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PubMed2024

Identification of four TTN variants in three families with fetal akinesia deformation sequence.

BACKGROUND: TTN is a complex gene with large genomic size and highly repetitive structure. Pathogenic variants in TTN have been reported to cause a range of skeletal muscle and cardiac disorders. Homozygous or compound heterozygous mutations tend to cause a wide spectrum of phenotypes with congenital or childhood onset. The onset and severity of the features were considered to be correlated with the types and location of the TTN variants. METHODS: Whole-exome sequencing was performed on three unrelated families presenting with fetal akinesia deformation sequence (FADS), mainly characterized by reduced fetal movements and limb contractures. Sanger sequencing was performed to confirm the variants. RT-PCR analysis was performed. RESULTS: TTN c.38,876-2 A > C, a meta transcript-only variant, with a second pathogenic or likely pathogenic variant in trans, was observed in five affected fetuses from the three families. Sanger sequencing showed that all the fetal variants were inherited from the parents. RT-PCR analysis showed two kinds of abnormal splicing, including intron 199 extension and skipping of 8 bases. CONCLUSIONS: Here we report on three unrelated families presenting with FADS caused by four TTN variants. In addition, our study demonstrates that pathogenic meta transcript-only TTN variant can lead to defects which is recognizable prenatally in a recessive manner.

Humans↗

PubMed2025

Genetic analysis of four cases of Poirier Bienvenu neurodevelopmental syndrome associated with CSNK2B variant.

BACKGROUND: CSNK2B deficiency underlies the pathogenesis of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS). In this study, we present four cases of pediatric seizures caused by de novo variants in CSNK2B, with the aim to reinforce the clinical and variant data pertaining to early genetic factors associated with epilepsy. METHODS: Trio whole exome sequencing were used to detect variants in the proband and her family members, and bioinformatics annotation was performed for the variant. Sanger sequencing and CSNK2B cDNA sequencing were employed to ascertain the carrier status of additional family members and evaluate the potential impact of variants on splicing. RESULTS: All four cases presented with epilepsy as the initial manifestation, accompanied by global developmental delay, particularly in language and motor developmental delay. Cases 1, 3 and 4 exhibited full-scale tonic-clonic seizures, while case 2 displayed myoclonic and typical absence seizures. Furthermore, case 2 demonstrated delayed growth and development compared to age-matched peers. No abnormality was detected in the head magnetic resonance imaging (MRI). Genetic analysis revealed novel heterozygous variants in the CSNK2B gene in all four cases, including c.175 + 1G > A, c.73-2A > G, c.291 + 1G > A and c.481delA. In case 2, reverse transcription analysis of CSNK2B mRNA revealed the retention of the 3' end sequence of Intron 2 and deletion of the 5' end sequence of Exon 3. In treatment, four case received a combination of one to three types of antiseizure medication and rehabilitation training individually. Case 1 continued to experience seizures to varying degrees, while cases 2-4 demonstrated effective seizure control. Overall motor and intellectual development improved in all four cases, however, there was slow recovery in language function. CONCLUSION: This study elucidates the molecular etiology of epilepsy in four cases with POBINDS and expands the mutational spectrum of pathogenic variants in the CSNK2B, highlighting their impact on splicing. The highly genetic heterogeneous phenotype of POBINDS relies on the detection of pathogenic variants in CSNK2B. Conventional antiseizure medication effectively control seizures, while rehabilitation treatment can significantly improve intelligence and motor function to varying degrees; however, language recovery tends to be relatively slow.

Humans↗

PubMed2004

Molecular screening and association analyses of the interleukin 6 receptor gene variants with type 2 diabetes, diabetic nephropathy, and insulin sensitivity.

IL-6 levels and polymorphisms have been implicated in type 2 diabetes mellitus (T2DM) and insulin resistance. The IL-6 receptor (IL-6R) comprises two subunits, IL-6R and gp130, of which IL-6R confers specificity to IL-6 action and is located in a region of replicated linkage to T2DM on chromosome 1q21. We screened this gene for variation in Northern European Caucasian and African-American ethnic groups. We identified 11 variants with a minor allele frequency over 5%, including two amino acid changes (D358A and V385I) and four variants in the 3' untranslated region. No variant was associated with obesity or measures of insulin sensitivity, but two single nucleotide polymorphisms in the 3' untranslated region showed a trend to an association with T2DM in all Caucasians, and three single nucleotide polymorphisms, including D358A, showed a trend (P < 0.06) to an association with T2DM among the subset of Northern European Caucasians. Variant V385I was unique to African-Americans and was significantly associated with diabetes and diabetic nephropathy (P < 0.05). Among individuals heterozygous for the four variants in the transcribed sequence, one allele was significantly overrepresented, thus suggesting the existence of a regulatory variant controlling mRNA stability or expression. IL-6R is not likely to explain the linkage to diabetes in this region, but our work supports a minor role of variants in T2DM risk and suggests that sequence variants may alter IL-6R mRNA levels and possibly levels of soluble IL-6R.

