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. 2024 Jun 20;25(12):6773.
doi: 10.3390/ijms25126773.

A Molecular Characterization of the Allelic Expression of the BRCA1 Founder Δ9-12 Pathogenic Variant and Its Potential Clinical Relevance in Hereditary Cancer

Julieta Dominguez-Ortiz  1   2   3 , Rosa M Álvarez-Gómez  3 , Rogelio Montiel-Manríquez  1 , Alberto Cedro-Tanda  4 , Nicolás Alcaraz  5 , Clementina Castro-Hernández  1 , Luis Bautista-Hinojosa  1 , Laura Contreras-Espinosa  1 , Leda Torres-Maldonado  6 , Verónica Fragoso-Ontiveros  3 , Yuliana Sánchez-Contreras  3 , Rodrigo González-Barrios  1 , Marcela Angélica De la Fuente-Hernández  3 , María de la Luz Mejía-Aguayo  3 , Ulises Juárez-Figueroa  6 , Alejandra Padua-Bracho  3 , Rodrigo Sosa-León  3 , Gabriela Obregon-Serrano  3 , Silvia Vidal-Millán  3 , Paulina María Núñez-Martínez  3 , Abraham Pedroza-Torres  3 , Sergio Nicasio-Arzeta  7 , Alfredo Rodríguez  6   8 , Fernando Luna  1 , Fernanda Cisneros-Soberanis  9 , Sara Frías  6   8 , Cristian Arriaga-Canon  1   10 , Luis A Herrera-Montalvo  1   10
Affiliations

A Molecular Characterization of the Allelic Expression of the BRCA1 Founder Δ9-12 Pathogenic Variant and Its Potential Clinical Relevance in Hereditary Cancer

Julieta Dominguez-Ortiz et al. Int J Mol Sci. .

Abstract

Hereditary breast and ovarian cancer (HBOC) syndrome is a genetic condition that increases the risk of breast cancer by 80% and that of ovarian cancer by 40%. The most common pathogenic variants (PVs) causing HBOC occur in the BRCA1 gene, with more than 3850 reported mutations in the gene sequence. The prevalence of specific PVs in BRCA1 has increased across populations due to the effect of founder mutations. Therefore, when a founder mutation is identified, it becomes key to improving cancer risk characterization and effective screening protocols. The only founder mutation described in the Mexican population is the deletion of exons 9 to 12 of BRCA1 (BRCA1Δ9-12), and its description focuses on the gene sequence, but no transcription profiles have been generated for individuals who carry this gene. In this study, we describe the transcription profiles of cancer patients and healthy individuals who were heterozygous for PV BRCA1Δ9-12 by analyzing the differential expression of both alleles compared with the homozygous BRCA1 control group using RT-qPCR, and we describe the isoforms produced by the BRCA1 wild-type and BRCA1Δ9-12 alleles using nanopore long-sequencing. Using the Kruskal-Wallis test, our results showed a similar transcript expression of the wild-type allele between the healthy heterozygous group and the homozygous BRCA1 control group. An association between the recurrence and increased expression of both alleles in HBOC patients was also observed. An analysis of the sequences indicated four wild-type isoforms with diagnostic potential for discerning individuals who carry the PV BRCA1Δ9-12 and identifying which of them has developed cancer.

