Nucleotide Sequence Homology Research Articles

Genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from South Korea, 2017.

SummaryPorcine epidemic diarrhoea virus (PEDV) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. Since the re‐emergence of massive PEDV outbreaks in South Korea in 2013−2014, domestic pig farms have continued to experience PED epidemics or endemics. This study represents the molecular characterization of PEDV isolates identified in diarrhoeic animals collected across the country in 2017. Initial sequencing analysis of the full‐length S genes revealed that 70% of the 2017 isolates (7/10) belong to the G2b subgroup, while the remaining isolates were classified as G1b. The data indicated that both variant G1b and global epidemic G2b strains were responsible for current PED outbreaks in South Korea. The 2017 G1b and G2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the S gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding Korean prototype G1b and G2b strains, respectively. In an interesting manner, one G2b‐like KNU‐1705 strain was found to possess a large 39‐nucleotide deletion in the ORF1a region theoretically encoding nonstructural protein 3. Phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global G1b or G2b strains but formed different branches within the same genogroup. These results indicate that PEDVs undergo continuous evolution in the field. In addition, one 2017 PEDV strain, KOR/KNU‐1705/2017, was successfully isolated and propagated in Vero cells. The antisera raised against the Korean prototype 2014 G2b strain efficiently neutralized KNU‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 PEDV strains. Our data advance the understanding of the molecular epidemiology and antigenicity of PEDV circulating in South Korea.

Detection of Co-Infection of Notocactus leninghausii f. cristatus with Six Virus Species in South Korea.

Co-infection with two virus species was previously reported in some cactus plants. Here, we showed that Notocactus leninghausii f. cristatus can be co-infected with six different viruses: cactus mild mottle virus (CMMoV)-Nl, cactus virus X (CVX)-Nl, pitaya virus X (PiVX)-Nl, rattail cactus necrosis-associated virus (RCNaV)-Nl, schlumbergera virus X (SchVX)-Nl, and zygocactus virus X (ZyVX)-Nl. The coat protein sequences of these viruses were compared with those of previously reported viruses. CMMoV-Nl, CVX-Nl, PiVX-Nl, RCNaV-Nl, SchVX-Nl, and ZyVX-Nl showed the greatest nucleotide sequence homology to CMMoV-Kr (99.8% identity, GenBank accession NC_011803), CVX-Jeju (77.5% identity, GenBank accession LC12841), PiVX-P37 (98.4% identity, GenBank accession NC_024458), RCNaV (99.4% identity, GenBank accession NC_016442), SchVX-K11 (95.7% identity, GenBank accession NC_011659), and ZyVX-B1 (97.9% identity, GenBank accession NC_006059), respectively. This study is the first report of co-infection with six virus species in N. leninghausii f. cristatus in South Korea.

VIRION DNAs STRUCTURE OF TEMPERATE ERWINIOPHAGES 49 AND 59

The aim was to conduct a comparative study of the phages 49 and 59 virion DNAs. Methods. Properties of phage DNA were studied using the standard restriction and bioinformatic analyses. Results. The genomes of erwiniaphages 49 and 59 encode 84 open reading frames.57 and 52 hypothetical proteins with unknown function were identified within the genome of phage 49 and 59, respectively. The search through NCBI databases indicates that phage 49 has no viral homologs among genomes of prokaryotic viruses. In case of phage 59, it is Cronobacter sakazakii bacteriophage ENT47670. It was identified that the phages 49 and 59 share 47 % homologies of nucleotide sequences. Most of identical open reading frames belong to the structural region of the genome and the host cell lysis module. It has been shown that the genomes of both bacteriophages are permuted and packaged according to the headful mechanism. Phage 59 is characterized by circular permutation of the genome, while phage 49 has discrete permutation. The virion DNA of phage 49 has an abnormal cleavage site for SmaI restriction enzyme that, unlike the rest SmaI-sites, is not completely hydrolyzed. Conclusions. Temperate bacteriophages 49 and 59 are not identical to the other known prokaryotic viruses and belong to unique phages of Siphoviridae family.