3' Untranslated Regions↗

PubMed2005

Association of mitochondrial deoxyribonucleic acid 16189 variant (T->C transition) with metabolic syndrome in Chinese adults.

OBJECTIVE: A common variant in mitochondrial DNA (mtDNA) at bp 16189 (T-->C transition) has been associated with small birth size, adulthood hyperglycemia, and insulin resistance in Caucasians. In this study, we investigated whether mtDNA 16189 variant is associated with metabolic syndrome in Chinese subjects. METHODS: Six hundred fifteen Chinese adults, aged 40 yr or older, were recruited in this study. The 16189 variant of mtDNA was detected using PCR and restriction enzyme digestion. Metabolic syndrome was diagnosed on modified National Cholesterol Education Program Adult Treatment Panel III guidelines, using body mass index (BMI) instead of waist circumference. An association study was performed with chi2 test and logistic regression analysis. RESULTS: The prevalence of the 16189 variant was higher in patients with metabolic syndrome than in those without: 44% (125 of 284) vs. 33.2% (110 of 331) (P = 0.006). The association between this 16189 variant of mtDNA and metabolic syndrome (P = 0.021) remained significant even after correcting for age and BMI. As to the individual traits, the prevalence of fasting plasma glucose of at least 110 mg/dl (> or =6.1 mmol/liter) [(51.5% (121 of 235) vs. 42.1% (160 of 380); P = 0.023], type 2 diabetes mellitus [48.1% (113 of 235) vs. 39.2% (149 of 380); P = 0.031], and hypertriglyceridemia [44.3% (104 of 235) vs. 35.8% (136 of 380); P = 0.037] were significantly higher in subjects harboring the 16189 variant of mtDNA than those with the wild type. However, the prevalence of hypertension [53.2% (125 of 235) vs. 47.6% (181 of 380); P = 0.180], BMI greater than 25 kg/m2 [48.5% (114 of 235) vs. 43.9% (167 of 380); P = 0.270], and low high-density lipoprotein cholesterol [61.3% (144 of 235) vs. 54.7% (208 of 380); P = 0.111] did not reach a significant difference between the two groups. Furthermore, there was a trend of increasing frequency of occurrence of the 16189 variant in individuals having an increasing number of components of metabolic syndrome (Ptrend < 0.005). CONCLUSION: Our data strongly suggest that mtDNA 16189 variant underlies susceptibility to metabolic syndrome in the Chinese population.

Aged↗

PubMed2006

A common mitochondrial DNA variant and increased body mass index as associated factors for development of type 2 diabetes: Additive effects of genetic and environmental factors.

OBJECTIVE: The suggested correlation between a T-to-C transition at the nucleotide 16189 in mitochondrial DNA (mtDNA) with increasing insulin resistance and adult-onset diabetes mellitus (DM) is debatable. METHODS: Our study examined mtDNA from 462 subjects with type 2 diabetes (T2DM) and 592 normoglycemic controls (non-DM). Each participant's body mass index (BMI), fasting plasma glucose, fasting insulin concentration, insulin resistance index, and beta-cell function were measured. Sequencing for mtDNA, focusing on exploration of the hypervariable polycytosine tract within the control region, was also conducted in all subjects. RESULTS: Prevalence of the mtDNA 16189 variant was significantly different between DM and non-DM subjects (39.2% vs. 30.7% respectively; P = 0.004). Increased incidence of DM was noted in those harboring the 16189 variant compared with those lacking the variant (multivariate odds ratio, 1.38; 95% confidence interval, 1.07-1.80). Moreover, increased BMI was identified as an aggravating factor for development of DM in subjects harboring the variant. Odds ratio determinations yielded 2.14 in overweight and 4.63 in obese subjects harboring the variant in comparison with subjects without (1.83 in overweight and 2.16 in obese subjects). This is consistent with a progressively increased prevalence of the mtDNA 16189 variant in the non-DM groups with higher fasting insulin concentration, insulin resistance index, and beta-cell function (all P(trend) < 0.005). CONCLUSION: The mtDNA 16189 variant can influence development of T2DM. The demonstrated dynamic between the 16189 variant and increased BMI exemplify an additive effect of genetic and environmental factors on the pathogenesis of T2DM.

