# Clinic Examination and Gene Diagnosis for a Birt–Hogg–Dubé Syndrome Family With a Novel flcn Frameshift Mutation Causing Nonsense-Mediated mRNA Degradation

## TL;DR

This study identifies a new FLCN gene mutation causing Birt–Hogg–Dubé syndrome and shows how it leads to disease through specific signaling pathways.

## Contribution

A novel FLCN frameshift mutation is identified and linked to BHD via NMD and specific signaling pathway activation.

## Key findings

- The FLCN frameshift mutation c.21_22del reduces mRNA and protein expression through NMD.
- The mutation causes BHD by activating AMPK, Wnt/β-catenin, and mTOR signaling pathways.
- The mutation leads to haploinsufficiency, contributing to BHD symptoms.

## Abstract

Background: Birt–Hogg–Dubé syndrome (BHD) was an autosomal dominant disorder caused by a mutation in the folliculin (FLCN) gene and characterized by benign cutaneous fibrofolliculomas in the head and neck, pulmonary cysts, spontaneous pneumothorax, and combined renal tumors.

Methods: This study reported a familial case presenting multiple pulmonary bullae, recurrent spontaneous pneumothorax, diffuse cystic lesions in both lungs, and renal cysts. To further clarify the diagnosis, next-generation sequencing (NGS) was performed in conjunction with the clinical diagnostic criteria for Birt–Hogg–Dubé. The eukaryotic recombinant expression vectors of pEGFP-C1-FLCN and knock-in FLCN mutation by CRISPR/Cas9 were conducted in 293 T and BEAS-2B cell lines. The mRNA and protein expression of the FLCN mutation were verified by fluorescence quantitative PCR and Western blot assay. Nonsense-mediated mRNA decay (NMD) assays and immunohistochemical assays were conducted to elucidate the pathogenicity of the mutation and explore potential mechanisms.

Results: A unique, novel, unspecified significance FLCN mutation NM_144997.7: c.21_22del (p. Cys8 Profs⁣∗28) in Exon 4 was detected in both patients. The results demonstrated that the newly identified FLCN frameshift mutation significantly decreased FLCN mRNA and protein expression. The NMD complex recognized and degraded mRNAs containing a premature termination codon (PTC) in the open reading frame of the FLCN frameshift mutation, resulting in haploinsufficiency and ultimately contributing to the manifestation of BHD. Protein expression on the AMP-activated protein kinase (AMPK), Wnt/β-catenin, and mammalian target of rapamycin (mTOR) signaling pathways by immunohistochemistry indicated that FLCN frameshift mutations were responsible for BHD through the activation of AMPK, Wnt/β-catenin, and mTOR signaling pathways.

Conclusion: The study demonstrated that a novel FLCN frameshift mutation was responsible for the pathogenesis of BHD and preliminarily demonstrated that FLCN causes BHD through the AMPK, Wnt/β-catenin, and mTOR signaling pathways.