Submission Details

FLII was evaluated for autosomal recessive dilated cardiomyopathy (DCM). In addition to DCM, FLII has also been reported as a possible candidate gene for Smith Magenis Syndrome at the time of review. This was considered in accordance with ClinGen lumping and splitting criteria. Due to distinct differences in disease mechanism, inheritance, and phenotype, the curation was split and curated for autosomal recessive, isolated idiopathic DCM. Human genetic evidence supporting this gene-disease relationship includes case-level data. At least four variants (nonsense, missense) have been reported in humans with DCM. A total of three cases from three independent families were reviewed from two primary publications. Two probands were from consanguineous families (Al-Hassnan et al. 2020 PMID: 32870709). These probands were homozygous for rare, missense FLII variants identified via whole exome sequencing after prioritizing analysis for variants in comprehensive list of 146 well-established cardiomyopathy associated genes was elusive. The third case was a proband from a non-consanguineous Dutch family (Ruijmbeek et al. 2023 PMID 37561591) who was identified to be compound heterozygous for a rare nonsense and rare missense variant in FLII with sequencing of parents confirming variants were in trans.

In addition, this gene-disease assertion is supported by expression data, mice and zebrafish animal models, and functional data. Following identification of human FLII from the Drosophila melanogaster homolog by Campbell et al. 1993, Northern blot analysis of various human tissues identified the second highest expression in cardiac tissue (Campbell et al., 1997 PMID: 9177775). Given germline homozygous FLII deletion is embryonically lethal in mice, a cardiac-specific knock-out (KO) was created postnatally with aMHC-Cre mediated expression with LoxP sites targeting exons 8 to 14 of FLII (Kuwabara et al. PMID: 37126682). Phenotypic analysis via echocardiograms at 16 weeks revealed significant increase in left ventricular body weight ratio and significant decrease in fractional shortening in cardiac-specific mutant mice versus aMHC-Cre controls. Additional phenotypic analysis of tamoxifen-induced cardiac specific Cre-mediated FLII KO (induced at 4 weeks) was completed in pre- and post-stress state. Echocardiographic findings at 11 weeks following 3 weeks of transverse aortic constriction stress recapitulated a DCM-concerning phenotype, supporting importance of FLII function in cardiac homeostasis (Kuwabara et al. PMID: 37126682). In zebrafish, Ruijmbeek et al., 2023 created compound heterozygous and homozygous zebrafish progeny corresponding to previously detailed human variants via CRISPR-CAS9 methodology. High-speed imaging technology at 120 hours post fertilization (hpf) identified significantly reduced ventricular contractility and ejection fraction in zebrafish mutants compared to controls (Ruijmbeek et al., 2023 PMID: 37561591). A variety of additional experiments in mutant mice and zebrafish support FLII function in cell adhesion complexes and myocardium organization. In HEK293T cells, co-immunoprecipitant analysis with overexpression of FLAG-tagged FLII followed by mass spectrometry identified actin as one of the interacting substrates (Kuwabara et al. 2023 PMID: 37126682). Confocal microscopy of isolated cardiomyocytes from mutant mice at 16 weeks of age compared to controls revealed abnormal shortening in the thin filament, while electron microscopy of KO mutant heart tissue at 6 months revealed disturbed organization of the intercalculated discs and sarcomere dysgenesis (Kuwabara et al., 2023 PMID: 37126682). In homozygous missense and truncating zebrafish compared to controls, confocal and transmission electron microscopy imaging at 120 hpf revealed disorganized, primitive cardiac trabeculae with irregular filament organization, myofibril orientation and maturation. Furthermore, localization studies for cell-adhesion proteins vinculin and cadherin2 through 3D confocal projections and quantitative pixel intensity plots at 60 hpf in isolated cardiomyocytes of homozygous truncating mutants compared to controls showed abnormally dispersed localization and reduced intensity (Ruijmbeek et al., 2023 PMID: 37561591).

In summary, there is moderate evidence to support this gene-disease relationship. More evidence is needed to establish the relationship of FLII with AR DCM definitively. This classification was approved by the ClinGen Dilated Cardiomyopathy Working Group on 6/14/2024 (SOP Version 10).