proliferative activity. Dysfunction in either mechanism can lead to abnormal prostate growth. This work is the first to examine genomic sequencing in NGRn BPH and provides data that fill known gaps in the understanding BPH and how it impacts men of African ancestry.

Keywords: African Ancestry; apoptosis; benign prostatic hyperplasia; germline variants; somatic variants; whole-exome sequencing.

Figure 1:. BPH and Normal Germline Variant Oncoplot.

Germline variants detected across 60 BPH and 11 unmatched normal samples were filtered against a list of non-benign variants catalogued by ClinVar. In at least two samples, 13 genes harboured variants. The top five most frequently mutated genes in BPH were BRCA1 (BRCA1 DNA Repair Associated) – 92%, HSD3B1 (Hydroxy-Delta-5-Steroid Dehydrogenase, 3 Beta- And Steroid Delta-lsomerase 1) – 85%, TP53 – 37%, PMS2 (PMS1 Homolog 2, Mismatch Repair System Component) – 23% and BARD1 (BRCA1 Associated RING Domain 1) – 20%. BPH – Benign Prostatic Hyperplasia

Figure 2:. Gene and Variant Level Germline Mutation Frequencies.

Gene and variant level germline mutation frequencies were compared between NGRn cohorts, unmatched normal (n = 11), BPH (n = 60) and PCa tumor (n = 45) samples. No significant differences in frequencies were detected at the, A) gene or B) variant level. NGRn – Nigerian, BPH – Benign Prostatic Hyperplasia, PCa – Prostate Cancer

Figure 3:. BPH Germline Gene and Variant Interactions.

Fisher exact tests were used to determine gene and variant pair interactions within BPH samples. A) TP53 and BRCA2 showed a statistically significant (p = 0.028) co-occurrence interaction with an odds ratio of 5.27. Six gene pairs showed interactions approaching significance (p <0.1). B) Nine variant pairs showed statistically significant (p < 0.05) interactions and 9 had interactions approaching (p ≤ 0.1) significance. The variant pairs with the most statistically significant interactions were, 1) TP53_rs 1800371 and BRIP1_rs145855459, 2) PALB2_rs57605939 and BRCA1_rs16942 and 3) BRCA1_rs16942 and BRCA1_rs16941. BPH – Benign Prostatic Hyperplasia

Figure 4:. BPH and Tumor Known Somatic Variant Oncoplot.

60 NGRn BPH and 45 NG Tumor were analyzed for somatic variants. Variants were filtered against a list of NG normal and non-benign ClinVar variants. In at least two BPH samples, 173 genes harbored variants. The top five most frequently mutated genes were MUC17 (Mucin 17 Cell Surface Associated) − 25%, DNAH9 (Dynein Axonemal Heavy Chain 9) − 22%, LMTK2 (Lemur Tyrosine Kinase 2) – 22%, DCHS2 (Dachsous Cadherin-Related 2) − 18% and TCERG1 (Transcription Elongation Regulator 1) − 18%. NGRn − Nigerian, BPH − Benign Prostatic Hyperplasia

Figure 5:. NGRn BPH Gene Interactions in Known PCa Associated Genes.

Within BPH samples, genes containing clinically actionable somatic variants were analyzed for the presence of gene pairs with co-occurring or mutually exclusive interactions. Pairwise Fisher’s exact tests showed five gene pairs with statistically significant (p < 0.05) co-occurring interactions and four interactions (three co-occurring and one mutually exclusive) approaching significance (p < 0.10). NGRn – Nigerian, BPH – Benign Prostatic Hyperplasia, PCa – Prostate Cancer

Figure 6:. BPH and Tumor Novel Somatic Variant Oncoplot.

60 NGRn BPH and 45 NGRn Tumor samples were analyzed for somatic variants not known to be clinically actionable. In at least two BPH samples, 279 genes harbored novel variants. The top five most frequently mutated genes were MUC19 (Mucin 19, Oligomeric) – 40%, FRG1B (LOC102724813) – 22%, RUNX2 (RUNX Family Transcription Factor 2) – 18%, PCDHA6 (Protocadherin Alpha 6) – 18% and DENND4B (DENN Domain Containing 4B) – 17%. NGRn – Nigerian, BPH – Benign Prostatic Hyperplasia

Figure 7:. NGRn BPH Gene Interactions in Novel PCa Associated Genes.

Within NGRn BPH samples, genes not known to contain clinically actionable somatic variants were analyzed for the presence of gene pairs with co-occurring or mutually exclusive interactions. Pairwise Fisher’s exact tests showed fourteen gene pairs with statistically significant (p < 0.05) interactions and four interactions approaching significance (p < 0.10). SORCS2/MUC19 was the only interaction identified as mutually exclusive. NGRn – Nigerian, BPH – Benign Prostatic Hyperplasia, PCa – Prostate Cancer

Figure 8:. NGRn BPH COSMIC Gene Signature.

Single nucleotide variations in the Nigerian BPH cohort were compared to known cancer related mutation signatures within the Catalogue of Somatic Mutations in Cancer (COSMIC). A) Nigerian BPH mutation patterns are similar to signatures 1 (cosign similarity ≥ 0.862) and 6 (cosign similarity ≥ 0.644). Both signatures are signatures are characterized by C>T substitutions at NpCpG trinucleotides and are associated with aging and dysfunctional DNA damage repair. B) 34 samples were composed predominantly of substitutions associated with signature 1. The remaining samples were predominantly signature 6. NGRn – Nigerian, BPH – Benign Prostatic Hyperplasia