Am J Transl Res 2018;10(5):1400-1412 www.ajtr.org /ISSN:1943-8141/AJTR0075362 Original Article Comparative analysis of the two extremes of FLNB-mutated autosomal dominant disease spectrum: from clinical phenotypes to cellular and molecular findings Qiming Xu1, Nan Wu1,3,4, Lijia Cui5, Mao Lin1, D Thirumal Kumar6, C George Priya Doss6, Zhihong Wu2,3,4, Jianxiong Shen1,3,4, Xiangjian Song7, Guixing Qiu1,3,4 Departments of 1Orthopedic Surgery, 2Central Laboratory, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China; 3Beijing Key Laboratory for Genetic Research of Skeletal Deformity, Beijing, China; 4Medical Research Center of Orthopedics, Chinese Academy of Medical Sciences, Beijing, China; 5Department of Endocrinology, Peking Union Medical College Hospital, Beijing, China; 6Department of Integrative Biology, Vellore Institute of Technology, Vellore, India; 7Department of Pediatric Orthopedics, Zhengzhou Orthopedic Hospital, Zhengzhou, Henan, China Received March 3, 2018; Accepted April 18, 2018; Epub May 15, 2018; Published May 30, 2018 Abstract: Non-randomly distributed missense mutations of Filamin B (FLNB) can lead to a spectrum of autoso- mal dominant-inherited skeletal malformations caused by bone hypoplasia, including Larsen syndrome (LS), ate- losteogenesi-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 mo- lecular 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 differ- ences 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. Keywords: FLNB, larsen syndrome, boomerang dysplasia, ATDC5 cell, apoptosis Introduction IM 108721) and boomerang dysplasia (BD, OMIM 112310). LS is at one extreme of the Filamin B (FLNB) constitutes a large dimeric spectrum with the mildest hypoplasia of skele- actin-binding protein to act as a cytoskeleton tal system, while BD is at the other extreme and multifunctional signal scaffold [1]. Nonran- with the severest hypoplasia of skeletal system domly-distributed missense mutations of FL- [2, 3]. LS presents skeletal deformities includ- NB can lead to a spectrum of autosomal-domi- ing scoliosis; cervical kyphosis; supernumerary nant inherited skeletal malformations caused carpal bones; dislocation of large joints; and by bone hypoplasia, including Larsen syndrome characteristic facial malformations, including (LS, OMIM 150250), atelosteogenesis-I (AO-I, prominent forehead, wide-spaced eyes and OMIM 108720), atelosteogenesis-III (AO-III, OM- depressed nasal bridge [4, 5]. BD is a lethal Comparative analysis of FLNB missense mutated disease spectrum skeletal dysplasia characterized by underdevel- difference in clinical phenotypes between LS opment and potential lack of ossification in ver- and BD. tebrae, the acetabulum, or in the long bones of limbs, [6]. Almost all fetuses with BD die late Material and methods in the prenatal stage, making it difficult to stu- dy the molecular mechanisms of FLNB in BD. Clinical and radiological investigation AO-I and AO-III express traits between those Our clinic recruited an eight-year old male with associated with LS and BD. In addition to the- diagnosis of LS. We recorded the medical his- se diseases with FLNB missense mutations, tory of the patient and his family, then condu- nonsense mutations of FLNB can lead to spon- cted physical and radiological examinations on dylocarpotarsal synostosis syndrome (SCT, body parts with potential skeletal malformation OMIM 272460), an autosomal recessive skele- (Figure 1). The morbidity of BD was much rarer tal malformation characterized by premature than LS. We chose a BD case with the mostly fusion in carpal and tarsal joints and between reported BD-associated mutation c.T512G the vertebrae leading to scoliosis and lordosis (p.Leu171Arg) from literature [2, 6] as the [10]. research object for BD. Various phenotypes of Mutations in FLNB are exclusively associated those chosen objects of LS and BD were com- with skeletal diseases [4], indicating a high his- pared in details (Table 1). tological specificity of FLNB mutations’ patho- Whole exome sequencing and Sanger se- genesis