Mutations of filamin B (FLNB) gene can lead to a spectrum of autosomal skeletal malformations including spondylocarpotarsal syndrome (SCT), Larsen syndrome (LRS), type I atelosteogenesis (AO1), type III atelosteogenesis (AO3), and boomerang dysplasia (BD). Among them, LRS is milder while BD causes a more severe phenotype. However, the molecular mechanism underlying the differences in clinical phenotypes of different FLNB variants has not been fully determined. Here, we presented two patients suffering from autosomal dominant LRS and autosomal recessive vitamin D-dependent rickets type IA (VDDR-IA). Whole-exome sequencing revealed two novel missense variants in FLNB, c.4846A>G (p.T1616A) and c.7022T>G (p.I2341R), which are located in repeat 15 and 22 of filamin B, respectively. The expression of FLNBI2341R in the muscle tissue from our LRS patient was remarkably increased. And in vitro studies showed that both variants led to a lack of filopodia and accumulation of the mutants in the perinuclear region in HEK293 cells. We also found that c.4846A>G (p.T1616A) and c.7022T>G (p.I2341R) regulated endochondral osteogenesis in different ways. c.4846A>G (p.T1616A) activated AKT pathways through inhibiting SHIP2, suppressed the Smad3 pathway, and impaired the expression of Runx2 in both Saos-2 and ATDC5 cells. c.7022T>G (p.I2341R) activated both AKT and Smad3 pathways and increased the expression of Runx2 in Saos-2 cells, while in ATDC5 cells it activated AKT pathways through inhibiting SHIP2, suppressed the Smad3 pathway, and reduced the expression of Runx2. Our study demonstrated the pathogenic mechanisms of two novel FLNB variants in two different clinical settings and proved that FLNB variants could not only directly cause skeletal malformations but also worsen skeletal symptoms in the setting of other skeletal diseases. Besides, FLNB variants differentially affect skeletal development which contributes to clinical heterogeneity of FLNB-related disorders.

Filamins (FLN) are a family of actin-binding proteins involved in regulating the cytoskeleton and signaling phenomenon by developing a network with F-actin and FLN-binding partners. The FLN family comprises three conserved isoforms in mammals: FLNA, FLNB, and FLNC. FLNB is a multidomain monomer protein with domains containing an actin-binding N-terminal domain (ABD 1-242), encompassing two calponin-homology domains (assigned CH1 and CH2). Primary variants in FLNB mostly occur in the domain (CH2) and surrounding the hinge-1 region. The four autosomal dominant disorders that are associated with FLNB variants are Larsen syndrome, atelosteogenesis type I (AOI), atelosteogenesis type III (AOIII), and boomerang dysplasia (BD). Despite the intense clustering of FLNB variants contributing to the LS-AO-BD disorders, the genotype-phenotype correlation is still enigmatic. In silico prediction tools and molecular dynamics simulation (MDS) approaches have offered the potential for variant classification and pathogenicity predictions. We retrieved 285 FLNB missense variants from the UniProt, ClinVar, and HGMD databases in the current study. Of these, five and 39 variants were located in the CH1 and CH2 domains, respectively. These variants were subjected to various pathogenicity and stability prediction tools, evolutionary and conservation analyses, and biophysical and physicochemical properties analyses. Molecular dynamics simulation (MDS) was performed on the three candidate variants in the CH2 domain (W148R, F161C, and L171R) that were predicted to be the most pathogenic. The MDS analysis results showed that these three variants are highly compact compared to the native protein, suggesting that they could affect the protein on the structural and functional levels. The computational approach demonstrates the differences between the FLNB mutants and the wild type in a structural and functional context. Our findings expand our knowledge on the genotype-phenotype correlation in FLNB-related LS-AO-BD disorders on the molecular level, which may pave the way for optimizing drug therapy by integrating precision medicine.

