S369x.com Reviews

S369X Mutation in KCNJ2

The s369x mutation in KCNJ2 disrupts the endoplasmic reticulum (ER)-to-Golgi export signal by truncating the C-terminal domain. This results in the mRNA that has a nonsense codon more than 50-55 nucleotides upstream of the splicing-generated exon-exon junction, which may trigger nonsense-mediated decay (NMD) or nonstop mRNA decay pathway.

Real-time RT-PCR quantification was used to identify possible mRNAs that may be prone to NMD or nonstop decay pathway. Interestingly, some mutations leading to mRNAs with premature termination codons, such as Q66X, L359X and S369X, were highly sensitive to NMD or nonstop mRNA decay, while others, such as V136fs75X and C432fs8X, had no effect on the mRNA level.

Phenotypes

Phenotypes of this rare gene variant vary from mild to severe. In one case (#H146), a 9-year-old boy exhibits mild coarsening of facial features, macrocephaly and moderate joint stiffness, but not enough to derail his otherwise unremarkable development.

The mutation also carries the unfortunate side effect of eliminating an endoplasmic reticulum (ER) to Golgi transport pathway. This is most apparent when comparing current recordings from Chinese hamster ovary cells coexpressing KCNJ2 WT and S369X subunits. However, when the mutant is fused to its wild-type cousin, the efficacy of the ER transport system is restored to a large extent and the currents are on par with those recorded from WT+S369X-transfected cells (-84 +- 14 pA/pF vs. -542 +- 46 pA/pF).

The most pronounced phenotypes of this rare gene variant are akin to the classic triad of dysmorphic features, periodic paralysis and electrocardiographic arrhythmia. In fact, the q-o-m has been coined as the Andersen Tawil syndrome by some experts.

Molecular Characterization

Using multiple techniques, we were able to molecularly characterize seventeen patients with mucopolysaccharidosis II (MPSII). We identified s369x alleles consisting of nonsense (Q66X, L359X, S369X), frameshift (C432fs, V136fs) mutations and a deletion (G394_X551) involving exon 9 in the gene KCNJ2 that encodes INWARD RECTIFIER POTASSIUM CHANNELS.

Among the mutations, we found that most of them introduced premature termination codons in mRNAs and were potentially sensitive to nonsense-mediated decay pathway. This mRNA surveillance mechanism is triggered by mutations that introduce a premature termination codon more than 50-55 nucleotides upstream of a splicing-generated exon-exon junction and is followed by nonstop decay, aimed at depleting cellular mRNAs lacking in-frame termination codons.

We also found that the S369X mutation leads to a loss of trafficking in the ER and a partial restoration of Kir2.1 subunit expression in the plasma membrane when coexpressed with WT. These findings suggest that a possible therapeutic approach might be enzyme enhancement therapy (EET). This strategy involves the use of pharmacological chaperones to rescue misfolded or unstable proteins.

Interactions with Other Proteins

Kir2.1 is a cytoplasmic C-terminal protein that exports from the endoplasmic reticulum (ER) to the Golgi apparatus via the ER-to-Golgi export signal. A truncation in the C-terminus of Kir2.1, induced by S369X mutations, eliminates this signal and reduces trafficking. However, this trafficking deficiency can be partially rescued by directly assembling with the WT protein, resulting in limited restoration of plasma membrane localization and channel function.

We also found that current recordings from Chinese hamster ovary cells transfected with KCNJ2-S369X subunits exhibited significantly smaller K(+) currents compared with those of KCNJ2 wild type (WT) (1 mg each) (-84 +- 14 pA/pF versus -542 +- 46 picoamperes per picofarad [pA/pF]; -140 mV; P0.0001). Coexpression of WT and S369X subunits did not show dominant negative suppression but resulted in larger currents than those of WT+S369X subunits (724 +- 98 pA/pF>-[84+542] pA/pF; 1 mg each; -140 mV). This suggests that S369X mutation may have a broader effect on channel trafficking than previously thought.

Clinical Significance

A mutation in the KCNJ2 gene, S369X, impedes trafficking and leads to a limited form of Andersen Tawil syndrome (ATS). Despite the fact that the S369X protein is truncated at its C-terminal, the mutant still possesses the requisite endoplasmic reticulum (ER) to Golgi transporter motif. This feature allows it to form a functional channel with the right pharmacological formulation. Current recording from Chinese hamster ovary cells expressing the mutant and WT subunits yielded a significantly smaller K(+) current in comparison to that of the wild type. A mild phenotype, consisting of dysmorphic features, periodic paralysis and cardiac arrhythmia, has been observed in this Taiwanese family.