A missense GDF5 variant causes brachydactyly type A1 and multiple‐synostoses syndrome 2

Abstract Objective This study aimed to identify the molecular defects and clinical manifestations in a Chinese family with brachydactyly (BD) type A1 (BDA1) and multiple‐synostoses syndrome 2 (SYNS2). Methods A Chinese family with BDA1 and SYNS2 was enrolled in this study. Whole‐exome sequencing was used to analyze the gene variants in the proband. The sequences of the candidate pathogenic variant in GDF5 was validated via Sanger sequencing. I‐TASSER and PyMOL were used to analyze the functional domains of the corresponding mutant proteins. Results The family was found to have an autosomal‐dominantly inherited combination of BDA1 and SYNS2 caused by the S475N variant in the GDF5 gene. The variant was located within the functional region, and the mutated residue was found to be highly conserved among species. Via bioinformatic analyses, we predicted this variant to be deleterious, which perturb the protein function. The substitution of the negatively charged amino acid S475 with the neutral N475 was predicted to disrupt the formation of salt bridges with Y487 and impair the structure, stability, and function of the protein, consequently, the abnormalities in cartilage and bone development ensue. Conclusions A single genetic variant (S475N) which disrupt the formation of salt bridges with Y487, in the interface of the antagonist‐ and receptor‐binding sites of GDF5 concurrently causes two pathological mechanisms. This is the first report of this variant, identified in a Chinese family with BDA1 and SYNS2.

first report of this variant, identified in a Chinese family with BDA1 and SYNS2.

K E Y W O R D S
brachydactyly type A1, GDF5, multiple-synostoses syndrome 2, variant, whole-exome sequencing Growth differentiation factor 5 (GDF5) is a member of the family of bone morphogenetic proteins (BMPs), which belongs to the transforming growth factor beta (TGFβ) superfamily. 1 GDF5 plays an important role in limb development.It determines the sizes of the initial cartilaginous condensations during joint development.2GDF5 regulates early chondrogenesis by interacting with two different types of BMP 1 receptors, namely BMPR1A and BMPR1B, with a higher affinity for BMPR1B among the two. 3][7][8][9][10][11][12] Some loss-of-function GDF5 variants result in reduced osteogenesis or abnormal bone development, such as brachydactyly (BD), whereas some gain-of-function GDF5 variants cause proximal symphalangism (SYM1, OMIM #185800) and multiple-synostoses syndrome 2 (SYNS2, OMIM #610017).BD is classified into five types (A-E) according to the affected phalanges, and type A comprises three sub-types (A1-A3). 13A variant of GDF5 has been shown to be related to BDA1 (OMIM #112500), BDA2 (OMIM #112600), and BDC (OMIM #113100). 6,14,15mozygous deletion variants in GDF5 lead to the development of extremely short fingers and limbs, which is associated with various types of acrochondrodysplasia. 16,17 In contrast to loss-of-function GDF5 variants, gain-of-function GDF5 variants increase the chondrogenous activity and are associated with SYM1 and SYNS2. 6,17SYM1 is characterized by proximal interphalangeal stiffness, in which the carpus and tarsus are fused.A variant of GDF5 causes joint adhesion in the elbows and knees, which is a feature of SYNS2.
Here, we describe a Chinese family with concurrent clinical features of BDA1 and SYNS2, which we found to result from a variant in the mature domain of GDF5 (S475N).
All the participants completed questionnaires regarding their medical and family history, and the collected information was supplemented by medical records.The diagnosis and classification of BDA1 and SYNS2 complied with the latest guidelines. 16All the participants signed informed consent for this study, which was approved by the Ethics Committee of the Central Hospital of Wuhan.

| DNA extraction and whole-exome sequencing (WES)
The clinical data of the family members were obtained, and 5 mL of fasting blood of each member was collected into a tube containing EDTA as the anticoagulant.Genomic DNA was isolated from these peripheral-blood samples by using a DNA Extraction Kit (TIANGEN, Beijing, China) according to the instructions of the manufacturer. 18S was performed on the proband.Agilent SureSelect Human All Exon V6 kit was used to capture the genomic sequences, followed by sequencing on the Illumina hiSeq2500 System.The reference genome was human GRCh37/hg19.

