Identification and in silico characterization of p.G380R substitution in FGFR3, associated with achondroplasia in a non-consanguineous Pakistani family

Achondroplasia (ACH) is inherited as an autosomal dominant trait of skeletal dysplasia with an estimated incidence ranging from 1:10,000 to 1: 70,000 live births [1, 2]. It is known to be the most common cause of dwarfism characterized by short stature that particularly affects the appendicular skeleton (upper and lower limbs) and to less extent, axial skeleton (skull, vertebrae, ribs) of the body [3‐5].

Clinically, ACH patients represent typical features including shortened arms and legs (especially the upper arm and thigh), bowed lower legs, disproportionately large head-to-body size, frontal bossing and midface retrusion or hypoplasia [5‐7]. During infancy, hypotonia is the most prominent characteristic caused by delayed and abnormal development of motor milestone [8]. Despite all these clinical manifestations, individuals with ACH have normal life span and intelligence factor [9].

Over the last two decades, dominant gain-of-function mutations of the specific site in fibroblast growth factor receptor 3 (FGFR3) have been shown implicated in human skeletal dysplasias, including achondroplasia (ACH), hypochondroplasia (HCH), thanatophoric dysplasia (TD) and severe achondroplasia, with developmental delay and acanthosis nigricans (SADDAN) [4, 10]. The FGFR3 is one of the four members of fibroblast growth factor receptor (FGFR) family, but differs from other FGFRs in its affinity for ligands and tissue distribution as it is mainly expressed in cartilage and brain [11, 12].

A typical FGFR contains an extracellular ligand-binding domain, a transmembrane region and an intracellular divided tyrosine kinase domain. Fibroblast growth factor (FGF) binds to an extracellular ligand-binding domain to initiate FGF/FGFR signaling that induces the expression of cell cycle suppression genes to negatively regulate bone development. However, mutations in the FGFR3 gene lead to a constitutively active FGFR3 protein. As a result, a cascade of uncontrollable signal transduction allows an aberrant expression of the suppression genes, hence development of short stature pathology [10].

Almost 98% of the ACH cases are caused by variation at nucleotide position 1138, with 97% involving a c.1138 G > A mutation and 1% involving a c.1138 G > C mutation [13, 14]. Both mutations substitute glycine with arginine (p.G380R) in the transmembrane domain of FGFR3 protein that leads to gain-of-function [4, 15]. Mostly these mutations are de novo (sporadic) as more than 80% of ACH cases are born to their average-statured parents [16]. Advanced paternal age is one of the major reasons that significantly contribute to de-novo mutations in the germ cells because of large number of cell divisions during spermatogenesis [17]. Moreover, the presence of guanine at nucleotide position 1138, which is a part of CpG dinucleotide island and the most mutable site in the human genome, can also explain the high incidence of spontaneous mutations in FGFR3 [18]. Other less frequent mutations are also identified in FGFR3 but are mainly associated with hypochondroplasia and thanatophoric dysplasia type I and II [19]. Therefore, in comparison to other genetic diseases, ACH is a genetically and phenotypically homogenous disorder where very few rather than hundreds of mutations are responsible [20, 21]. In this study a non-consanguineous Pakistani family involving two affected generations, was clinically and genetically characterized for skeletal dysplasia. Genetic analysis revealed a heterozygous dominant mutation in FGFR3 affecting the protein stability and dimerization efficiency, leading to ACH in a Pakistani family.

Achondroplasia, primarily a failure of endochondral ossification in the growth plate of cartilage, defined to be associated with pathogenic mutations in the transmembrane segment of FGFR3 gene [32, 33]. To date, eight different pathogenic mutations of FGFR3 transmembrane segment have been shown implicated in cancer and growth disorders including ACH; however, exact pathogenetic mechanism of the disease is still unclear. In the present study, we screened a non-consanguineous Pakistani family with ACH and identified a highly recurrent heterozygous c.1138 G > A mutation of FGFR3. The single nucleotide substitution not only replaces glycine with arginine at codon position 380 (p.G380R) but also generates a unique restriction site for SfcI endonuclease. An electrophoretic banding pattern of SfcI digestion supports the dominant mode of disease segregation in the family. The presence of heterozygous c.1138 G > A mutation in all affected individuals, but none of the phenotypically normal members of the family along with hundred ethnically-matched healthy controls confirms the previously reported association of mutation with ACH in this Pakistani family.