Analysis of population genetic diversity of mosquitoes from Shandong Province based on mitochondrial DNA cytochrome oxidase subunit I gene fragment

To explore the characteristics of gene sequence of mtDNA-COⅠ of Culex pipiens pallens from different geographical regions in Shandong Province and different resistant strains from the lab and five common mosquito species, and analyze the genetic diversity of these mosquitoes. Adult mosquitoes were collected from Jinan, Jining, Qingdao cities and other places in Shandong Province. The sensitive, dichlorvos-resistant, pyrethroid-resistant and propoxur-resistant strains were reared in the lab. Five species of mosquito (Cx. pipiens pallens, Cx. tritaeniorhynchus, Anopheles sinensis, Aedes albopictus, and Armigeres subalbatus) were collected from Jining City and identified in the lab. mtDNA-COⅠwas specifically amplified by PCR and sequenced. The gene sequences were compared and analyzed by the biological information systems, and the phylogenetic tree was constructed. The amplified mtDNA-COⅠfragments of Cx. pipiens pallens from eight different cities and four different resistant strains were 528 bp in length, with 67.4% A+T contents and two mutation sites. The nucleotide sequence homology among the different geographic strains was 99.95% and the gene sequences of the four resistant strains were the same, showing a high homogeny. The amplified mtDNA-COⅠfragments of the five species of mosquitoes were 528 bp with 408 conserved sites, 120 variable sites, 42 parsimony informative sites and 78 singleton sites. The A+T contents were between 65.7% and 68.0%. The nucleotide sequence homology among the different mosquito species was between 86.17% and 92.05%, and the molecular identification was consistent with the traditional morphological identification. The molecular phylogenetic study showed that the different species were clustered at their own branch at the species and genus levels, while genera Armigeres was distantly related to the others. mtDNA-COⅠcould not serve as the molecular marker to analyze the population genetic variation and phylogenesis of Cx. pipiens pallens from different geographical regions and different resistant strains, but it has species and genus specificities, which could be used for the identification of the mosquito species and genus.

Phylogenetic and genomic characterization of a novel atypical porcine pestivirus in China.

Atypical porcine pestivirus (APPV) has been considered a novel pestivirus and causative agent of congenital tremor type A-II. An APPV CH-GX2016 strain was characterized from newly born piglets with clinical symptoms of congenital tremor in Guangxi, China. The genome of APPV CH-GX 2016 strain was 11,475bp in length and encoded a polyprotein composed of the 3,635 amino acids. This genome sequence exhibited 88.0% to 90.8% nucleotide sequence homology with other APPV reference sequences in GenBank. Phylogenetic analysis further showed that APPV CH-GX is a novel pestivirus compared with previously described classical pestivirus strains. Therefore, APPV is present in pigs in China.

Characterization and Epidemiology of Pigeon Paramyxovirus Type-1 Viruses (PPMV-1) Isolated in Macedonia.

We have characterized in this study 10 PPMV-1 isolated from domestic pigeons and one PPMV-1 isolated from a feral pigeon in the period 2007-2012, using both classical methods (HI test and ICPI test) and molecular methods (RT-qPCR, RT-PCR, and nucleotide sequencing). Using phylogenetic analysis of partial fusion gene sequences, these viruses clustered with recent European PPMV-1 isolates (EU/re) within the genotype VIb/1. All isolates possessed virulent cleavage site motifs with variable morbidity and mortality in pigeons. The intracerebral pathogenecity indices of the five isolates ranged from 0.59 to 1.53. The repetitive isolation of PPMV-1 viruses for several consecutive years led toward establishing enzootic presence of the disease in pigeons. A high nucleotide sequence homology between the Macedonian isolates and EU/re isolates was shown. Co-circulation of different isolates in the same holdings was detected. This is the first study to extensively describe the molecular epidemiology of PPMV-1 isolated in Macedonia.

Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Coxsackievirus B3 (CVB3) is a member of the genus Enterovirus within the family Picornaviridae and is an important pathogen of viral myocarditis, which accounts for more than 50% viral myocarditis cases. VP1 is major capsid protein that this region has a low homology in both amino acid and nucleotide sequences among Enteroviruses. Therefore we have chosen this region for designed a set of RT-LAMP primers for CVB3 detection. For this the total RNA was extracted from 24-h post infected-HeLa cells with complete cytopathic effect (CPE), and applied to a one-step reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using CVB3-specific primers. The optimization of RT-LAMP reaction was carried out with three variables factors including MgSO4 concentration, temperature and time of incubation. Amplification was analyzed by using 2% agarose gel electrophoresis and ethidium bromide and SYBR Green staining. Our results were shown the ladder-like pattern of the VP1 gene amplification. The LAMP reaction mix was optimized and the best result observed at 4mM MgSO4 and 60°C for 90min incubation. RT-LAMP had high sensitivity and specificity for detection of CVB3 infection. This method can be used as a rapid and easy diagnostic test for detection of CVB3 in clinical laboratories.

Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection

The present study reports outbreaks of tilapia lake virus (TiLV), an emerging pathogen causing syncytial hepatitis of tilapia (SHT), in farmed tilapia in Thailand. Occurrence of the virus was confirmed by RT-PCR and nucleotide sequence homology to the TiLV from Israel. Diseased fish exhibited typical histopathological features of syncytial giant cells in the liver examined through H&E and semi-thin sections. Presence of intracytoplasmic viral particles was revealed by TEM. In situ hybridization using a specific DIG-labeled probe derived from a partial genome segment 3 of TiLV genome revealed multiple tissues tropism of the virus including liver, kidney, brain, spleen, gills and connective tissue of muscle. An alternative semi-nested RT-PCR protocol has been developed in this study for disease diagnosis. Additionally, comparative genetic analysis revealed genetic variations of Thailand-originated virus to the Israel TiLV strains, sharing 96.28 to 97.52% nucleotide identity and 97.35 to 98.84% amino acid identity. Outbreaks of TiLV in different continents might signal a serious threat to tilapia aquaculture globally.

Isolation and Identification of an Algicidal Bacterium Against Microcystis Aeruginosa

Water blooms have become a worldwide and serious environmental problem. Algicidal bacteria have attracted wide attention as possible agents for inhibiting algal water blooms recently. The aim was to isolation alage-lysin bacterium against Microcystis aeruginosa, so as to provide reference for the control of bloom. In this study, one strain of algae-lysing bacterium H2 was isolated from Chaohu Lake. To identification the H2 strain, its morphological characteristics and 16S rDNA gene sequence were studied. The results showed that the algicidal bacterium H2 is a Gram-negative bacterium. The 16S rDNA nucleotide sequence homology of strain H2 with many strains of Chryseobacterium sp all reached above 95 %. So H2 was identified as Chryseobacterium sp by morphology and homology research based on 16S rDNA.

Von Willebrand Factor Interacts with Surface-Bound C1q and Induces Platelet Rolling.

Premature atherosclerosis and thrombotic complications are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the high incidence of these complications cannot be explained by traditional risk factors alone, suggesting direct effects of an activated immune system on hemostasis. The unexpected nucleotide sequence homology between SLE patient-derived autoantibodies against complement C1q (Fab anti-C1q) and von Willebrand factor (VWF) led us to investigate a potential interaction between the complement and hemostatic systems on the level of initiating molecules. VWF was found to bind to surface-bound C1q under static conditions. The binding could specifically be inhibited by Fab anti-C1q and C1q-derived peptides. Under shear stress the C1q-VWF interaction was enhanced, resembling the binding of VWF to collagen I. Additionally, we could show that C1q-VWF complexes induced platelet rolling and firm adhesion. Furthermore, we observed VWF binding to C1q-positive apoptotic microparticles and cholesterol crystals, as well as increased VWF deposition in C1q-positive glomeruli of SLE patients compared with control nephropathy. We show, to our knowledge for the first time, binding of VWF to C1q and thus a direct interaction between starter molecules of hemostasis and the classical pathway of complement. This direct interaction might contribute to the pathogenic mechanisms in complement-mediated, inflammatory diseases.