Adult↗

PubMed1995

Occurrence and biological properties of a common genetic variant of luteinizing hormone.

We have characterized the frequency and selected biological properties of a variant form of LH caused by two point mutations in the gene of the LH beta-subunit. Detection of the LH variant (or polymorphism) is based on aberrant immunoreactivity; it is not detected by a monoclonal antibody (Mab) recognizing a specific epitope in the LH alpha/beta-dimer (assay 1), but an assay using two LH beta-specific Mab recognizes this LH form normally (assay 2). Hence, the ratio of LH measured by assays 1 and 2 is 1.18-2.10 (range of mean +/- 2 SD) in wild-type subjects, 0.54-0.98 in heterozygotes, and below 0.15 in homozygotes with regard to the mutant LH beta allele. Analysis of sera from 249 healthy male and female subjects of Finnish origin revealed a frequency of 24.1% heterozygotes and 3.6% homozygotes for the mutation, with similar proportions in each sex. The ratio of in vitro bioactivity to immunoreactivity (assay 2) of the variant LH was significantly (P < 0.01) increased (2.9 +/- 0.1; n = 11) compared to that of wild-type LH (2.2 +/- 0.1; n = 13). No difference was observed in LH pulsatility, measured from blood samples collected at 5-min intervals for 5 h, between three male and three female subjects homozygous for the LH variant and three matched male and three female controls with wild-type LH. Likewise, the responses of LH immunoreactivity (assay 2) to GnRH stimulation were similar with both types of LH. The half-time of the variant LH in rat circulation from both sexes was significantly shorter than that of LH from control subjects (males, 25.5 +/- 3.8 vs. 48.3 +/- 2.7 min, respectively; P < 0.01; n = 3). Upon isoelectric focusing of peripheral serum samples, the isoform distribution of the variant LH was similar to that of wild-type LH. In conclusion, the LH variant discovered by us appears to occur with high frequency in the Finnish population (28% homo- or heterozygotes). It has increased in vitro bioactivity and a decreased half-time in vivo. These differences are compatible with a putative extra carbohydrate chain in the LH beta-chain, as one of the two mutations introduces an extra glycosylation signal. The subjects homozygous for the LH polymorphism are apparently healthy. However, the altered bioactivity and in vivo kinetics of the LH variant may induce subtle changes in LH action, either predisposing the affected individuals to or protecting them from disease conditions related to LH action.

Adult↗

PubMed1997

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Amino Acid Sequence↗

PubMed1997

Tumor-specific expression of alternatively spliced estrogen receptor messenger ribonucleic acid variants in human pituitary adenomas.

The well documented mitogenic and hormone regulatory effects of estrogen (E2) on pituitary cells are mediated via its nuclear receptor (ER), a cellular homolog of v-erbA oncogene. ER isoforms generated by alternative exon splicing, termed ER variants delta 2ER to delta 7ER, have been identified in breast cancer and have been postulated to have important pathogenetic and clinical implications in tumorigenesis and/or development of hormone resistance. Because pituitary tumors, particularly prolactinomas, are known to be E2-dependent, we investigated alternatively spliced ER variant messenger ribonucleic acid expression in 40 human pituitary tumors of various phenotypes and normal pituitary tissues, using reverse transcription-PCR and Southern blot analyses. Nine of 11 prolactinomas readily expressed multiple ER variants (delta 2ER, delta 4ER, 5ER, and delta 7ER), whereas 6 of 11 tumors showed faint expression of delta 3ER. Four of 7 glycoprotein hormone-producing tumors that synthesized FSH beta expressed delta 2ER, delta 5ER, and delta 7ER. In 9 GH- and 10 ACTH-secreting tumors examined, the expression of normal and variant ER was restricted to tumors that also exhibited scattered PRL immunoreactivity. Variant and normal ER were not found in three null cell tumors (oncocytomas) that showed negative immunoreactivity for all pituitary hormones or their subunits. In contrast, only delta 4ER and delta 7ER were uniformly detected in normal pituitaries. delta 6ER was not detected in any normal or neoplastic pituitary specimen studied. We conclude that multiple alternatively spliced ER variants are coexpressed with normal ER in a tumor phenotype-specific manner. In addition, ER variants delta 2ER and delta 5ER were found to be tumor specific. Future functional studies will be required to determine whether coexpression of multiple ER variants along with normal ER confers a pathophysiological role in pituitary hormone regulation and/or tumor cell proliferation.

