Homozygous Allele Research Articles

Association of SCN5A gene polymorphism with dilated cardiomyopathy

Subjects and methods. The study included patients with IDC (group 1; n=111, 89.2% men, average age 51.7±9.7 years) and ICM (group 2; n=110, 91.5% men, average age 58.7±8.4 years). All patients (IDC and ICM) underwent coronary angiography. Based on the anamnesis data and instrumental studies, those patients who could be said to have no risk factors for the development of dilatation of the heart cavities were identified in the group 1. And those patients who were reliably diagnosed with coronary artery disease were in the group 2, that is, dilatation of the heart cavities is due to a previous myocardial infarction, existing angina pectoris. The control group (n=121, average age 53.6±4.8 years) included patients who had no manifestations of cardiovascular diseases. The patients underwent laboratory and instrumental studies, as well as molecular and genetic studies of the A/G polymorphism of the SCN5A gene (rs1805124).Results. In the group with IDC 51.4% of patients were carriers of the common homozygous AA genotype, the heterozygous AG genotype-40.5%, and the rare homozygous GG genotype-8.1%. In the control group 63.3% of patients were identified as carriers of a homozygous genotype by a common allele, and 33.5% were carriers heterozygous genotype, and homozygous genotype for a rare allele – 3.2%. The analysis revealed a statistically significant decrease in the frequency of carrying the homozygous AA genotype in patients with IDC compared to the control group of the rs1805124 polymorphism of the SCN5A gene. In the group of patients with ICM, the А allele (69.5% vs. 80.1%, p=0.003) and the AA genotype (50.9% vs. 63.3%, p=0.030) were significantly less common than in the control group. The rare homozygous GG genotype was statically more common in patients with ICM compared to the control group (11.8% vs. 3.2%, p=0.004). Also, the G allele in the group of patients with ICM was detected statically significantly more often than in the control group (30.5% vs. 19.9%, p= 0.003).Conclusion. The polymorphic locus rs1805124 of the SCN5A gene is associated with both IDC and ICM. Homozygous genotype AA and allele A are conditionally protective factors for the development of these conditions in men.

Characterization of the human ABO genotypes and their association to common inflammatory and cardiovascular diseases in the UK Biobank

The ABO gene contains three major alleles that encodes different antigens; A, B, and O, which determine an individual's blood group. Previous studies have primarily focused on identifying associations between ABO blood groups and diseases risk. Here, we sought to test for association between ABO genotypes (OO, OA, AA; OB, BB, and AB) and a large set of common inflammatory and cardiovascular diseases in UK Biobank as well as disease-related protein biomarkers in NSPHS. We first tested for association by conducting a likelihood ratio test, testing whether ABO contributed significantly to the risk for 24 diseases, and 438 plasma proteins. For phenotypes with FDR < 0.05, we tested for pair-wise differences between genetically determined ABO genotypes using logistic or linear regression. Our study confirmed previous findings of a strong association between ABO and cardiovascular disease, identified associations for both type 1 and type 2 diabetes, and provide additional evidence of significant differences between heterozygous and homozygous allele carriers for pulmonary embolism, deep vein thrombosis, but also for von Willebrand factor levels. Furthermore, the results indicated an additive effect between genotypes, even between the two most common A subgroups, A1 and A2. Additionally, we found that ABO contributed significantly to 39 plasma proteins, of which 23 have never been linked to the ABO locus before. These results show the need of incorporating ABO genotype information in the consultation and management of patients at risk, rather than classifying patients into blood groups.

Possible Protective Effect of LOXL1 Variant in the Cohort of Chernobyl Catastrophe Clean-Up Workers.