| Sanger sequencing
Sanger sequencing was performed to confirm that the proband and his family carried the candidate variant in GDF5.The target DNA segments were amplified via polymerase chain reaction (PCR).The primers for GDF5 were as follows: forward primer, 5 0 -GCAGACGGGCAGCAATCC-3 0 ; and reverse primer,  3 | RESULTS

| Clinical characteristics
The basic characteristics of the affected family members are displayed in Table 1, and the family pedigree is shown in Figure 1 T A B L E 2 Evolutionary conservation analysis for the variant in GDF5.and son presented with features of BDA1, namely lack of the middle and proximal interphalangeal joints of the second, third, fourth, and fifth fingers and toes; shortened proximal phalanx of the first toe; fused tarsal bones; bone fusion in both elbow joints; and elbow valgus deformity.Additionally, all the fingers of the daughter lacked a bone germinal center (Figure 1-II and Table 1).
GDF5 was found to be a candidate pathogenic gene in the proband, who did not carry any other known brachydactyly-related pathogenic variant.The variant was located on chromosome 20, position 34 021 789, G1424A, NM_000557, p.S475N, namely, rs121909347.
This variant was analyzed for evolutionary conservation, and the results showed that it was located in a highly conserved region among multiple animal species (Table 2).Via multiple bioinformatics software, including SIFT, Polyphen2, LRT, FATHMM, and REVEL, the variant was predicted to perturb the protein function.At present, there are no relevant records about the variant frequency of this variant in major databases, such as 1000 g, esp6500, and GnomAD.Sanger sequencing the G1424A in GDF5 of other family members revealed that the affected relatives (III-1 and III-2) have the same variant, and the non-affected relatives (I-1, II-1, and II-3) have no variant (Figure 2).

| Prediction of the protein structure
The tertiary structures of the wild-type and mutant GDF5 proteins were predicted using I-TASSER (Figure 3).The S475N variant is highlighted on the 3D model of GDF5 (Figure 3A).GDF5 S475N is located within the finger of the GDF5 dimer.The substitution of the negatively charged amino acid S475 with the neutral N475 disrupts the formation of salt bridges with Y487 (Figure 3B, C).Therefore, the GDF5 S475N variant destroys the connection between S475 and Y487, presumably impairing the stability, structure, and function of the protein and causing its degradation.

| DISCUSSION
Here, we described a novel GDF5 variant in a Chinese family with BDA1 and SYNS2.Our whole-exome sequencing revealed that the proband had a variant in the GDF5 gene.The variant was located within the functional region, and the mutated residue was found to be highly conserved among species.Via bioinformatic analyses, we predicted this variant to be deleterious, which perturb the protein function.This variant disrupts the binding site of GDF5 at the 487th residue.The stability of the protein and the affinity of the protein for its protein may be changed, consequently, the abnormalities in cartilage and bone development ensue.
The proband analyzed here presented with carpal/tarsal fusion, proximal interphalangeal joint adhesion, humerus radius connection, and progressive conductive deafness, which are characteristic features of SYNS.To date, the molecular mechanism underlying this progressive chondrogenic and osteoblastic disorder has remained unknown.
The single-nucleotide polymorphism (SNP) and insertiondeletion (InDel) variants in the proband were identified by comparing the obtained genomic sequences with those in the following publicly available databases: 1000 Genomes, Exome Aggregation Consortium, Single-Nucleotide Polymorphism Database, NHLBI Exome Sequencing Project, and Genome Aggregation Database.Several in silico prediction tools (SIFT, PolyPhen2_HVAR, Polyphen2_HDIV, LRT, and FATHMM) were used to predict the effects of identified variants on protein structure and function.
5 0 -CAAGCGACCCAGCAAGAACC-3 0 .The PCR conditions were as follows: 95 C for 5 min; 35 cycles of 95 C for 1 min, 59 C for 30 s, then 72 C for 10 min.After the PCR product was purified, it was sequenced using an ABI3730XL Automatic Sequencer (Applied Biosystems, Foster City, CA).

F
I G U R E 3 3D presentation and structural comparison of the wild-type and mutant (S475N) GDF5 proteins.(A) 3D presentation of the GDF5 protein (prepared and visualized using I-TASSER and PyMol, respectively).The S475N variant is highlighted in pink.(B) The GDF5 S475 polypeptide.(C) The GDF5 N475 polypeptide.