Adenoma↗

PubMed1999

Mutation analysis reveals novel sequence variants in NTRK1 in sporadic human medullary thyroid carcinoma.

Tyrosine kinase NTRK1 is expressed in neural and nonneuronal tissues. Like RET, NTRK1 is often activated by rearrangements that involve one of at least five other genes in papillary thyroid carcinoma (PTC). Because of similarities in involvement of the two tyrosine kinases RET (rearranged during transfection) and NTRK1 in the pathogenesis of PTC, the obvious parallels between RET and NTRK1 and between PTC and medullary thyroid carcinoma (MTC), NTRK1 seemed to be an excellent candidate gene to play a role in the genesis of MTC. Single-strand conformational polymorphism analysis of 16 exons of NTRK1, from 31 sporadic MTC, revealed variants in five exons (exons 4 and 14-17). Sequence analysis demonstrated one sequence variant each in exons 4, 14, 16, and 17, and four different variants in exon 15. Differential restriction enzyme digestion specific for each variant confirmed the sequencing results. All variants were also present in the corresponding germline DNA. Interestingly, the sequence variants at codon 604 (c1810C>T) and codon 613 (c1838G>T) ofexon 15 always occurred together and might represent linkage disequilibrium. The frequencies of the sequence variants in germline DNA from patients with sporadic MTC did not differ significantly from those in a race-matched control group. Although we did not find any somatic mutations of NTRK1 in sporadic MTC, the single-strand conformational polymorphism conditions reported here, together with the knowledge of the frequency of various sequence variants, may help in future mutation analyses of DNA from other neural and nonneural tissues.

Carcinoma, Medullary↗

PubMed2002

Role of calpain-10 gene variants in familial type 2 diabetes in Caucasians.

The calpain-10 gene (CAPN10) has been implicated in type 2 diabetes (T2DM) susceptibility by both linkage and association in a Hispanic population from Starr County Texas. Common intronic variants seem to alter CAPN10 mRNA levels and were associated with insulin resistance but not diabetes in Pima Indians. The role of these variants in Caucasian populations is less clear. We found some evidence for linkage of T2DM to chromosome 2q approximately 20 cM proximal to the NIDDM1/CAPN10 locus. To test the hypothesis that CAPN10 is a diabetes susceptibility locus in Caucasian families at high risk for T2DM, we examined the influence of the three previously implicated CAPN10 variants on both diabetes risk and measures of insulin sensitivity and glucose homeostasis. We genotyped approximately 700 members of 63 families for 3 variants (SNP-43, SNP-19, and SNP-63). We tested each variant separately and as haplotype combinations for altered transmission from parents to affected children (transmission disequilibrium test), and we tested for an effect of each variant individually on measures of glucose and insulin during a glucose tolerance test in nondiabetic family members. Finally, we looked for an effect of each variant on measures of insulin sensitivity (S(I)) and insulin secretion estimated by frequently sampled iv glucose tolerance test and Minimal Model analysis. We could not confirm an increase in risk for T2DM susceptibility for any variant or for any haplotype combination, although we found marginal evidence for an increased risk of the 111/221 haplotype combination (P = 0.036) after ascertainment correction. However, both SNP-19 and SNP-63 increased fasting and/or postchallenge insulin levels, consistent with reduced insulin sensitivity. Furthermore, SNP-19 had modest effects on insulin sensitivity measured by homeostatic model, and on postchallenge glucose. The reduction in insulin sensitivity was confirmed by analysis of the subset of individuals who underwent iv glucose tolerance tests, where SNP-19 significantly altered the insulin sensitivity index. CAPN10 cannot be considered a major diabetes susceptibility gene in our population and seems unlikely to explain the observed linkage findings. However, CAPN10 influences insulin sensitivity and glucose homeostasis in nondiabetic members of kindreds at high risk for T2DM.