Ionising radiation (IR) is an environmental factor known to alter genomes and therefore challenge organisms to adapt. Lithuanian clean-up workers of the Chernobyl nuclear disaster (LCWC) experienced high doses of IR, leading to different consequences. This study aims to characterise a unique protective genomic variation in a relatively healthy LCWC group. This variation influenced their individual reaction to IR and potentially protects against certain diseases such as exfoliation syndrome and glaucoma. Clinical and IR dosage data were collected using a questionnaire to characterise the cohort of 93 LCWC. Genome-wide genotyping using Illumina beadchip technology was performed. The control group included 466 unrelated, self-reported healthy individuals of Lithuanian descent. Genotypes were filtered out from the microarray dataset using a catalogue of SNPs. The data were used to perform association, linkage disequilibrium, and epistasis analysis. Phenotype data analysis showed the distribution of the most common disease groups among the LCWC. A genomic variant of statistical significance (Fishers’ exact test, p = 0.019), rs3825942, was identified in LOXL1 (NM_005576.4:c.458G>A). Linkage disequilibrium and epistasis analysis for this variant identified the genes LHFPL3, GALNT6, PIH1D1, ANKS1B, and METRNL as potentially involved in the etiopathogenesis of exfoliation syndrome and glaucoma, which were not previously associated with the disease. The LOXL1 variant is mostly considered a risk factor in the development of exfoliation syndrome and glaucoma. The influence of recent positive selection, the phenomenon of allele-flipping, and the fact that only individuals with the homozygous reference allele have glaucoma in the cohort of the LCWC suggest otherwise. The identification of rs3825942 and other potentially protective genomic variants may be useful for further analysis of the genetic architecture and etiopathogenetic mechanisms of other multifactorial diseases.

The Relationship between Stress, Personality, Dopamine Transporter, and Serotonin Transporter Level with Internet Addiction in Secondary School Students in Padang

BACKGROUND: The unpleasant stress can cause a variety of mental disorder that will lead children to run away from real life and one of the way is using excessive internet. In the past few years, more interested in studying the negative effect of the internet. The development of internet has been very rapid and used to play games and make friendship, but it also has negative impact on people’s lives. Young refers to some of the symptoms of internet addiction that resembles the symptoms of substance addiction, causing physical and psychological problems. Several studies have tried to show a relationship between dopamine and internet addiction by measuring the level of dopamine transporter (DAT). Lee et al. (2008) showed that one homozygous short allele from the serotonin transporter (SERt) gene 5-hydroxytryptamine transporter (5 HTT)-linked polymorphic region is more frequent in excessive internet users. AIM: The aim of this study was to investigate the relationship between stress, personality, DAT, and SERt level with internet addiction in secondary school students. METHODS: Methods applied in this study were a cross-sectional comparative design conducted in secondary school students in January 2016–March 2017 in Padang. The stress level was assessed using the depression anxiety and stress scale. Personality tests were measured using Minnesota Multiphasic Personality Inventory for Adolescent. Internet addiction was assessed using the Young Internet Addiction Test. Enzyme-linked immunosorbent assay was used to determine peripheral blood DAT and SERt or 5 HTT levels from the students who were diagnosed with stress. RESULTS: The results showed that personality was negatively associated with internet addiction (P = 0.271). Positive significant relationship of internet addiction was showed by association with levels of DAT (P = 0.001) and SERt (P = 0.001) in blood serum. DAT levels showed high relationship effect of stressed students become addicted to the internet. CONCLUSION: It is suggested that students were addicted to the internet as a coping mechanism against stress. Personality questionnaire tests failed to identify students who became addicted to the internet. The peripheral blood DAT and SERt level was predictive to identify students addicted to internet use. The present study supported the hypothesis that the DAT plays an important role in internet addiction.

Detection of Subclinical Paroxysmal Atrial Fibrillation and Its Correlation with Candidate Genes in Patients with Cryptogenic Ischemic Stroke and TIA

