McArdle disease is an autosomal recessive disorder caused by the absence of muscle glycogen phosphorylase, which leads to blocked muscle glycogen breakdown. We used three different cellular models to evaluate the efficiency of different read-through agents (including amlexanox, Ataluren, RTC13 and G418) in McArdle disease. The first model consisted of HeLa cells transfected with two different GFP-PYGM constructs presenting the Pygm p.R50X mutation (GFP-PYGM p.R50X and PYGM Ex1-GFP p.R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the PYGM Ex1-GFP p.R50X construct. As these plasmids encode murine Pygm cDNA without any intron sequence, their transfection in cells would allow for analysis of the efficacy of readthrough agents with no concomitant nonsense-mediated decay interference. The third model consisted of skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p.R50X mutation in the Pygm gene). We found no evidence of read-through at detectable levels in any of the models evaluated. We performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction, identifying a potential role for nucleotide positions −9, −8, −3, −2, +13 and +14 (the first nucleotide of the stop codon is assigned as +1). The Pygm p.R50X mutation presents TGA as a stop codon, G nucleotides at positions −1 and −9, and a C nucleotide at −3, which potentially generate a good context for read-through induction, counteracted by the presence of C at −2 and its absence at +4.
Divalent metal-iron transporter 1 (DMT1) is a mammalian iron transporter encoded by the SLC11A2 gene. DMT1 has a vital role in iron homeostasis by mediating iron uptake in the intestine and kidneys and by recovering iron from recycling endosomes after transferrin endocytosis. Mutations in SLC11A2 cause an ultra-rare hypochromic microcytic anemia with iron overload (AHMIO1), which has been described in eight patients so far. Here, we report two novel cases of this disease. The first proband is homozygous for a new SLC11A2 splicing variant (c.762 + 35A > G), becoming the first ever patient reported with a SLC11A2 splicing mutation in homozygosity. Splicing studies performed in this work confirm its pathogenicity. The second proband harbors the previously reported DMT1 G75R mutation in homozygosis. Functional studies with the G75R mutation in HuTu 80 cells demonstrate that this mutation results in improper DMT1 accumulation in lysosomes, which correlates with a significant decrease in DMT1 levels in patient-derived lymphoblast cell lines (LCLs). We also suggest that recombinant erythropoietin would be an adequate therapeutic approach for AHMIO1 patients as it improves their anemic state and may possibly contribute to mobilizing excessive hepatic iron.
An autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III) is caused by a missense mutation in the KIF23 gene whose protein product, mitotic kinesin-like protein (MKLP1), is part of the centralspindlin complex involved in cytokinesis. Several case reports suggested the existence of an autosomal recessive inheritance form of CDA III so far not genetically characterized. By means of whole exome sequencing in a Spanish CDA III family with healthy parents, we identified in the male proband a novel homozygous missense mutation p.Pro432Ser in the RACGAP1 gene, which encodes for the RACGAP1 protein (Rac GTPase-activating protein 1, also known as MgcRacGAP or CYK-4), the partner of MKLP1 in the centralspindlin complex. A second CDA III Spanish patient has a different rare and novel homozygous missense mutation, p.Thr220Ala, in the RACGAP1 gene. Both patients presented with macrocytic anemia, aberrant multinucleated erythroblasts in the bone marrow typically seen in CDA III cases, no iron overload and skull defects secondary to severe anemia. Silencing of RACGAP1 using siRNA in HeLa cells mimics the cytokinesis defect observed in the bone marrow of our patients. Both mutations disrupt normal cytokinesis and alter the GTPase balance in patients' cells. We conclude that the autosomal recessive form of CDA type III is caused by mutations in the RACGAP1 gene, encoding for RACGAP1 protein, which is the partner of MKLP1 in the centralspindlin complex critical for cytokinesis and now both proteins are associated with CDA type III. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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