Alleles↗

PubMed2006

Glycosylation variant analysis of recombinant human tissue plasminogen activator produced in urea-cycle-enzyme-expressing Chinese hamster ovary (CHO) cell line.

Tissue plasminogen activator (tPA) was produced in ornithine transcarbamoylase (OTC) cells by introducing the tPA gene into OTC cells. OTC cells were originally derived from Chinese hamster ovary (CHO) cells and express the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and OTC. To investigate glycosylation variants, tPA variants produced in serum-supplemented culture medium of OTC-tPA cells were separated by lysine-Sepharose 4B chromatography. Unlike in previous studies that used lysine-Sepharose chromatography, two peaks were identified to correspond to eluted glycosylation variants type I and II and type II and the percentages of the type I and type II variants were found to be 23% and 77%, respectively. The biological activities of the type I and II and type II variants were twofold that of the Third International tPA Standard (98/714) produced in the CHO cell line, and the activity of type II variant was 12.6% higher than that of the type I and II variants. These results demonstrate that tPA produced in urea-cycle-enzyme-producing OTC cells have a very high biological activity and the percentage of type II variant which is very valuable for the biopharmaceutical industry is higher than that of any report using CHO cells.

Animals↗

PubMed2025

NAVIP: Unraveling the influence of neighboring small sequence variants on functional impact prediction.

Once a suitable reference sequence has been generated, intra-species variation is often assessed by re-sequencing. Variant calling processes can reveal all differences between strains, accessions, genotypes, or individuals. These variants can be enriched with predictions about their functional implications based on available structural annotations, i.e., gene models. Although these functional impact predictions on a per-variant basis are often accurate, some challenging cases require the simultaneous incorporation of multiple adjacent variants into this prediction process. Examples include neighboring variants which modify each other's functional impact. The Neighborhood-Aware Variant Impact Predictor (NAVIP) considers all variants within a given protein coding sequence when predicting the effect. As a proof of concept, variants between the Arabidopsis thaliana accessions Columbia-0 and Niederzenz-1 were annotated. NAVIP is freely available on GitHub (https://github.com/bpucker/NAVIP) and accessible through a web server (https://pbb-tools.de).

Arabidopsis↗

PubMed2026

A portable recalibration workflow for reference-based variant calling in non-human genomes.

A key computational step in reference-based variant calling is distinguishing true genetic variants from sequencing errors. Advanced tools and workflows have been developed to handle this by computational modelling of technical errors from the sequencing machines. However, these recalibration workflows have largely been evaluated for human data only and its exact applicability for non-human data remains unknown. Here, we conducted a systematic evaluation of variant calling on human, rice, sheep, and chickpea data, and found that existing workflows introduce unexpected statistical bias, thus leading to suboptimal variant calls for non-human data. To address this problem, we present simple guidelines for constructing a "pseudo-"database (pseudoDB) of genetic variants as a scalable and portable solution for recalibration and variant calling. With human data, our pseudoDB-based workflow performs comparably to existing dbSNP-based GATK3 workflows and those using DeepVariant, Strelka2, and FreeBayes. We extend this to other non-human genomes, namely cattle, brown bear, swan goose, African oil palm, Komodo dragon, and stevia, altogether resulting in the identification of up to 242.0% unique genetic variants. The majority of newly identified variants are within the non-coding regions, hinting at the rich diversity of genome regulation in the non-human population. Our pseudoDB-based workflow is agnostic to reference genomes and modular for easy integration with other computational workflows for human and non-human resequencing data.

Humans↗

PubMed2023

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling.

BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Animals↗

PubMed2024

Infection kinetics, syncytia formation, and inflammatory biomarkers as predictive indicators for the pathogenicity of SARS-CoV-2 Variants of Concern in Calu-3 cells.

