Genetic Screening and Functional Characterization ofPDGFRBMutations Associated with Basal Ganglia Calcification of Unknown Etiology

Abstract

Three causal genes for Idiopathic Basal Ganglia Calcification (IBGC) have been identified. Most recently, mutations in PDGFRB, encoding a member of the platelet-derived growth factor receptor family type β, and PDGFB, encoding PDGF-B, the specific ligand of PDGFRβ, were found implicating the PDGF-B/PDGFRβ pathway in abnormal brain calcification. In this study we aimed to identify and study mutations in PDGFRB and PDGFB in a series of 26 patients from the Mayo Clinic Florida Brain Bank with moderate to severe basal ganglia calcification (BCG) of unknown etiology. No mutations in PDGFB were found. However, we identified one mutation in PDGFRB, p.R695C located in the tyrosine kinase domain, in one BGC patient. We further studied the function of p.R695C mutant PDGFRβ and two previously reported mutants, p.L658P and p.R987W PDGFRβ in cell culture. We show that, in response to PDGF-BB stimulation, the p.L658P mutation completely suppresses PDGFRβ autophosphorylation whereas the p.R695C mutation results in partial loss of autophosphorylation. For the p.R987W mutation, our data suggest a different mechanism involving reduced protein levels. These genetic and functional studies provide the first insight into the pathogenic mechanisms associated with PDGFRB mutations and provide further support for a pathogenic role of PDGFRB mutations in BGC.

A Point Mutation in PDGFRB Causes Autosomal-Dominant Penttinen Syndrome

“…Cell culture, vector transfection, and western blotting were performed as described. 7 As expected, the wild-type construct generated three proteins detectable by an anti-PDGFRB antibody (clone 28E1, Cell Signaling Technology) and of approximately 180 kDa, 160 kDa, and 130 kDa, representing the mature, transmembrane form of the protein and two incompletely post-translationally processed forms, respectively 8 ( Figure 4A, Table S1). Total PDGFRB was calculated by adding the intensity of~180,~160, and~130 kDa bands.…”

“…5 We went on to functionally evaluate the predicted protein alteration in the PDGFRB product. We compared the function of the p.Val665Ala Penttinen syndrome variant to a loss-offunction p.Leu658Pro variant identified in individuals with idiopathic basal ganglia calcification, type 4 (IBGC4 [MIM: 615007]) 7 and to wild-type PDGFRB. Human PDGFRB cDNA c.1973T>C (p.Leu658Pro) and wild-type pCMV constructs have been described previously.…”

“…Human PDGFRB cDNA c.1973T>C (p.Leu658Pro) and wild-type pCMV constructs have been described previously. 7 The c.1994T>C (p.Val665Ala) mutation identified here was introduced independently into the wild-type PDGFRB pCMV construct with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) and mutagenesis primers (5 0 -CACCTGAACGTGGcCAACCTGT TGGG-3 0 [forward] and 5 0 -CCCAACAGGTTGgCCACGTT CAGGTG-3 0 [reverse], mutation indicated in lowercase). We transfected each of the mutant and the wild-type constructs into HeLa cells.…”

Primary familial brain calcification: update on molecular genetics

“…It has been hypothesized that the loss of function of PDGFRb could lead to the impairment of the blood brain barrier (BBB) integrity, [2] c.1802C[T p.Ser601Leu Exon 11 [2,22] c.1828_1831delTCC p.Ser610Alafs*17 Exon 11 [22] c.1909A[C p.Ser637Arg Exon 11 [37] g.42275321_42329908del Whole gene [24] Neurol Sci causing vascular and perivascular calcium accumulation [3]. Moreover, a deficient PDGF-b signalling is highly damaging to VSMCs and pericytes, resulting in complete lack of pericytes or pericyte hypoplasia, endothelial hyperplasia, increased vessel diameter, increased vascular permeability and vessel instability [4,27]. Alternatively, it has been suggested that mutations in PDGFRB gene might be activating mutations, impairing the PDGFRb-PiT-1 signalling and inducing VSMCs mineralization.…”

“…All mutations, p.Leu658Pro, p.Arg695Cys, p.Arg987Trp and p.Glu1071Val, cause the substitution of conserved amino acids and were predicted to be pathogenic. Cell culture experiments showed that variants in the tyrosine-kinase domain (from 562 to 953 aa) reduce the receptor levels and the autophosphorylation [27]. It has been demonstrated that the missense mutation p.Leu658Pro reduces the kinase activity, while p.Arg987Trp mutation causes a rapid degradation of the receptor and impairs the activation of STAT3, a transcription activator, blocking the downstream signalling.…”