Etiological monitoring and analysis of cases of acute flaccid paralysis in Hebei Province in 2011-2014

Objective: To identify poliomyelitis(polio)virus, the VP1 gene, and its nucleotide sequence in fecal samples from patients with acute flaccid paralysis(AFP)in Hebei Province in 2011-2014. Methods: A surveillance system for AFP was established in Hebei Province in 2011-2014 and registered in 2014. Stool samples, each weighing 5 g, were collected from 1 504, 15-year-old symptomatic patients with AFP, resulting in a total of 3 001 samples(1 497 patients provided duplicate samples and 7 provided single specimens). Poliovirus nucleic acid was extracted, the RNA was reverse transcribed, and a VP1 gene fragment was amplified with real-time PCR. The PCR products were sequenced to construct a phylogenetic tree and check the relatedness of the strains to the Sabin vaccine strain. A χ2 test was used to compare the differences in the incidence of infection in different years. Results: Poliovirus was isolated from 50(1.7%)of the 3 001 stool samples, 10 of which were type Ⅰ strains, 15 were type Ⅱ strains, 16 were type Ⅲ strains, and 9 were mixed-type strains. The positive rates for poliovirus in the years 2011-2014 were 1.0%(9/890), 1.5%(12/824), 2.2%(17/770), and 2.3%(12/517), respectively(χ2=2.24, P=0.525). Analyses of the VP1 nucleotide and amino acid sequence homologies revealed that the type Ⅰ, type Ⅱ, and type Ⅲ poliovirus strains shared nucleotide sequence homologies with the Sabin vaccine strain of 98.8%- 100%, 99.1%- 100%, and 99.2%- 100%, respectively, and amino acid sequence homologies of 98.6%- 100%, 98.3%-100% and 98.6%-100%, respectively. A VP1-based phylogenetic analysis showed that the variation rates for the poliovirus type Ⅰ, type Ⅱ, and type Ⅲ strains were 0.66%, 0.66%, and 0.55%, respectively. Conclusion: Only one poliovirus strain was detected in Hebei Province in 2011-2014, except for the type Ⅱ vaccine-derived poliovirus. The remaining strains were all similar to the Sabin vaccine strain, with high VP1 homology.

Endemic Variation of H9N2 Avian Influenza Virus in China.

Forty-two H9N2 subtype AIV strains were isolated from vaccinated commercial chickens in China from 2012 to 2015. Their HA genes had nucleotide sequence homology from 86.7% to 99.7%, and similarity to the classic vaccine strain was 88.6%-92.6%. A comparison was carried out with published HA genes (410 H9 strains) and whole genomes (306 strains) isolated in China during 2012-2015. Interestingly, 99.1% (448/452) of Chinese H9N2 AIV belonged to lineage h9.4.2, and 98.5% (445/452) of the viruses belonged to h9.4.2.5. Meanwhile, 99.6% (443/445) of lineage 9.4.2.5 viruses had PSRSSR↓GLF instead of PARSSR↓GLF motifs in the HA cleavage sites; 98.2% (444/452) of HA genes showed human receptor binding associated mutation Q226L. A total of 96.8% (337/348) of the viruses had three amino-acid deletions at 63-65 in the NA stalk, associated with enhanced virulence in chickens and mice; 97.1% (338/348) of M2 proteins had the S31N mutation associated with adamantane resistance in humans. Two H9 viruses isolated in this study were highly homologous to the human-origin H9N2 virus reported in 2013. The isolates were divided into four different genotypes (U, S, V, and W). Genotype S was the major one, accounting for 94.8% (330/348). Genotypes V and W were new reassortment genotypes, with genotype W recombined with the PB2 gene originating from the new wild waterfowl-like lineage. According to the cross-HI antibody titer data, HA gene evolution, and isolation history, the isolates were divided into A, B, and C antigenic groups successively. All the antigenic group viruses were found to circulate throughout China. This study emphasizes the importance of updated vaccine and strengthened veterinary biosecurity on poultry farms and trade markets.

Genetic features of livestock-associated Staphylococcus aureus ST9 isolates from Chinese pigs that carry the lsa(E) gene for quinupristin/dalfopristin resistance

Whole-genome sequencing (WGS) was used to investigate the genetic features of the recently identified lsa(E) gene in porcine S. aureus ST9 isolates. Three quinupristin/dalfopristin-resistant isolates harboring the lsa(E) gene (two MRSA and one MSSA) were sequenced. Phylogenetic analysis of 184S. aureus genomes showed that ST9 porcine isolates belong to a distinct sequence cluster. Further analysis showed that all isolates were deficient in the recently described type IV restriction-modification system and SCCmec type XII was identified in the two MRSA isolates, which included a rare class C2 mec gene complex. A 24kb ΨSCC fragment was found in the MRSA and MSSA isolates sharing 99% nucleotide sequence homology with the ΨSCCJCSC6690 (O-2) element of a ST9 MRSA isolate from Thailand (accession number AB705453). Comparison of these ST9 isolates with 181 publically available S. aureus genomes identified 24 genes present in all (100%) ST9 isolates, that were absent from the most closely related human isolate. Our analysis suggests that the sequenced quinupristin/dalfopristin-resistant ST9 lineage represent a reservoir of mobile genetic elements associated with resistance and virulence features.

