PMC10200209_01
Neuroimaging of the patient with GRN mutation. A) MRI showing left-side atrophy in the frontal, temporal, and parietal lobes. B) FDG-PET showing left-side hypometabolism in the frontal, temporal, and parietal lobes. C) AV45-PET showing absence of Abeta deposition. D) APN1607 PET showing absence of tau deposition.
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We observed marked lateral atrophy (Fig. 1A) and hypometabolism (Fig. 1B) in the left cortex in the frontal, temporal, and parietal lobes by MRI and 18F-FDG-PET. To exclude early-onset Alzheimer's disease (AD), we conducted amyloid (AV45; Fig. 1C) and tau (APN1607; Fig. 1D) PET scanning but found no evidence of pathologic protein deposition. The translation of gene transcripts can be disrupted by mutations in splicing elements or splicing signal sequences or by the introduction of premature termination codons (Supplementary Figure 1A). The 45-bp deletion at the intron 10/exon 11 boundary of the GRN gene was expected to affect the 5' splice site of exon 11 and result in exon skipping (Supplementary Figure 1B). The results of the bioinformatic analysis showed that the deletion may also lead to a frameshift and introduction of a premature stop codon (p.Val473Argfs*8) (SupplementaryFigure 1C).
Genetic analysis of the GRN gene. A) Pedigree of the patient with GRN mutation. B) PCR amplification products across the deletion at the intron 10/exon 11 boundary of GRN from the patient and a wild-type control were examined by agarose gel electrophoresis. The 367-bp band corresponds to the wild-type allele. A 322-bp fragment corresponding to the heterozygous mutated allele with the 45-bp deletion was amplified from the gDNA of the patient. C) Left panel: Agarose gel electrophoresis of the long-range RT-PCR products revealing only the 2081-bp cDNA fragment corresponding to the predicted canonical transcripts in both the patient and control subject. Right panel: Agarose gel electrophoresis of shorter RT-PCR products showing only the 1071-bp band expected in both the patient and control. cDNA, complementary DNA; gDNA, genomic DNA. D) Upper lane: Sequence analysis of the gDNA PCR product of the patient revealed a c.1414-14_1444delCCCTTCCCCGCCAGGCTGTGTGCTGCGAGGATCGCCAGCACTGCT mutation in heterozygous state. Lower lane: Sanger sequencing of the RT-PCR products of the patient revealed the canonical transcripts. We found a substantial reduction in the amount of mutant allele which is virtually absent. E) GRN gene expression was analyzed by qRT-PCR using total RNA extracted from peripheral leukocytes of the patient with primer sets corresponding to sequences within exons 4-5 (left panel) and exons 10-11 (right panel). A -~50% decrease in the GRN product (normalized to RPS17 level) was observed in the patient compared with the same product in the 3 wild-type control subjects. Data are expressed as Mean+-SD of 3 independent experiments. **p < 0.01 (Mann-Whitney U test). F) Plasma PGRN levels (ng/ml) in the patient carrying the GRN Val473fs mutation (red triangle), 5 FTD patients without GRN mutations (blue squares), and 5 healthy control subjects (green circles). Each data point represents an individual. Black horizontal lines indicate the median plasma PGRN levels of FTD patients without GRN mutations and healthy controls.
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The patient's pedigree is shown in Fig. 2A. The results confirmed a heterozygous 45-bp deletion in the GRN gene in the proband (Fig. 2B, D). Sanger sequencing of the RT-PCR products from both directions showed only canonical transcripts (Fig. 2D lower lane). Agarose gel electrophoresis of long-range RT-PCR products (exon 0 UTR-exon 12 UTR) revealed only the expected 2081-bp band in both the patient and control, with no abnormal bands (Fig. 2C). Agarose gel electrophoresis of these RT-PCR products revealed only the expected 1071-bp band in both the patient and control and Sanger sequencing in both directions confirmed only canonical transcripts in the patient (Fig. 2C). To corroborate these results, we quantified mRNA levels in the patient's leukocytes by qRT-PCR; the results showed that the GRN transcript level was approximately 0.5-fold lower in the patient compared with the 3 wild-type controls (Fig. 2E). PGRN levels in the patient carrying the Val473fs mutation, 5 FTD patients without GRN mutations, and 5 healthy control subjects were measured by ELISA (Fig. 2F). PGRN level in the patient with the Val473fs mutation (74.15 ng/ml) was one-third of the level in the 10 non-mutation carriers (Mean+-SD, 216.40+-21.01 ng/ml, and range, 142.47-325.80 ng/ml).
Schematic representation of GRN mutations in China. The schematic representation of the 12 coding exons (green boxes) and UTR of GRN shows all pathogenic mutations identified in Chinese patients reported to date. Most of the mutations cause premature termination of the coding sequence, leading to the degradation of the mutant GRN mRNA by nonsense-mediated decay. The c.1414-14_1444del mutation identified in this study is indicated in red.
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Figure 3 illustrates the GRN mutations identified in China, and related clinical and genetic information is shown in Table 1.