Functional analysis of 6 variations in FOXL2

Area of Science:

• Genetics and Molecular Biology
• Cell Biology
• Developmental Biology

Background:

• The FOXL2 gene encodes a forkhead transcriptional factor crucial for cellular functions.
• Pathogenic variants in FOXL2 are associated with various developmental disorders, particularly affecting ovarian development.
• Understanding the functional impact of these variants is essential for elucidating disease mechanisms.

Purpose of the Study:

• To investigate the functional consequences of pathogenic variants in the FOXL2 gene.
• To analyze the effects of specific FOXL2 variants on protein localization and gene expression.
• To explore the potential impact of these alterations on ovarian development.

Main Methods:

• Experimental analysis of six FOXL2 variants (double missense, three deletions, nonsense, duplication) in HeLa cells.
• Confocal microscopy to determine subcellular localization of mutant FOXL2 proteins.
• Dual luciferase reporter assays and quantitative PCR to evaluate gene expression modulation.

Main Results:

• Deletion variants showed nuclear localization; double missense variant exhibited diffuse cellular localization; nonsense and duplication variants localized to the cytoplasm.
• Mutant proteins from p.Ala229Leufs43, p.Glu72, and p.Ala225_Ala234dup variants significantly reduced inhibitory effects on StAR gene transcription.
• Indel and nonsense variants, excluding the double missense variant, markedly reduced inhibitory effects on CCDN2 and INHBB gene expression.

Conclusions:

• The double missense variant does not have a combined inhibitory effect on gene expression.
• Despite varied subcellular localization, all mutant FOXL2 proteins likely impact downstream gene expression via a common pathway.
• Mutant FOXL2 proteins may disrupt ovarian development through diverse mechanisms beyond StAR gene regulation.