Abstract Introduction: Cardioembolic etiology is assumed to be the most frequent cause of cryptogenic strokes. The detection of subclinical paroxysmal atrial fibrillation (AF) is important in the correct choice of preventive treatment. The aim of this prospective study was to detect the incidence of AF in patients with a cryptogenic stroke or transient ischemic attack (TIA) and to evaluate the association between the presence of AF and selected single-nucleotide polymorphisms (SNP). Methods: Patients with a cryptogenic stroke/ TIA (n=100) and a control group (n=15) of volunteers without significant cardiovascular disease were included in the study during the period of 2014 to 2019. To detect AF they underwent 12 months of ECG monitoring using an implanted loop recorder (ILR). Genotyping for SNPs rs10033464, rs2200733, rs225132, and rs2106261 was performed by a high resolution melting analysis. Results: We found AF to be present in 24 (24%) patients with a cryptogenic stroke/TIA, versus no subjects in the control group. The SNPs rs2106261, rs2200733, rs225132, and rs10033464 were not found to be associated with AF in our study (p=0.240; 1.000; 0.887; 0.589). However, a weak trend for a higher frequency of rs2106261 risk allele A homozygotes was observed in the patients with AF compared to the patients without AF (0.416 vs. 0.263, p=0.073). Homozygotes for allele A of rs2106261 were also present in a significantly higher frequency in AF patients compared to the controls (0.416 vs. 0.133, p = 0.012). Conclusion: In our study paroxysmal AF was a probable etiological factor in 24% of patients with cryptogenic ischemic stroke / TIA during the 12 months of monitoring. The homozygous allele A of rs2106261 was identified to be the possible genetic risk factor of AF, but this should be verified in larger cohorts. The study has been registered at www.clinicaltrials.gov, identifier NCT02216370.

Thiopurine pharmacogenomics and pregnancy in inflammatory bowel disease

The thiopurine drugs azathioprine and 6-mercaptopurine are widely used for the maintenance of clinical remission in steroid-dependent inflammatory bowel disease (IBD). Thiopurines are recommended to be continued throughout pregnancy in IBD patients, but conclusive safety data in pregnant patients remain still insufficient. On the other hand, a strong association between a genetic variant of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15 p.Arg139Cys) and thiopurine-induced myelotoxicity has been identified. Pharmacokinetic studies have revealed that thiopurine metabolism is altered in pregnant IBD patients and suggested that the fetus may be exposed to the active-thiopurine metabolite, 6-thioguaninetriphosphate, in the uterus. A recent study using knock-in mice harboring the p.Arg138Cys mutation which corresponds to human p.Arg139Cys showed that oral administration of 6-MP at clinical dose induces a severe toxic effect on the fetus harboring the homozygous or heterozygous risk allele. This suggests that NUDT15 genotyping may be required in both women with IBD who are planning pregnancy (or pregnant) and their partner to avoid adverse outcomes for their infant. The risk to the fetus due to maternal thiopurine use is minimal but there are some concerns that are yet to be clarified. In particular, a pharmacogenomic approach to the fetus is considered necessary.

The effect of dietary total antioxidant capacity (DTAC) and Caveolin-1 gene variant interaction on cardiovascular risk factors among overweight and obese women: A cross-sectional investigation

Previous studies have shown that the Caveolin-1 (CAV-1) gene variant may be associated with Cardiovascular disease (CVD) risk. Moreover, dietary total antioxidant capacity (DTAC) has been shown to potentially elicit favorable effects on CVD risk. Therefore, this study sought to investigate the effect of DTAC and CAV-1 interaction on CVD risk factors. This cross-sectional study consisted of 352 women, with overweight and/or obesity, aged 18-48years from Iran. A food frequency questionnaire (FFQ), with 147 items, was used to assess dietary intake. The CAV-1 rs 3807992 and anthropometric data were measured by the PCR-RFLP method and bioelectrical impedance analysis (BIA), respectively. Serum profiles were measured by standard protocols. Participants were also divided into two groups based on DTAC score and rs3807992 genotype. The mean age of the participants was 37.34±9.11 and 36.01±9.12 years for homozygous (GG) and minor allele carriers (AG+AA) respectively.The mean±SD of insulin, total cholesterol (TC),high-density lipoprotein (HDL), low-density lipoprotein (LDL) and TG of participants were 1.21±0.23, 185.3±35.77, 46.58±10.86, 95.3±24.12 and 118.1±58.88, respectively. There was a significant difference between genotypes for physical activity (P=0.05), HDL (P<0.001), insulin (P=0.04), CRI-I (TC/HDL-C) (P=0.01), and CRI-II (LDL-C/HDL-C) (P=0.04). Our findings also showed, after controlling for confounding factors, significant interactions between DTAC score and the A allele carrier group on TC (Pinteraction=0.001), LDL (Pinteraction=0.001), insulin (Pinteraction=0.08), HOMA-IR (Pinteraction=0.03), AC ((TC - HDL - C)/HDL - C) (Pinteraction=0.001), and CHOLINDEX (LDL-C-HDL-C) (Pinteraction=0.02). The results of the present study indicate that high DTAC intake may modify the odds of risk factors for CVD in AA and AG genotypes of rs 3807992. These results highlight that diet, gene variants, and their interaction, should be considered in CVD risk assessment.

