PMC8138311_01
(A) HB-PFBC pedigree. Filled symbols represent family members affected with brain calcification, including both symptomatic (black) and asymptomatic (gray) cases. The black arrow indicates the proband and question marks indicate individuals whose blood samples and brain imaging were both not available. delC: c.806delC; +: wild-type allele; Cal, calcification; non-Cal, no calcification; NA, not available. (B) Brain computed tomography (CT) images of affected individuals in the HB-PFBC family. High radiopacity and density areas represent brain calcification. (C) The c.806delC mutation of SLC20A2 in the HB-PFBC proband and frequency diagram of protein conservation analysis for mutated Pro269 site. (D)
SLC20A2 and SLC20A1 mRNA expression in the peripheral leukocytes of the HB-PFBC family members. Red bar indicates the patient, and blue indicates normal people. (E) Brain CT and MRI axial T2*GRE images of the GD-PFBC patient. (F) The c.1154delG mutation of SLC20A2 in the GD-PFBC patient.
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F1
Brain CT imaging revealed bilateral symmetric calcification in the caudate nuclei, lentiform nuclei, thalami, and dentate nuclei of the cerebellum (Figure 1E). T2*GRE also showed bilateral symmetric hypointense signal changes in the aforementioned brain regions (Figure 1E). The patient was eventually diagnosed with Fahr's disease with hypertriglyceridemia and hyperhomocysteinemia. We identified a novel c.1154delG mutation at exon 8 of SLC20A2 (NM_001257180.2) (Figure 1F), in an evolutionarily conserved region, and predicted the formation of an NMD-directed degraded transcript or a truncated protein [p.(Ser385Ilefs*70)] in this patient.
PMC8138311_02
(A) Brain CT images of the HLJ-PFBC patient. (B) The PDGFB c.456+1G>C mutation in the HLJ-PFBC patient and normal control. (C) The impact of the PDGFB c.456+1G>C mutation on its mRNA splicing revealed by TA-cloning sequencing and cDNA-PCR sequencing in the HLJ-PFBC patient and normal control. (D) The overall and wild-type allele-specific expression of PDGFB in the peripheral leukocytes of the HLJ-PFBC patient (red) and normal controls revealed by SYBR Green dye- and TaqMan probe-based assays. Control-mix indicated a mixture of cDNA templates from six other normal controls. (E) Fusion protein expression of the PDGFB-Flag construct in the HEK293T cell line. The c.329T>C and c.356T>C of PDGFB are two previously reported PFBC-causative mutations. CHX (cycloheximide), a protein synthesis inhibitor; MG132, a ubiquitin-proteasome inhibitor. (F) The sketch of the mRNA splicing process of PDGFB c.456+1G>C.
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F2
Her physical examination revealed slightly elevated blood pressure (130/100 mmHg), and biochemical examination showed normal levels of serum calcium, phosphate, magnesium, calcitonin, and PTH. However, cranial CT revealed bilateral and symmetrical calcification in the basal ganglia, thalamus, and cerebellum (Figure 2A). A splice donor mutation (c.456+1G>C), located at the initiation of intron 4 of PDGFB (NM_002608.4), was suspected to be the causative mutation in this patient (Figure 2B). TA-cloning sequencing revealed that the c.456+1G>C mutation resulted in an in-frame deletion of 21 bp of nucleotides in vivo (Figure 2C). The relatively lower numbers of mutant TA clones (mt/wt clones = 22/44) and lower heights of cDNA sequencing peaks indicated that the newly deleted transcript was either undergoing the NMD process, the compensatory effect of the wild-type allele, or both, in the peripheral blood. Quantitative real-time PCR using SYBR Green dye and TaqMan probes revealed that the overall expression of PDGFB mRNA was increased by 2.47-fold when compared with age- and sex-matched controls, while the expression of the wild-type allele was increased by more than 3-fold (1.55 vs. 0.50%) (Figure 2D). Mutant PDGFB protein might be more stable than wild-type PDGFB and may induce the compensatory expression of wild-type protein in the HEK293T cell line (Figure 2E). The c.456+1G>C mutation effect on the PDGFB mRNA splicing process is illustrated in Figure 2F.