Impact of BRCA mutations, age, surgical indication, and hormone status on the molecular phenotype of the human Fallopian tube

Abstract

The human Fallopian tube (FT) is an important organ in the female reproductive system and has been implicated as a site of origin for pelvic serous cancers, including high-grade serous tubo-ovarian carcinoma (HGSC). We have generated comprehensive whole-genome bisulfite sequencing, RNA-seq, and proteomic data of over 100 human FTs, with detailed clinical covariate annotations. Our results challenge existing paradigms that extensive epigenetic, transcriptomic and proteomic alterations exist in the FTs from women carrying heterozygous germline BRCA1/2 pathogenic variants. We find minimal differences between BRCA1/2 carriers and non-carriers prior to loss of heterozygosity. Covariates such as age and surgical indication can confound BRCA1/2-related differences reported in the literature, mainly through their impact on cell composition. We systematically document and highlight the degree of variations across normal human FT, defining five groups capturing major cellular and molecular changes across various reproductive stages, pregnancy, and aging. We are able to associate gene, protein, and epigenetic changes with these and other clinical covariates, but not heterozygous BRCA1/2 mutation status. This sheds new light into prevention and early detection of tumorigenesis in populations at high-risk for ovarian cancer.

Fig. 1. Overview of the study design and sample cohort.

a Left: a total of 125 benign Fallopian tubes were collected: control FTs with no known pathogenic mutations (non-BRCAm; N = 70), pathogenic germline BRCA1 mutation carriers (BRCAm; N = 28), and pathogenic germline BRCA2 carriers (N = 27). Middle: Some sections from fimbria were imaged following H&E staining. Right: DNA, RNA, and protein extraction from FFPE to generate whole-genome bisulfite (WGBS), mRNA, and protein quantification matrices. b Distribution of 105 FTs which have data for one or more of the -omics assays. Sample number is shown by BRCA1/2 germline status colored by menopause status. From these 105 FTs, 92 have high quality data for all three assays. c Age distribution at the time of surgery was not different by BRCA status (F test P value = 0.54 from ANOVA model; BRCA1m n = 28; BRCA2m n = 27; non-BRCAm n = 70). d Distribution of postpartum samples and self-reported race by BRCA status; Black FTs were overrepresented in non-BRCAm and postpartum FTs were present exclusively in the non-BRCAm group. e, f Pathogenic germline mutations in our cohort were validated in the WGBS data. Mutations in BRCA1 included 11 frameshift, five missense, one splice site, six nonsense, and one large deletion. Mutations in BRCA2 included 13 frameshift, one missense, one splice site, and four nonsense mutations. The specific types of mutations were not known for one BRCA1 and one BRCA2 patient, and one BRCA1 sample had a large deletion spanning multiple exons; mutations for these 3 samples are not shown. Portions of this figure were created in BioRender. Beddows, I. (2025) https://BioRender.com/r66m378.

Fig. 2. DNA methylation profiles of the cohort.

a Region-centered binned averages show high DNA methylation levels for heterochromatin (top row), lack of methylation at transcriptional start sites (TSS; middle row), and nucleosome positioning around CTCF binding sites (bottom row). No difference in methylation by BRCA status at these features was detected. b Multiscale DNA methylation averages along chromosome 17 from 10 kb to 10 Mb showing no BRCA-associated differences in methylation at any of these scales. c Heatmap showing DNA methylation levels at loci known to be hypermethylated in STIC or HGSC (rows) for samples of different origins (columns). Regardless of study origin and BRCA status, benign FTs do not show gain of methylation at these loci.

Fig. 3. Differential gene and protein expression between non-BRCAm and BRCAm FTs.

a Volcano plot for all genes between BRCAm and non-BRCAm adjusting for stromal fraction and excluding postpartum samples. X-axis shows the fold change and y-axis shows the −log10(P value) for each gene from quasi-likelihood F tests. b As in (a) but for proteomics data with P values from moderated t tests. c Heatmap of 117 of 159 differentially expressed genes shown in (a) comparing non-BRCAm and BRCAm. Not shown are 42 genes with less than one counts per million (CPM) in 95% of samples. Supplementary Fig. 5a shows all 159 DEGs and their average log CPM values. Rows are grouped by up in non-BRCAm (top) or up in non-BRCAm (bottom); d Gene ontology (GO) enrichment for genes up in non-BRCAm showing the top 10 enriched terms all of which are related to immunoglobin mediated immunity. P values are from Fisher exact tests. e GO enrichment terms which are up in BRCAm shown in (c) showing relatively lower enrichment by P value for terms mostly involving core metabolism. P values from Fisher exact test. f Twenty-five protein products from DEGs were identified showing immune hot samples but otherwise few consistent changes. g Boxplot of BRCA1 expression by BRCA1/2 mutation status showing no difference between groups (BRCA1 n = 22; BRCA1 n = 19; non-BRCAm n = 53). Y-axis is log2(CPM). h as in (g) but for BRCA2 expression. P values derived from F test conducted with ANOVA models.