Fetal magnetic resonance imaging (MRI) is obtained for prenatal diagnosis and prognostication of skeletal dysplasias; however, related literature is limited. The purpose of this study was to define the utility of fetal MRI for skeletal dysplasias and to report MRI findings associated with specific diagnoses. This retrospective study was approved by the institutional review board; informed consent was waived. Women referred for suspected fetal skeletal dysplasia who underwent MRI between January 2003 and December 2018 were included. Definitive diagnoses were determined by genetic testing, autopsy, physical examination and/or postnatal/postmortem imaging. Fetal MRI examinations and reports were reviewed. Descriptive statistics were used to summarize imaging findings. Eighty-nine women were referred for fetal MRI for possible skeletal dysplasia. Forty-three (48%) were determined to have a diagnosis other than skeletal dysplasia and nine were excluded for lack of specific skeletal dysplasia diagnosis. Thirty-seven cases of skeletal dysplasia with available fetal MRI and specific diagnosis were included for analysis. Diagnoses included achondrogenesis (n=2), achondroplasia (n=5), Boomerang dysplasia (n=1), campomelic dysplasia (n=2), Jeune syndrome (n=1), Kniest dysplasia (n=1), osteogenesis imperfecta (n=15) and thanatophoric dysplasia (n=10). A specific skeletal dysplasia diagnosis was mentioned in 17/37 (46%) of MRI imaging reports and correct for 14/17 (82%). MRI findings were reported for each specific skeletal dysplasia diagnosis. Fetal MRI is a useful diagnostic tool for skeletal dyplasias and excluded the diagnosis in nearly half of referred pregnancies. In addition to providing fetal lung volumes, fetal MRI demonstrates findings of the brain in achondroplasia and thanatophoric dysplasia, of the spine in achondroplasia and achondrogenesis, of the calvarium in osteogenesis imperfecta and thanatophoric dysplasia, and of the cartilage in Kniest dysplasia.

The location and/or type of variants in FLNB result in a spectrum of osteochondrodysplasias ranging from mild forms, like spondylocarpotarsal synostosis syndrome and Larsen syndrome, to severe perinatal lethal forms, such as atelosteogenesis I and III and Boomerang dysplasia. Spondylocarpotarsal synostosis syndrome is characterized by disproportionate short stature, vertebral anomalies and fusion of carpal and tarsal bones. Biallelic loss-of-function variants in FLNB are known to cause spondylocarpotarsal synostosis syndrome and 9 families and 9 pathogenic variants have been reported so far. We report clinical features of 10 additional patients from 7 families with spondylocarpotarsal synostosis syndrome due to 7 novel deleterious variants in FLNB, thus expanding the clinical and molecular repertoire of spondylocarpotarsal synostosis syndrome. Our report validates key clinical (fused thoracic vertebrae and carpal and tarsal coalition) and molecular (truncating variants in FLNB) characteristics of this condition.

Non-randomly distributed missense mutations of Filamin B (FLNB) can lead to a spectrum of autosomal dominant-inherited skeletal malformations caused by bone hypoplasia, including Larsen syndrome (LS), atelosteogenesi-I (AO-I), atelosteogenesi-I (AO-III) and boomerang dysplasia (BD). Among this spectrum of diseases, LS causes a milder hypoplasia of the skeletal system, compared to BD's much more severe symptoms. Previous studies revealed limited molecular mechanisms of FLNB-related diseases but most of them were carried out with HEK293 cells from the kidney which could not reproduce FLNB's specificity to skeletal tissues. Instead, we elected to use ATDC5, a chondrogenic stem cell line widely used to study endochondral osteogenesis. In this study, we established FLNB-transfected ATDC5 cell model. We reported a pedigree of LS with mutation of FLNBG1586R and reviewed a case of BD with mutation of FLNBL171R . Using the ATDC5 cell model above, we compared cellular and molecular phenotypes of BD-associated FLNBL171R and LS-associated FLNBG1586R . We found that while both phenotypes had an increased expression of Runx2, FLNBL171R-expressing ATDC5 cells presented globular aggregation of FLNB protein and increased cellular apoptosis rate while FLNBG1586R-expressing ATDC5 cells presented evenly distributed FLNB protein and decreased cellular migration. These findings support our explanation for the cause of differences in clinical phenotypes between LS and BD. Our study makes a comparative analysis of two extremes of the FLNB-mutated autosomal dominant spectrum, relating known clinical phenotypes to our new cellular and molecular findings. These results indicated next steps for future research on the role of FLNB in the physiological process of endochondral osteogenesis.