Whole genome and exome sequencing realignment supports the assignment of KCNJ12, KCNJ17, and KCNJ18 paralogous genes in thyrotoxic periodic paralysis locus: functional characterization of two polymorphic Kir2.6 isoforms

Abstract

Next-generation sequencing (NGS) has enriched the understanding of the human genome. However, homologous or repetitive sequences shared among genes frequently produce dubious alignments and can puzzle NGS mutation analysis, especially for paralogous potassium channels. Potassium inward rectifier (Kir) channels are important to establish the resting membrane potential and regulating the muscle excitability. Mutations in Kir channels cause disorders affecting the heart and skeletal muscle, such as arrhythmia and periodic paralysis. Recently, a susceptibility muscle channelopathy-thyrotoxic periodic paralysis (TPP)-has been related to Kir2.6 channel (KCNJ18 gene). Due to their high nucleotide sequence homology, variants found in the potassium channels Kir2.6 and Kir2.5 have been mistakenly attributable to Kir2.2 polymorphisms or mutations. We aimed at elucidating nucleotide misalignments by performing realignment of whole exome sequencing (WES) and whole genome sequencing (WGS) reads to specific Kir2.2, Kir2.5, and Kir2.6 cDNA sequences using BWA-MEM/GATK pipeline. WES/WGS reads correctly aligned 26.9/43.2, 37.6/31.0, and 35.4/25.8 % to Kir2.2, Kir2.5, and Kir2.6, respectively. Realignment was able to reduce over 94 % of misalignments. No putative mutations of Kir2.6 were identified for the three TPP patients included in the cohort of 36 healthy controls using either WES or WGS. We also distinguished sequences for a single Kir2.2, a single Kir2.5 sequence, and two Kir2.6 isoforms, which haplotypes were named RRAI and QHEV, based on changes at 39, 40, 56, and 249 residues. Electrophysiology records on both Kir2.6_RRAI and _QHEV showed typical rectifying currents. In our study, the reduction of misalignments allowed the elucidation of paralogous gene sequences and two distinct Kir2.6 haplotypes, and pointed the need for checking the frequency of these polymorphisms in other populations with different genetic background.

Increased KCNJ18 promoter activity as a mechanism in atypical normokalemic periodic paralysis

“…However, the four-fold SNV in Kir2.6, which is located on a single allele of our patient, has been recently identified as a natural variant, which apparently has no functional difference to wild-type channels. 13 …”

“…The four-fold SNV in Kir2.6 R39Q/R40H/A56E/I249V was previously studied under high extracellular potassium concentration (20 mM), so only inward currents could be analyzed and not the physiologically relevant outward conductance and physiologic rectification pattern. 13 Therefore, we recorded the variant under physiologic saline conditions by injecting similar amounts of cRNA in X laevis oocytes and recording currents 48 hours after injection. In TEVC recordings, the current rectification was indistinguishable between the two KCNJ18 variants ( figure 1, A and B ).…”

Novel lincRNA Susceptibility Gene and Its Role in Etiopathogenesis of Thyrotoxic Periodic Paralysis

“…Edge BioSystems (Gaithersburg, MD) was used to perform exome sequencing, variant calling, and annotation. The sequences were aligned to the GRCh37/hg19 human genome assembly using the CLC Bio Genomic Workbench (Qiagen Bioinformatics, Redwood City, CA) and studied using the whole exome sequencing (WES) pipeline for paralogous KCNJ genes, as previously reported [ 9 ].…”

A Missense Mutation in <em>KCNJ12</em> Was Strongly Associated with Beef Cattle Stature

“…Thus the KCNJ12 gene possibly involved in the regulation of muscle membrane properties and excitation-contraction coupling [3]. Expectedly, mutations in Kir channels can cause disorders affecting the heart and skeletal muscle, such as arrhythmia and periodic paralysis in human [4]. For example, non-synonymous coding single nucleotide polymorphisms (SNPs) of KCNJ12 are associated with pathogenesis of Rhabdomyosarcomas (RSCs) [5].…”