Association between a novel mutation in SLC20A2 and familial idiopathic basal ganglia calcification

Abstract

Familial idiopathic basal ganglia calcification (FIBGC) is a rare, autosomal dominant disorder involving bilateral calcification of the basal ganglia. To identify gene mutations related to a Chinese FIBGC lineage, we evaluated available individuals in the family using CT scans. DNA was extracted from the peripheral blood of available family members, and both exonic and flanking intronic sequences of the SLC20A2 gene were amplified by PCR and then sequenced. Non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to confirm the presence of mutations. Allele imbalances of the SLC20A2 gene or relative quantity of SLC20A2 transcripts were evaluated using qRT-PCR. A novel heterozygous single base-pair deletion (c.510delA) within the SLC20A2 gene was identified. This deletion mutation was found to co-segregate with basal ganglia calcification in all of the affected family members but was not detected in unaffected individuals or in 167 unrelated Han Chinese controls. The mutation will cause a frameshift, producing a truncated SLC20A2 protein with a premature termination codon, most likely leading to the complete loss of function of the SLC20A2 protein. This mutation may also lead to a reduction in SLC20A2 mRNA expression by approximately 30% in cells from affected individuals. In conclusion, we identified a novel mutation in SLC20A2 that is linked to FIBGC. In addition to the loss of function at the protein level, decreasing the expression of SLC20A2 mRNA may be another mechanism that can regulate SLC20A2 function in IBGC individuals. We propose that the regional expression pattern of SLC20A1 and SLC20A2 might explain the unique calcification pattern observed in FIBGC patients.

Figure 1. Study subjects and imaging evaluation.

(A) Pedigree of family PJ-IBGC. The proband is indicated by an arrow. Filled symbols represent affected individuals, including both symptomatic (black) and asymptomatic (gray). (B) CT scan of the affected individuals. (C) MRS examination of a 4-year-old girl. I 1: a 54-year-old man; II 1: a 37-year-old woman; II:3: a 24-year-old man; III 1: an 11-year-old girl; III 2: a 4-year-old girl.

Figure 2. Identification of the c.510delA SLC20A2 mutation.

(A) Sequencing chromatogram showing the heterozygous c.510delA mutation in SLC20A2 (right) and the wild type sequence (left). (B) Heteroduplex mobility assay of the 171 bp PCR product derived from all the affected individuals and some unrelated normal individuals. After denaturation (95°C), re-annealed reactions were run under non-denaturing conditions. M, 100 bp DNA marker; N, normal individuals. The bands were visualized by silver-staining. (C) Schematic diagram of the wild-type and mutant SLC20A2 proteins. Purple regions represent the L183–V483 fragment of SLC20A2, which are important for the Pi transportation activity. The blue circle indicates the mutated amino acid residue. Amino-acid residues of the novel C-terminal peptides in the p.R172fsX19 mutant are given with the 19 new residues in red. The structure model was drawn using TOPO2 software (http://www.sacs.ucsf.edu/TOPO2/).

Figure 3. mRNA expression analysis of the SLC20A2 mutation.

(A) Sequencing showing an imbalance of the c.510delA (p.R172fsX19) mutant and wild-type alleles in cDNA templates. (B) Relative quantity (mean ± SD) of SLC20A2 transcripts derived from real-time quantitative polymerase chain reactions in 3 affected individuals and a normal individual.