The identified genetic variation was further tested through in silico analysis to calculate its impact on protein structure, stability and function attributed to disease under study. Comparative in silico analysis of varied features of wild-type and mutant FGFR3 proteins predicted significant conformation changes in the secondary protein structure. As a consequence, decreased protein stability with altered interaction and function can be expected. The Identified mutation has previously been described to be implicated in persistent ligand-independent activation of the FGFR3 protein, a deviation from its normal function as a negative bone growth regulator [4, 18]. In the present study, it was calculated through bioinformatics that the identified mutation decreases the protein stability, hence disrupted the normal functioning of FGFR3 protein completely or partially and categorized as deleterious ACH mutation.

Under normal circumstances, FGF/FGFR3 signaling initiates lateral dimerization of FGFR3 monomers to take on autophosphorylation of tyrosine residues in the intracellular domain of FGFR3 [34]. The transmembrane domain plays a critical role in this dimerization and/or activation of receptor tyrosine kinases by controlling orientation of the intracellular kinase domain [35, 36]. Once activated, FGFR3 phosphorylate cytoplasmic target proteins, leading to the activation of intracellular downstream signaling cascade. Signaling Pathways such as mitogen-activated protein kinase (Ras/MAPK), phosphoinositide 3-kinase/Akt, phospholipase C and protein kinase C pathways inhibit chondrocyte proliferation through STAT1 signaling by inducing the expression of cell cycle suppressor genes such as CDK inhibitor p21, hence negatively regulate bone growth [10, 37].

In contrast, both the deficiency of FGFR3 or presence of gain-of-function mutations in FGFR3 are reportedly implicated in prolonged bone growth or short stature respectively, suggesting the role of FGFR3 as limiting factor rather than a promoting agent [10, 38, 39]. Among the known mutations of FGFR3, c.1138 G > A substitution (p.G380R) is the most recurrent point mutation ever known in human genome accounts for almost 98% ACH cases [18, 40‐42]. It is considered that p.G380R change causes hydrogen bonding between two arginine side chains that stabilizes FGFR3 dimerization in the cell membrane, leading to a constitutive ligand-independent activation of the FGFR3 [43]. Thus, uncontrollable signal transduction promotes the inhibition of cartilage growth and development by aberrantly inducing the expression of cell cycle suppressor genes [35, 43]. Many other studies have hypothesized that point mutations in the FGFR3 transmembrane regions can induce conformational rearrangements of the dimer that can affect its association strength in different states and hence represent a general mechanism of activation of such receptors [40, 43]. However, the exact mechanism of FGFR3-mediated signal transduction in health and disease is still unknown.

We report a common FGFR3 substitution mutation p.G380R in three patients from a non-consanguineous Pakistani family. In view of previous reports we assume that the identified mutation might have altered the dimerization efficiency of transmembrane region leading to uncontrolled signal transduction and expression of cell cycle suppressor genes. This might clarify the decreased cell proliferation in the growth plate and would justify the basis for reduced endochondral bone growth and disproportionate short stature in ACH. Further detailed genotype-phenotype studies are expected to provide a better understanding of the role of FGFR3 in skeletal development. Our findings agree with the world-wide sharing of p.G380R mutation among achondroplasia subjects and extend the body of evidence that supports the role of FGFR3 gene in ACH. Moreover, like many other countries, Sfc1 restriction site in FGFR3 can be used as a molecular diagnosis marker for ACH in Pakistan.