Abstract 256: Sequence conservation of BRCA1 intron-exon junctions
Authors: Ishfaq Talia , Salik Maryam , Jafry Uzma , Ahmad Zaain , Fackenthal James D. 
Published: July 2021

Abstract
Introduction: Clinical sequencing of BRCA1 and BRCA2 exons and intron-exon boundaries reveals sequence variants of uncertain clinical significance (VUSs). Many BRCA1/2 sequence variants near intron-exon boundaries are potentially pathogenic by disrupting normal splicing patterns. However, molecular analysis of these potential effects is complicated by naturally occurring alternative splicing products. It is not yet clear which of these are required for normal gene functions. We therefore aligned intron-exon boundary sequence strings for 21 BRCA1 exons in order to 1) determine whether VUSs near intron-exon boundaries disrupt conserved sequences, and 2) determine whether sequences flanking exons involved in alternative splicing events are sufficiently conserved.
Methods: Sequence strings representing intron-exon junction regions were prepared for 21 BRCA1 exons. Each string consisted of 20 bases of 5' intron, the 5'-most 20 bases of each exon, the 3'-most 20 bases of each exon, and 20 bases of the 3' intron. Strings were similarly prepared for chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo abelii), baboon (Papio anubis), and Rhesus macaque (Macaca mulatta). Each string was aligned using the MUSCLE multiple sequence alignment method.
Results: Splice junction sequence conservation was from 68%-100% conserved (mean % conservation, 92.7%; median, 95%). In almost all cases, sequence differences were transitions or differing numbers of thymines in poly-T tracts. The two most divergent intron-exon junction regions flanked exons 17 and 18 (70% and 68% similar, respectively). The human exon 16-17 junction is not conserved in in gorilla, and the human exon 18-19 junction is not conserved in macaques. These differences account for the divergence among primates at these sites. Seven alternative splicing events in human BRCA1 are classified as “predominant,” and all are predicted to retain the translational reading frame. All of these alternative splicing events occur in nearly perfectly conserved splicing environments, suggesting they may occur in other species as well and possibly contribute to gene function. Seven clinically identified VUSs that fall within the human BRCA1 splice junction region are wild type base differences in other primates. c.231G&gt;A from human exon 5 is seen in baboon, c.441+18C&gt;T in human intron 6 is seen in orangutan, c.455T&gt;C in exon 7 is seen in orangutan, c.547+7G&gt;A in intron 7 is seen in macaque and baboon, and c.4097-10G&gt;A, c.4097-20C&gt;T, and c.4115G&gt;A, all in or near exon 11, are seen in macaque, baboon, and orangutan. These observations suggest these VUSs are likely to be benign polymorphisms and not pathogenic variants. We note, however, that c.547+7G&gt;A disrupts a candidate intron splicing enhancer as predicted by RegRNA 2.0 and believe this variant warrants further study.
Citation Format: Talia Ishfaq, Maryam Salik, Uzma Jafry, Zaain Ahmad, James D. Fackenthal. Sequence conservation of BRCA1 intron-exon junctions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 256.