Heterozygous variants of Extracellular Matrix (ECM) protein Fibulin-2 (FBLN2) in 6 human males with Prune Belly Syndrome (PBS)
Shaheer Ali, BS, Thomas J. Egeland, MS, Shane F. Batie, MD, Jeremy A. Mathews, MS, Brandi L. Cantarel, PhD, Steven M. Harrison, PhD, Nida I. Karra, PhD, Nathalia G. Amado, PhD, Linda A. Baker, MD.
University of Texas Southwestern Medical Center, Dallas, TX, USA.
BACKGROUND: PBS is a rare congenital myopathy with altered ECM deposition. FBLN2 is a small, fibrous, integrin- and fibronectin-binding ECM glycoprotein that interacts with cell surface components and assembles and stabilizes supramolecular ECM complexes during fibrogenesis, angiogenesis, and organogenesis (skeletal, cardiac and smooth muscle, cartilage, nerves, and bladder). Its upregulation imparts poor prognosis in bladder cancer and rare de novo FBLN2 variants are associated with pulmonary arterial hypertension and atrioventricular septal defects. We hypothesized that FBLN2 could be causal in PBS.
METHODS: With IRB-approval, probands and family members were enrolled and phenotyped. Paired-end Whole Exome Sequencing (WES) on lymphocyte DNA was done using the Illumina SureSelect kit and HiSeq2500 sequencer, with data pipeline analysis. Each rare variant with minor allele frequency <0.001 was in silico evaluated and defined as encoding a significantly dysfunction protein if ≥3 criteria were met: SIFT < 0.1, Polyphen2 > 0.9, CADD score > 20, GERP > 4, MutationTaster = "A" or "DC", and Mutation Assessor= "M" or "H" (Fig.1A). Qualifying variants were confirmed by Sanger sequencing and screened in ClinVar database for prior disease association. Familial segregation was investigated on available samples. Mouse and human bladder (BL) and abdominal wall (AW) mRNA or protein expression was analyzed by qPCR and Western blot (WB).
RESULTS: 7 missense FBLN2 variants were found in 6 unrelated PBS males, respectively (Fig 1A,B). All probands are heterozygous (het). PBS-4 has 2 FBLN2 compound het variants, however cis versus trans state is not available. None of these variants are known to cause human genetic diseases. On the FBLN2 protein structure (Fig 1C), 3 PBS variants alter the N-terminal cys-rich and cys-free domains, 3 change the EGF-like domains, and 1 disturbs the Fibulin-type domain. Transcript and protein expression analysis (Fig 1D,E) revealed that Fbln2 is expressed in human BL and AW. Similarly, mouse Fbln2 mRNA is highly expressed in BL and AW during development and in adult ages.
CONCLUSIONS: By screening for rare or novel, protein-damaging DNA variants, we identified 7 het missense FBLN2 mutations in 6 PBS probands. Different mechanisms of FBLN2 disruption, tissue specific regulation, and unique mutant protein interactions may yield distinct patterns of FBLN2 protein dysfunction. Our data suggest that PBS is an oligogenic and complex disorder that can be explained in part by heterozygous mutations in the fibrotic ECM protein FBLN2. 
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