The ongoing COVID-19 pandemic has led to the emergence of new SARS-CoV-2 variants as a result of continued host-virus interaction and viral genome mutations. These variants have been associated with varying levels of transmissibility and disease severity. We investigated the phenotypic profiles of six SARS-CoV-2 variants (WT, D614G, Alpha, Beta, Delta, and Omicron) in Calu-3 cells, a human lung epithelial cell line. In our model demonstrated that all variants, except for Omicron, had higher efficiency in virus entry compared to the wild-type. The Delta variant had the greatest phenotypic advantage in terms of early infection kinetics and marked syncytia formation, which could facilitate cell-to-cell spreading, while the Omicron variant displayed slower replication and fewer syncytia formation. We also identified the Delta variant as the strongest inducer of inflammatory biomarkers, including pro-inflammatory cytokines/chemokines (IP-10/CXCL10, TNF-α, and IL-6), anti-inflammatory cytokine (IL-1RA), and growth factors (FGF-2 and VEGF-A), while these inflammatory mediators were not significantly elevated with Omicron infection. These findings are consistent with the observations that there was a generally more pronounced inflammatory response and angiogenesis activity within the lungs of COVID-19 patients as well as more severe symptoms and higher mortality rate during the Delta wave, as compared to less severe symptoms and lower mortality observed during the current Omicron wave in Thailand. Our findings suggest that early infectivity kinetics, enhanced syncytia formation, and specific inflammatory mediator production may serve as predictive indicators for the virulence potential of future SARS-CoV-2 variants.

Humans↗

PubMed2026

Correlation assessment of SARS-CoV-2 variants and their subvariants present in clinical and wastewater samples in Oregon, USA (February 7, 2021 - February 26, 2022) using the Freyja bioinformatics approach.

BACKGROUND: Wastewater surveillance is a valuable tool for monitoring SARS-CoV-2 at the community level. As the virus diversified into many variants and subvariants that share overlapping mutations, resolving them accurately from wastewater becomes a key bioinformatic challenge. OBJECTIVES AND AIMS: This study evaluated two distinct bioinformatic approaches, multilocus sequence typing (MLST) and Freyja, for identifying SARS-CoV-2 variants and subvariants in Oregon wastewater samples collected from February 2021 to February 2022. METHODS: The MLST approach identified SARS-CoV-2 variants using unique mutations curated from clinical samples. In contrast, the Freyja approach resolved variant and subvariant abundances using genome wide mutation profiles weighted by sequencing depth. In this study, the variant and subvariants relative abundances produced by both approaches were compared against those observed in clinical surveillance data. RESULTS: Both approaches identified SARS-CoV-2 variants at relative abundances that agreed closely with those observed in clinical surveillance data. However, only the Freyja approach identified over 200 Delta subvariants, divided into three clades (21A, 21I and 21J) and two levels (Level 1 and 2) based on Pango subvariants. Delta subvariants showed strong agreement at Level 1 subvariants (rs = 0.892-0.944), while agreement at Level 2 subvariants was inconsistent (rs = 0.324-0.903). CONCLUSIONS: The Freyja approach provided enhanced resolution of SARS-CoV-2 variants and subvariants in wastewater, at abundances that agreed with clinical surveillance. This added resolution is a critical advantage for public health surveillance as SARS-CoV-2 continues to evolve and share mutations across variants and subvariants.

Oregon↗

PubMed2005

STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies. METHODS: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA. RESULTS: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta, delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4 variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1, beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains. CONCLUSIONS: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.

Animals↗

PubMed1995

Presence of alternatively spliced-estrogen receptor mRNA variants in normal human uterine endometrium and endometrial cancer.

Presence of alternatively spliced-estrogen receptor (ER) mRNA variants has been revealed in the breast cancer tissues. The ER variants transcribed from these mRNA variants were supposed to cause changes in the estrogen responsiveness of breast cancer. Although uterine endometrial cancer also has an estrogen-dependent profile, these ER mRNA variants have not yet been reported in the tumor. In the present study, we attempted to detect the exon 7 deletion- (del.7-) and exon 5 deletion (del.5) ER mRNA variants in normal human uterine endometrium (hEM) and uterine endometrial cancer tissue (hEC) by the use of reverse transcription-polymerase chain reaction-Southern blotting (RT-PCR-SB) with the PCR primers: hE4 (forward), hE6 (reverse), and hE8 (reverse), which were located in exons 4, 6, and 8, respectively. Two major products were generated from RNAs of both hEM and hEC with primers hE4 and hE8. The nucleotide sequence of the longer product was identical to exon 4-8 of human ER cDNA, whereas that of the shorter one completely deleted exon 7. Moreover, when the RT-PCR was done with the primers hE4 and hE6, the shorter product lacking exon 5 was detected with the longer one having the same sequence as exon 4-6 of human ER cDNA. Since the RT-PCR-SB with primers hE4 and hE8 produced a very low or undetectable level of the signals corresponding to del.5 ER mRNA variant, the level of del.7 ER mRNA variant seemed to be higher than that of del.5 ER mRNA variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