blz His brother individual B II is months old He was

His brother, individual B.II-4, is 11 months old. He was born at term with intrauterine growth retardation. His birth weight was 2.2 kg (<2nd percentile), his length was 47 cm (10th percentile), and his head circumference was 36 cm (75th percentile). He failed to thrive in the first months of life. He was found to have a coarctation of the aorta, which was surgically corrected at the age of 7 weeks. An initial renal ultrasound revealed multiple echogenic foci within the medulla of the left kidney, suggestive of nephrocalcinosis, which resolved on follow-up imaging. At the current age of 11 months, he has global developmental delay. He has a head lag and cannot sit unless supported. He can roll over and reach for objects. He can fix and follow visually. He can smile but does not seem to recognize his mother. He has not had any seizures. On his most recent examination (age 11 months), his weight was 7.5 kg (<3rd percentile), but he was gaining weight very slowly. His height was 72 cm (25th percentile), and head circumference was 45 cm (25th percentile). He had dysmorphic features similar to those of his affected brother. He had a flat, triangular face with a smooth philtrum, thin lips, widely spaced eyes, a depressed nasal bridge, and a facial hemangioma. He was hypotonic and hyporeflexic. The exome of individual B.II-3 was clinically sequenced with the Roche Nimblegen VCRome v.2.1 exome capture kit and Illumina HiSeq 2000 at the Whole Genome Laboratory at Baylor College of Medicine and analyzed as previously described. No pathogenic or likely pathogenic variants that could explain the disease were reported. Interestingly, we found that B.II-3 bears a homozygous c.526C>T (p. Arg176Trp) variant in SLC45A1 (GenBank: NM_001080397.1). This variant is very rare (MAF = 0.00001660 in the ExAC Browser) and predicted to be damaging by PolyPhen-2 (score = 0.989) and SIFT (score = 0.000). Segregation of the variant was verified by Sanger sequencing in the family. B.II-4 was also found to be homozygous, whereas his unaffected siblings and parents were heterozygous for this variant (Figure 1A). SLC45A1, also known as PAST-A and DNB5, is predominantly expressed in the developing and adult brain, including the blz and cerebellum.3, 7 Several observations suggest that SLC45A1 functions as a glucose transporter. First of all, its primary structure displays classic features of glucose transporters belonging to the major facilitator superfamily, including the presence of 12 putative membrane-spanning helices, a long cytoplasmic loop between TM6 and TM7, and several motifs associated with the transport of sugar. Moreover, transfection studies have established that SLC45A1 is a membrane-associated protein that possesses glucose and galactose (but not sucrose or fructose) transport activity. The uptake of glucose in this assay was pH dependent, such that it increased significantly at low pH value. Finally, the Drosophila gene slc45-1, a homolog of SLC45A1 (and an ortholog of SLC45A2), also encodes a membrane protein that can transport sugar. Collectively, these observations strongly suggest that SLC45A1 is a H+ and glucose symporter. Both identified variants, c.526C>T (p. Arg176Trp) and c.629C>T (p.Ala210Val), are located in exon 3 of SLC45A1 (GenBank: NM_001080397.1). Arg176 is located in the extracellular loop just after the third transmembrane domain, whereas Ala210 is positioned at the beginning of the intracellular loop just after the fourth transmembrane domain (Figure 1B). Arg176 and Ala210, as well as their flanking amino acids, are conserved in mammals, amphibians, and birds (Figure 1C). We sought to biologically validate these variants by examining their effect on the glucose transport activity of SLC45A1. SLC45A1-encoding cDNA was inserted into the pFN21A expression vector (Promega). Each missense mutation (c.526C>T and c.629C>T) was introduced to a separate SLC45A1 plasmid with QuikChange Multi Site-Directed Mutagenesis Kits (Agilent Technologies). We sequenced the entire constructs to verify the presence of the mutations and the absence of artifacts. We transfected COS-7 cells with the indicated plasmid by using the cationic lipid-based transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific). The glucose transport assay was performed as described by Shimokawa et al. In brief, 64 hr after transfection, cells were washed with KRPH buffer (20 mM HEPES [pH 7.4], 5 mM Na2HPO4, 1.25 mM MgSO4, 1.25 mM CaCl2, 136 mM NaCl, and 4.7 mM KCl). Glucose uptake was determined by incubation with KRPH buffer (pH 6.8) containing 50 μM 3H-labeled 2-deoxy-d-glucose (5–10 Ci/mmol; NET328A250UC, lot no. 1684189, PerkinElmer) for 30 min. Then, the cells were washed with KRPH buffer and solubilized in 1% Triton X-100. The radioactivity incorporated into the cells was measured with a liquid scintillation counter.