Fig. 4. Cellular landscapes of normal human Fallopian tube samples.

a Gene expression heatmap for known markers of various cell types present in the FT (Supplementary Data 4). Rows represent individual markers and columns represent individual FTs; markers are grouped by their cell type, and samples are clustered freely within premenopause, postmenopause or postpartum. A luteal, follicular, and inactive/stroma-rich group within the premenopause samples consistent with molecular and clinical data are indicated by arrows pointing to the root node. b Heatmap of an expanded panel of secretory epithelial cell markers for normal FT (this study, right) and normal endometrium (GSE132711, left). From these markers and those in (a), there is a visible high stroma subgroup within the premenopausal FTs (root node designated with an arrow) that contains most of the inactive endometrium samples. This high stroma subgroup is associated with relatively lower secretory cell marker expression than other premenopause samples. The remaining epithelium-high premenopausal FT samples showed gene expression changes similar to endometrium (i.e., ESR1 and PGR), and were divided into a proliferative and a secretory cluster (root node designated with an arrow). These patterns of expression corresponded to the pathology-determined endometrium state from sample-matched endometrium. Postmenopause and postpartum samples do not show menstrual cycling but do have distinct patterns of secretory epithelial marker expression.

Fig. 5. Five molecular states of normal human Fallopian tube.

a FTs were imaged following H&E staining for all samples where available to examine cell type composition. H&E slides for five representative samples from the five expression groups identified in Fig. 4 are shown. Left panels show a cross-sectional cut of a representative distal FT (scale bar 500 μm) and right panels are a zoom in on epithelial, stromal, and immune cells within these same slides (scale bars represent 50 µm). b, c Marker genes for the five expression groups were identified independently in the RNA and protein. Features with both a 1:1 match in the RNA and protein and FDR < 0.05 for the same marker cluster in both assays are shown for gene expression (b) and protein expression (c). These markers are grouped by the intersect of which sample group they mark (Supplementary Fig. 9a, right). Samples and features are clustered based on their RNA expression in (b), and this same order is used for the corresponding protein products (c).

Fig. 6. Aging in the normal Fallopian tube.

a Clinical age at time of salpingectomy (x-axis) correlates with predicted age from the Horvath methylation clock. A linear line of best fit is shown as a black line; shading around the best fit line indicates the 95% confidence interval as done with ggplot2 function geom_smooth(method = “lm”). A Spearman rank correlation coefficient was found to be significant with p = 1.2e-9. b Differences between predicted and clinical age (as indication of accelerated or decelerated aging) are not different by BRCA status (F test p = 0.57 from ANOVA model; BRCA1m n = 25; BRCA2m n = 19; non-BRCAm n = 59). The y-axis is the difference between predicted and clinical age with horizontal lines representing the first quartile, median, and third quartile of each x-axis group. c DNA methylation at solo-WCGWs, which lose methylation during aging, are not different by BRCA1/2 status F test p = 0.12 from ANOVA model; BRCA1m n = 25; BRCA2m n = 19; non-BRCAm n = 59). Horizontal lines representing the first quartile, median, and third quartile. d Average DNA methylation at ERalpha binding sites from JASPAR database (y-axis) positively correlates with stroma fraction (x-axis) as determined by Spearman rank correlation (p = 2.3e-22; rho = 0.78). LOESS smoothed lines are displayed for each of the reproductive status groups with the 95% confidence interval represented by the shaded region e. When restricted to samples with more than 70% of epithelium, solo-WCGW methylation is inversely associated with age at time of sampling, as expected for a more homogeneous population. Spearman rank correlation coefficient rho was −1 with p = 0.017. f Multi-scale plot showing DNA methylation profile at solo-WCGWs for the petit arm of chromosome 6 for two samples with high epithelium and different age (20 years and 39 years) and the oldest sample with high stromal content. Mega-base DNA methylation loss is associated with age in the epithelium in normal human FT, but not in the stroma compartment.