In 2010, an 8 year old female experiencing obstructive apnea, episodes of vomiting and dehydration, ketotic hypoglycemia with illness, fatigue, exercise intolerance, dilated cardiomyopathy (left ventricle dilation), and seizure-like episodes was referred to the NIH Undiagnosed Diseases Program. To identify potentially pathogenic DNA sequence variants coding mutations in the patient’s genome, exome sequencing was performed on the patient, on both unaffected parents, and on 3 unaffected siblings. A frameshift truncating variant in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1, was identified. As a result of the deleterious nature of the variant, the NKCC1 mutation received the highest priority. The mutant cDNA harboring an 11 bp deletion in exon 22 was created for expression in Xenopus laevis oocytes. In contrast to the wild-type cotransporter, which demonstrated a significant bumetanide-sensitive and hypertonic-stimulated K+ uptake, the mutant cotransporter was non-functional. Western blot analysis of oocytes expressing a c-myc tagged mutant cotransporter revealed a molecular size consistent with the formation of a dimer, while very little signal was observed at the size of a truncated monomer. This is in contrast to oocytes expressing wild-type cotransporters which expectedly showed monomer and dimer forms of the cotransporter. Co-expression of mutant NKCC1 with wild-type NKCC1 in oocytes did not result in a dominant-negative effect. K+ influx measurements in fibroblasts isolated from the patient revealed that 50% of the K+ influx was ouabain-sensitive or mediated by the Na+/K+ pump, whereas 41% of the flux was bumetanide-sensitive or mediated by NKCC1. Addition of ouabain and bumetanide resulted in 87% reduction in K+ influx. When the cells were exposed to hypertonicity, there was no activation of NKCC1, neither in wild-type fibroblasts nor fibroblasts isolated from patient. The bumetanide-sensitivity, however, disappeared in mutant but not wild-type fibroblasts, indicating some deleterious effect of the mutant cotransporter. Western blot analysis of protein lysates from patient’s fibroblasts also showed a larger amount of dimer when compared to HEK293 cells which were used as controls. Expression of fluorescently-tagged mutant and wild-type transporters in Hela cells revealed that the wild-type transporter helps the mutant reach the plasma membrane. Altogether, these data indicate that the mutation leads to a non-functional allele without some deleterious effect on the function of the cotransporter (bumetanide insensitivity under hypertonicity). Whether the mutant protein exerts deleterious effects on other cellular functions still needs to be explored.