to the skeletal system. Multiple studies quencing have attempted to explain the pathogenesis of FLNB mutations in skeletal malformation [3], The study was approved by the Ethics Com- including delay of ossification in growth plate of mittee of Peking Union Medical College Hos- long bone [11], hypo-mobility of chondrocytes pital. With informed consent from the patient [12] and disturbance of proliferation; and dif- and his family members, we extracted DNA ferentiation and apoptosis in chondrocytes [13- from peripheral blood samples of the pati- 15]. However, most of these studies were focused on nonsense mutations associated ent, and his father, mother and sister using with SCT. Little literature has explained the Puregene Blood kit. Whole exome sequencing pathogenic mechanisms of FLNB missense was performed using the Illumina HiSeq 2000 mutations in skeletal malformations due to (Illumina, San Diego, CA), reaching an average complexity of this spectrum of diseases. Mo- coverage of 116X. Reads were mapped to reover, those studies were mostly carried out in UCSC human genome reference assembly 19 HEK293 cells from the kidney, which may not (hg 19). Variant was called using BWA softwa- be affected by FLNB in the same way as skele- re (0.7.5a-r405). To identify pathogenic muta- tal tissues. tions, the variants were further compared to three databases including ClinVar, OMIM and In this study, we examine whether FLNB mis- HGMD; single nucleotide polymorphisms (SNP) sense variants cause the difference between among the normal population was excluded LS and BD at cellular and molecular levels. based five population-based databases, includ- The target variants of LS were selected as ing 1000 Genomes Project Dataset, Exome c.4756G>A (p.Gly1586Arg) in FLNB, which was Sequencing Project, Exome Aggregation Con- found in our LS patient, and another LS case sortium dataset and Allele Frequency Net Da- reported by Krakow et al. [10], and the target tabase of Japan Tokyo and Netherlands. Mu- mutation of BD was c.512T>G (p.Leu171Arg) in tations found in FLNB were further confirmed FLNB, which was reported in a male fetus diag- using Sanger sequencing. Exon 14 in FLNB nosed as BD [2, 6]. After transfecting FLNB was amplified using polymerase chain reaction plasmid to ATDC5 cell line, we compared distri- (PCR), and sequenced in an Applied Biosystem bution patterns of these two FLNB variants in 3730xl DNA Analyzer. cytoplasm, properties of cellular shape, cell migration, and apoptosis, and expression of Plasmid construction and transfection Runx2 and Smad3 in endochondral osteogen- esis. The cellular and molecular findings in our The wild-type FLNB plasmid was kindly donated study sketched a logical chain to explain the by Stephen P. Robertson from Otago University, 1401 Am J Transl Res 2018;10(5):1400-1412 Comparative analysis of FLNB missense mutated disease spectrum Figure 1. Clinical manifestation and family tree of the patient with Larsen syndrome. Whole spine X-ray and cervical spine X-ray revealed severe scoliosis (A) and cervical kyphosis with dysplasia of C4 and C5 vertebrae (B); hand and carpal joint X-ray showed supernumerary carpal bones and spatulate thumb; there should be eight carpal bones in a normal wrist while thirteen carpal bones were found in the wrist of this patient (C); gross anatomical pictures revealed serious back curves, short stature and varus deformities of elbow on both sides (D); The father (III2), uncle (III1), grandfather (II1) and great-grandfather (I1) had unusual faces, spatulate distal phalanges and varus deformities in both elbows similar to the patient (IV1) (E); A FLNB missense mutation (NM_001457.3, c.4756G>A (p.Gly1586Arg)) was identified in both the patient and his father, and was not found in the mother and sister (F). Dunedin, New Zealand [2]. The full-length FL- University. Both HEK293 and ATDC5 cells NB cDNA (reference sequence NM_001457.3), were grown in DMEM containing 10% FCS was assembled with EGFP on the C-terminal under 37°C with 5% CO2. Antibiotics-free media (from