Recurrent Lobar Hemorrhages and Multiple Cortical Superficial Siderosis in a Patient of Alzheimer's Disease With Homozygous APOE ε2 Allele Presenting Hypobetalipoproteinemia and Pathological Findings of 18F-THK5351 Positron Emission Tomography: A Case Report

In Alzheimer's disease, the apolipoprotein E gene (APOE) ε2 allele is a protective genetic factor, whereas the APOE ε4 allele is a genetic risk factor. However, both the APOE ε2 and the APOE ε4 alleles are genetic risk factors for lobar intracerebral hemorrhage. The reasons for the high prevalence of lobar intracerebral hemorrhage and the low prevalence of Alzheimer's disease with the APOE ε2 allele remains unknown. Here, we describe the case of a 79-year-old Japanese female with Alzheimer's disease, homozygous for the APOE ε2 allele. This patient presented with recurrent lobar hemorrhages and multiple cortical superficial siderosis. The findings on the 11C-labeled Pittsburgh Compound B-positron emission tomography (PET) were characteristic of Alzheimer's disease. 18F-THK5351 PET revealed that the accumulation of 18F-THK 5351 in the right pyramidal tract at the pontine level, the cerebral peduncle of the midbrain, and the internal capsule, reflecting the lesions of the previous lobar intracerebral hemorrhage in the right frontal lobe. Moreover, 18F-THK5351 accumulated in the bilateral globus pallidum, amygdala, caudate nuclei, and the substantia nigra of the midbrain, which were probably off-target reaction, by binding to monoamine oxidase B (MAO-B). 18F-THK5351 were also detected in the periphery of prior lobar hemorrhages and a cortical subarachnoid hemorrhage, as well as in some, but not all, areas affected by cortical siderosis. Besides, 18F-THK5351 retentions were observed in the bilateral medial temporal cortices and several cortical areas without cerebral amyloid angiopathy or prior hemorrhages, possibly where tau might accumulate. This is the first report of a patient with Alzheimer's disease, carrying homozygous APOE ε2 allele and presenting with recurrent lobar hemorrhages, multiple cortical superficial siderosis, and immunohistochemically vascular amyloid β. The 18F-THK5351 PET findings suggested MAO-B concentrated regions, astroglial activation, Waller degeneration of the pyramidal tract, neuroinflammation due to CAA related hemorrhages, and possible tau accumulation.

Genetic relationships and identification of core germplasm among rice photoperiod- and thermo-sensitive genic male sterile lines

BackgroundHarnessing heterosis is one of the major approaches to increase rice yield and has made a great contribution to food security. The identification and selection of outstanding parental genotypes especially among male sterile lines is a key step for exploiting heterosis. Two-line hybrid system is based on the discovery and application of photoperiod- and thermo-sensitive genic sensitive male sterile (PTGMS) materials. The development of wide-range of male sterile lines from a common gene pool leads to a narrower genetic diversity, which is vulnerable to biotic and abiotic stress. Hence, it is valuable to ascertain the genetic background of PTGMS lines and to understand their relationships in order to select and design a future breeding strategy.ResultsA collection of 118 male sterile rice lines and 13 conventional breeding lines from the major rice growing regions of China was evaluated and screened against the photosensitive (pms3) and temperature sensitive male sterility (tms5) genes. The total gene pool was divided into four major populations as P1 possessing the pms3, P2 possessing tms5, P3 possessing both pms3 and tms5 genes, and P4 containing conventional breeding lines without any male sterility allele. The high genetic purity was revealed by homozygous alleles in all populations. The population admixture, principle components and the phylogenetic analysis revealed the close relations of P2 and P3 with P4. The population differentiation analysis showed that P1 has the highest differentiation coefficient. The lines from P1 were observed as the ancestors of other three populations in a phylogenetic tree, while the lines in P2 and P3 showed a close genetic relation with conventional lines. A core collection of top 10% lines with maximum within and among populations genetic diversity was constructed for future research and breeding efforts.ConclusionThe low genetic diversity and close genetic relationship among PTGMS lines in P2, P3 and P4 populations suggest a selection sweep and they might result from a backcrossing with common ancestors including the pure lines of P1. The core collection from PTGMS panel updated with new diverse germplasm will serve best for further two-line hybrid breeding.