Keywords: BRCA1; BRCA1Δ9–12; allele differential expression; founder mutation; hereditary breast and ovarian cancer; isoform; nanopore sequencing; pathogenic variants.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Scheme 1
Scheme 1
Schematic description of experimental design. (A) Total RNA was extracted from peripheral blood lymphocytes obtained from 20 healthy individuals who were homozygous for the BRCA1WT allele, corresponding to the control group (C); 10 healthy individuals who were heterozygous for the BRCA1Δ9–12 variant (HH); and 10 cancer patients who were heterozygous for the BRCA1Δ9–12 variant (CaH), the last two as study groups. (B) A gene expression analysis between alleles in the three groups was performed with a real-time PCR (RT–qPCR) and ΔCT analysis. (C) A transcription variant analysis of the alleles of the three groups was performed using the Oxford Nanopore target sequencing. Each individual was marked with a barcode, allowing for tracking in each sample. (D) Isoform profiling of the expression pattern in the studied groups.
Figure 1
Figure 1
The expression of the BRCA1WT allele is increased in comparison to that of the BRCA1Δ9–12 allele in healthy and cancer-heterozygous patients. (A) A depiction of the 24 exons composing the BRCA1WT transcript (green). Primers for the amplification of the wild-type product recognize the junction of exons 6 and 7 (forward) and exon 10 (reverse), as depicted with yellow squares. RT–PCR produced a 210 bp amplification product of the wild-type transcript. (B) The deletion of exons 9–12 of the BRCA1 gene (green) leads to a mutant BRCA1Δ9–12 transcript (yellow). The primers used for amplification of the mutant transcript recognized the junction of exons 6 and 7 (forward) and the junction of exons 8 and 13 (reverse). RT–PCR produced a 127 bp amplification product of the BRCA1Δ9–12 transcript. (C) Endpoint PCR amplicons from 10 control samples (C1–C10) amplified with a mixture of one forward primer and two different reverse primers. The presence of a single 210 bp band indicates homozygosity for the BRCA1WT allele. GAPDH was used as a housekeeping control gene. (D) Endpoint PCR amplicons of samples from five patients with cancer (CA1–CA5) and five healthy heterozygotes (P1–P5) amplified with a mixture of one forward primer and two different reverse primers. The presence of two bands at 210 bp and 127 bp indicates heterozygosity for BRCA1 and amplification of the BRCA1WT and BRCA1Δ9–12 transcripts. GAPDH was used as a housekeeping control gene; GAPDH controls from CaH and HH samples were run on different gels. (E) Sanger sequencing electropherogram of a BRCA1WT control sample, corroborating the joining of exons 8 and 9 in the amplification product. (F) Sanger sequencing electropherogram of a BRCA1Δ9–12 sample, corroborating the joining of exons 8 and 13 in the amplification product. (G) A comparison of the differential expression between the BRCA1 alleles among the three groups of samples evaluated by RT–PCR and analyzed using ∆CT. There were differences between all the box data of the groups except for the BRCA1WT allele in the HH group, which showed expression levels similar to those of the homozygous control group. All bar comparisons were performed between alleles, not between study groups. The range of differences is indicated by the following values: * < 0.05, *** < 0.001. In the case of no difference, ns—not significant (p > 0.05) was used.
Figure 2
Figure 2
Differential expression of both BRCA1 transcripts is associated with recurrence in patients. The relative expression of the samples (n = 9) was assessed using the ΔCT method, and the differences between groups were evaluated using the Wilcoxon test in conjunction with a Bonferroni adjustment. All analyses were performed using R; statistical significance was set at p < 0.05. Statistically significant differences are marked with * p < 0.05. A red square indicates where an overexpression is observed. (A) Association between clinical characteristics and relative expression of BRCA1WT allele. * p = 0.032 is noted in the red square. (B) Association between clinical characteristics and relative expression of BRCA1Δ9–12 allele variant. * p = 0.016 is noted in the red square.
Figure 3
Figure 3
Complete amplification of BRCA1 variants for nanopore sequencing. (A) The location of the BRCA1WT transcript (green) and its exons are indicated by primers targeting exons 2 and 24. The BRCA1WT sequence product corresponds to 5802 bp. (B) The BRCA1Δ9–12 transcript (yellow) with the deleted exons shows the same primer localization at exons 2 and 24, with a decreased length of the amplified product of 2164 bp. (C) An endpoint PCR of the control samples (representative samples C3 and C4), healthy heterozygous samples (representative sample P6), and cancer patient samples (representative sample CA9) showing the transcripts and isoforms amplified with the primers described in A and B, with emphasis on the 5802 bp and 2164 bp amplicons. (D) Sanger sequencing corroboration of the BRCA1WT sample C3 sequence (left) from the purified band corresponding to an expected size of 5802 bp. The chromatogram of the BRCA1WT amplification showed the splicing of exons 8 and 9. Sanger sequencing of the sequences of BRCA1WT and BRCA1Δ9–12 in the sample CA9 (right) of the purified bands corresponding to expected sizes of 5802 bp and 2164 bp, confirming in the chromatograms the join of exons 8 and 9 in the BRCA1WT and exons 8 and 13 in BRCA1Δ9–12 amplicons.
Figure 4
Figure 4
Identification of differential isoform expression. The samples are color-coded as Ctrl (green), HH (yellow), and CaH (red). (A) Principal component analysis (PCA) of WT and 9–12 isoform expression between Ctr, HH, and CaH samples. The plot shows the two principal components. The Ctr samples differentiated from the HH and CaH samples on the PC1 axis with a greater variance (35.49%). (B) Principal component analysis in 3D (PCA 3D), where the samples are visualized in the plot with a third axis, PC3, where HH samples aggregate in the positive values of the axis, separating from CaH samples, which aggregate in the negative values of the axis. The P7 sample can be observed separately from the Ctr group on the PC2 axis. (C) Heatmap of dissimilarity matrix for clustering BRCA1 isoform expression. The color intensity on the map corresponds to the Euclidean distance of samples based on normalized counts of the BRCA1 isoforms, where white indicates high similarity and dark blue indicates low similarity in the isoform’s expression across individuals. (D) Heatmap of isoform z scores of the top differentially expressed isoforms between groups.
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Grants and funding

This research was funded by “Programa presupuestario Anexo 13 del Decreto de PEF: 309 Clínica de Cáncer Hereditario” at the National Cancer Institute of Mexico and Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT). Julieta Dominguez-Ortiz is a Ph.D. student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and she received fellowship 465336 from the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT), Mexico, with CVU 412880.