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  • To evaluate SLC A abundance after transfection

    2021-09-16

    To evaluate SLC45A1 abundance after transfection, we performed western blots. Transfected COS-7 Fosinopril sodium were lysed and separated for membrane fraction. Equal amounts of proteins from membrane fractions were resolved by SDS-PAGE, transferred to a nitrocellulose filter, and immunoblotted with an anti-SLC45A1 antibody (1:1,000; ab123314, lot no. GR86331-1, Abcam). We re-probed blots with an anti-β-actin antibody (1: 1,000; 4967L, Cell Signaling Technology) to monitor the quantity and integrity of cell-surface protein. SLC45A1 and β-actin were detected by chemiluminescence with an ECL system (GE Healthcare) and visualized with a Lumino Graph imaging analyzer (ATTO). These experiments showed that the levels of SLC45A1 variants were similar to wild-type (WT) levels, suggesting that none of the variants tested affect the stability of the protein (Figure 2A). The accumulation of WT SLC45A1 in COS-7 cells led to 4.5-fold more glucose transport activity than in mock-transfected cells (Figure 2B). The glucose transport activity of the p.Arg176Trp and p.Ala210Val SLC45A1 variants was significantly decreased by approximately 50% (p = 0.013) and 33% (p = 0.008), respectively, in comparison with that of intact SLC45A1. These results indicate that Arg176 and Ala210 are critical for the glucose transport activity of SLC45A1. Anazi et al. recently reported a homozygous c.167T>C (p.Ile56Thr) missense mutation in SLC45A1 (or c.269T>C [p.Ile90Thr]; GenBank: NM_001080397.2) in a 5-year-old girl with developmental delay, ataxia, and Dandy-Walker malformation. Although this phenotype somewhat overlaps that of our affected individuals, none of our individuals had cerebellar abnormalities. Unlike the variants we reported herein, which lie in the boundaries of the transmembrane regions of SLC45A1, the p.Ile56Thr variant is located the N-terminal intracellular domain. Moreover, we found that this variant had no significant effect on the glucose transport activity of SLC45A1 (Figure 2). Thus, p.Ile56Thr either is Fosinopril sodium not pathogenic or affects the function of the protein in a manner that is not measured by our assay. Combined with functional evidence that these mutations affect protein function, the presence of rare homozygous variants segregating in affected members of two unrelated families with similar phenotypes strongly suggests that the variants identified in SLC45A1 are pathogenic. The main clinical features of the four affected individuals are moderate to severe ID, epilepsy, and associated neuropsychiatric features (see Table 1 for a summary of clinical features). Indeed, three individuals have neuropsychiatric disorders, and the fourth individual is too young for symptoms to be apparent (11 months). Two individuals have autistic traits, and two have varying degrees of anxiety and obsessive-compulsive traits. Extra-neurologic features were observed: cardiac abnormalities were noted in two individuals (coarctation of the aorta and PDA), and renal abnormalities were noted in two siblings (renal agenesis and nephrocalcinosis). It is unclear at this time whether these features are related to SLC45A1 mutations. Interestingly, bilateral nephrocalcinosis has been previously observed in one individual with epileptic encephalopathy associated with a de novo mutation in SLC1A2. Of note, the individuals from family B, who display more severe developmental delay and ID, carry a missense mutation that results in a greater impairment of glucose transport, as measured in our in vitro assay (Figure 2B). SLC45A1 was initially identified in the context of a screen for genes upregulated by hypercapnia in the ventral medullary surface (VMS) of the medullary oblongata. The VMS is the site of chemosensitive neurons that sense acidosis and hypercapnia and induce hyperventilation. In vitro, SLC45A1 possesses glucose transport activity that is enhanced by acidification. All together, these observations suggest that SLC45A1 might play a role in the regulation of respiration, possibly by controlling energy production in VMS neurons. Additional work would be needed to further confirm these findings. Although our affected individuals did not show any gross abnormalities of breathing, we cannot exclude the possibility that they display subtle aberrations of respiratory control. SLC45A1 also localizes to the cortex. Its function in this part of the brain remains unknown. However, it appears likely that SLC45A1 also functions as a glucose transporter in this area of the brain in view of its structure and activity in transfected cells.