title: 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.
authors.affiliation: Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669, 11° andar, São Paulo, SP, 04039-032, Brazil.|Laboratory of Structural and Functional Neurobiology, Department of Biophysics, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Rolf M
authors.initials: RM
authors.lastname: Paninka
authors.affiliation: Department of Psychobiology, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Diego R
authors.initials: DR
authors.lastname: Mazzotti
authors.affiliation: Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669, 11° andar, São Paulo, SP, 04039-032, Brazil.
authors.forename: Marina M L
authors.initials: MM
authors.lastname: Kizys
authors.affiliation: Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669, 11° andar, São Paulo, SP, 04039-032, Brazil.
authors.forename: Angela C
authors.initials: AC
authors.lastname: Vidi
authors.affiliation: Department of Psychobiology, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Hélio
authors.initials: H
authors.lastname: Rodrigues
authors.affiliation: Laboratory of Integrative Biology and Metabolism, Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Silas P
authors.initials: SP
authors.lastname: Silva
authors.affiliation: Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669, 11° andar, São Paulo, SP, 04039-032, Brazil.
authors.forename: Ilda S
authors.initials: IS
authors.lastname: Kunii
authors.affiliation: Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669, 11° andar, São Paulo, SP, 04039-032, Brazil.
authors.forename: Gilberto K
authors.initials: GK
authors.lastname: Furuzawa
authors.affiliation: Laboratory of Structural and Functional Neurobiology, Department of Biophysics, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Manoel
authors.initials: M
authors.lastname: Arcisio-Miranda
authors.affiliation: Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669, 11° andar, São Paulo, SP, 04039-032, Brazil. mrdsilva@unifesp.br.
authors.forename: Magnus R
authors.initials: MR
authors.lastname: Dias-da-Silva
mesh_terms: D053447:Channelopathies / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods; D059472:Exome; D020022:Genetic Predisposition to Disease; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C000613708:KCNJ17 protein, human; C546860:KCNJ18 protein, human; C489463:Kir2.2 channel; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms
keywords: Electrophysiology; Kir2.6; Potassium inward rectifier channel; Thyrotoxic periodic paralysis; WES; WGS