# RNA-seq Splicing Profile of the CDH1 Gene and Its Impact on the Clinical Pathogenicity Classification of CDH1 Variants: A Description of Alternative and Pathogenic Splicing Patterns

## TL;DR

This study explores how the CDH1 gene is spliced in different ways and how these splicing patterns can help classify CDH1 gene variants as pathogenic or not.

## Contribution

The study identifies novel CDH1 isoforms and provides a framework for interpreting splicing variants in clinical practice.

## Key findings

- Eleven alternative splicing events were identified, including novel isoforms from intron 2.
- A splicing variant skipping exon 11 leads to a disrupted reading frame and high expression in negative CDH1 controls.
- Four distinct transcripts from a duplication event were identified, leading to truncated proteins and pathogenic classification.

## Abstract

The CDH1 gene encodes for E-cadherin, a key protein involved in cell adhesion. CDH1 gene is frequently studied in the routine practice, to investigate a possible predisposition to breast and diffuse gastric cancers and to manage cancer prevention in the family. The point mutation and large rearrangement are studied. Herein, we presented a qualitative and quantitative description of alternative splicing events of CDH1 gene using a short-read RNAseq approach. The aim of this work was to establish the CDH1 alternative splicing profile as a basis for the interpretation of the possible clinical impact of CDH1 splicing variants. We highlighted novel CDH1 isoforms and presented an evaluation of known alternative CDH1 isoforms. We validated our approach by classifying three pathogenic CDH1 complex splicing variants. Our study findings suggest an interesting possible implication of CDH1 intron 2 in CDH1 isoform regulation.

Background/Objectives: CDH1 gene is widely studied, as pathogenic variants are involved in diffuse gastric cancers and lobular breast cancers. CDH1 genotype contributes to the management of clinical practice recommendations for cancer prevention. We proposed a qualitative and quantitative description of CDH1 alternative splicing profile on lymphoblastoid cell lines (LCLs). The aim of this description was to allow a comprehensive interpretation of the effect of variants of uncertain significance (VUS) on CDH1 splicing. Methods: We studied, using RNAseq, the splicing profile of 22 LCLs (untreated and treated with puromycin) with no pathogenic variant on CDH1 and evaluated the effect on CDH1 splicing of four VUS. Results: We highlighted a total of eleven alternative splicing events including four junctions starting from intron 2, defining novel isoforms of CDH1. We also identified an isoform causing the skip of exon 11 and leading to a disruption of the reading frame with high levels of expression on negative CDH1 control LCLs, confirmed by ddPCR. Splicing RNAseq results for CDH1 VUS: c.1008+1G>A and c.1936+5G>A showed complex splicing patterns but allowed their classification as pathogenic. We studied CDH1 VUS exon 4 to exon 11 duplication with RNA analysis combined with Bionano optical genome mapping. Depending on alternative splicing of proximal and distal exons 11 within the duplication, we identified four distinct transcripts, leading to truncated proteins, classifying the duplication as pathogenic. Conclusions:
CDH1 has a complex alternative splicing profile characterized by a dynamic splicing of intron 2 making CDH1 a good candidate for a study using long-read RNAseq.