Novel GAA sequence variant c.1211 A > G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease |
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Authors: | MI Nilsson MA Kroos AJ Reuser E Hatcher M Akhtar ME McCready MA Tarnopolsky |
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Institution: | 1. Department of Pediatrics, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8 3Z5;2. Department of Pathology and Molecular Medicine, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8 3Z5;3. Department of Clinical Genetics and Pediatrics, Erasmus MC University Medical Center, Rotterdam, Netherlands;4. Center for Lysosomal and Metabolic Disease, Erasmus MC University Medical Center, Rotterdam, Netherlands |
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Abstract: | Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO = c.1211A > G and c.1798C > T; AO = c.1211A > G and c.692 + 5G > T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A > G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A > G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C > T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692 + 5G > T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A > G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C > T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692 + 5G > T (vs. c.1798C > T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother). |
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Keywords: | 4-MU 4-methylumbelliferone A adenosine AO adult onset Asp aspartic acid Arg arginine C cytidine cDNA DNA complementary to RNA CON control(s) CK creatine kinase CRIM cross-reactive immunologic material Cys cysteine ERT enzyme replacement therapy FEV forced expiratory volume FVC forced vital capacity G guanosine GAA acid α-glucosidase (gene) GAA acid α-glucosidase (protein) Gly glycine GSD II glycogen storage disorder type II HET heterozygous HRP horseradish peroxidase IO infantile onset IVS intervening sequence kDa kilodalton(s) NaAc sodium acetate PCR polymerase chain reaction RT room temperature T thymidine TRIS tris-buffered saline 3&rsquo UTR three prime untranslated region WB western blotting |
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