MULTIFUNCin: A Multifunctional Protein Cue Induces Habitat Selection by, and Predation on, Barnacles

Foundation species provide critical resources to ecological community members and are major determinants of biodiversity. The barnacle Balanus glandula is one such species and dominates space among the higher reaches on wave-swept shores. Here, we show that B. glandula produces a 199.6-kDa glycoprotein (named "MULTIFUNCin"), and following secretion, a 390-kDa homodimer in its native state. MULTIFUNCin expression is localized in the epidermis, cuticle, and new shell material. Consequently, this molecule can specify upon contact the immediate presence of a live barnacle. Shared, conserved domains place MULTIFUNCin in the α2-macroglobulin (A2M) subgroup of the thioester-containing protein family. Although previously undescribed, MULTIFUNCin shares 78% nucleotide sequence homology with a settlement-inducing pheromone (SIP) of the barnacle, Amphibalanus amphitrite Based on this and further evidence, we propose that the two proteins are orthologues and evolved ancestrally in structural and immunological roles. More recently, they became exploited as chemical cues for con- and heterospecific organisms, alike. MULTIFUNCin and SIP both induce habitat selection (settlement) by conspecific barnacle larvae. In addition, MULTIFUNCin acts as a potent feeding stimulant to major barnacle predators (sea stars and several whelk species). Promoting immigration via settlement on the one hand, and death via predation on the other, MULTIFUNCin simultaneously mediates opposing demographic processes toward structuring both predator and prey populations. As a multifunctional protein cue, MULTIFUNCin provides valuable sensory information, conveys different messages to different species, and drives complex biotic interactions.

Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA.

Hsp27 gene in Drosophila ananassae subgroup was split by a recently acquired intron.

In Drosophila, heat shock protein 27 (Hsp27) is a critical single-copy intron-free nuclear gene involved in the defense response against fungi and bacteria, and is a regulator of adult lifespan. In the present study, 33 homologous Hsp27 nucleotide sequences from different Drosophila species were amplified by PCR and reverse transcription PCR, and the phylogenetic relationships were analysed using neighbour-joining, maximum-likelihood and Bayesian methods. The phylogenetic topologies from analysis with different algorithms were similar, suggesting that the Hsp27 gene was split by a recently acquired intron during the evolution of the Drosophila ananassae subgroup.

Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence.

Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous pairing of intact DNA molecules is observed.

Molecular comparison of Slc11a1 and Slc11a2 genes of swamp- and riverine-type water buffaloes.

Solute-linked carrier 11a and 11a2 (Slc) have been associated with disease resistance and/or susceptibility across animal species. These genes have an important mechanism in the regulation against intracellular infection. This study analysed the genetic characteristic of Slc 11a and 11a2 in swamp-type and riverine-type water buffaloes to understand their immunological distinction. Characterization of Slc11a1 and Slc11a2 genes from swamp- and riverine-type water buffaloes was carried out by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of Slc11a1 and Slc11a2 contained an open reading frame of 1647 and 1723 nucleotides, encoding 549 and 574 amino acids, respectively. Nucleotide sequence homology of both Slc11a1 and Slc11a2 had 99% in swamp and riverine type, which gives almost identical polypeptide. However, Slc11a1 and Slc11a2 have substitutions of 5 and 1 amino acid residues, correspondingly. These substitutions suggest as a potential gene markers for resistance and/or susceptibility to intracellular infection. Furthermore, phylogenetic analysis confirmed the degree of relationship between the bubaline species and justifies the distinctness of each breed by the bootstrap value generated.

MNSs genotyping by MALDI‐TOF MS shows high concordance with serology, allows gene copy number testing and reveals new St(a) alleles

Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA, GYPB and GYPE. Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB:GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs.

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.

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 WESorWGS. 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.