Journal of Pathology publish this investigation article

March 1^st, 2022

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here, we present a systematic functional analysis of PALB2 splice-site variants detected in ~113,000 women of the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the ACMG/AMP (American College of Medical Genetics and Genomics and the Association for Molecular Pathology) variant classification scheme. Up to 23 variants were classified as Pathogenic/Likely Pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C and c.211+1G>A) were classified as variants of unknown significance since they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG/AMP rationale. This article is protected by copyright. All rights reserved

Valenzuela-Palomo A., Bueno-Martinez E., Sanoguera-Miralles L., Lorca V., Fraile-Bethencourt E., Esteban-Sanchez A., Gomez S., Carvalho J., Allen J., Garcia-Alvarez A., Perez-Segura P., Dorling L., Easton DF., Devilee P., Vreeswijk MPG., de la Hoya M. and Velasco EA.

Splicing predictions, minigene analyses and ACMG/AMP clinical classification of 42 germline PALB2 splice-site variants

Splicing predictions, minigene analyses and ACMG/AMP clinical classification of 42 germline PALB2 splice-site variants

	Grupo de Splicing y Cáncer. Instituto de Biología y Genética Molecular (IBGM). Consejo Superior de Investigaciones Científicas (CSIC).
	Laboratorio de Oncología Molecular. Hospital Universitario Clínico San Carlos. Salud Madrid. Comunidad de Madrid.
	Knight Cancer Research Building.
	Servicio de Identificación y Caracterización Microbiana (ICM). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).
	Centre for Cancer Genetic Epidemiology. Department of Public Health and Primary Care. University of Cambridge.
	Department of Human Genetics. Leiden University Medical Centre.