Abstract LB020: Epigenomic tumor evolution modeling with single-cell methylation data profiling

Abstract The heritability of methylation patterns in tumor cells, as shown in recent studies, suggests that tumor heterogeneity and progression can be interpreted and predicted in the context of methylation changes. To elucidate methylation-based evolution trajectory in tumors, we introduce a novel computational method for methylation phylogeny reconstruction leveraging single cell bisulfite treated whole genome sequencing data (scBS-seq), incorporating additional copy number information inferred independently from matched single cell RNA sequencing (scRNA-seq) data, when available. We validate our method with the scBS-seq data of multi-regionally sampled colorectal cancer cells, and demonstrate that the cell lineages constructed by our method strongly correlate with original sampling regions. Our method consists of three components: (i) noise-minimizing site selection, (ii) likelihood-based sequencing error correction, and (iii) pairwise expected distance calculation for cells, all designed to mitigate the effect of noise and uncertainty due to data sparsity commonly observed in scBS-seq data. In (i), we present an integer linear program-based biclustering formulation to select a set of CpG-sites and cells so that the number of CpG-sites with non-zero coverage in the selected cells is maximized. This procedure filters out cells with read information in too few sites and CpG-sites with read information in too few cells. In (ii), we address the sequencing errors commonly encountered in currently available platforms with a maximum log likelihood approach to correct likely sequencing errors in scBS-seq reads, incorporating CpG-site copy number information in case it can be orthogonally obtained. Given the copy number and read information for a site in a cell, together with the overall sequencing error probability, we compute the log likelihood for all possible underlying allele statuses. If the mixed read statuses at the CpG-site for the cell are more likely due to sequencing error on homozygous alleles as opposed to the presence of alleles mixed methylation statuses, we correct the reads of the minority methylation status to the majority one. In (iii), we introduce a formulation to estimate distances between any pair of cells. As scBS-seq data is typically characterized by shallow read coverage, there is rarely read count evidence for two (or more, depending on CNV status) alleles at a CpG-site. Since allele-specific methylation has been shown to have increased frequency in cancer tissues, given the reads at a CpG-site, it is especially important to consider the possibility of unobserved alleles and their methylation status when determining the CpG-site's possible methylation zygosities. Our method incorporates copy number information when available, and for each CpG-site in a cell, we compute a probability distribution across all possible methylation zygosities. Then, given specific distance values between pairs of distinct zygosities and the likelihood of each possible zygosity for each shared CpG-site in both cells, we compute the expected total distance between any pair of cells as the mean of expected distances across all shared CpG-sites. We leverage such pairwise distances in methylation phylogeny construction. Citation Format: Xuan C. Li, Yuelin Liu, Farid Rashidi, Salem Malikic, Stephen M. Mount, Eytan Ruppin, Kenneth Aldape, Cenk Sahinalp. Epigenomic tumor evolution modeling with single-cell methylation data profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB020.

ANALYSIS OF THE ASSOCIATION OF BRONCHIAL ASTHMA CLINICAL COURSE WITH Arg16Gly POLYMORPHIC VARIANT IN THE β2-ADRENOCEPTOR GENE

Relevance. The relevance of the study of Arg16Gly polymorphism of the β2-adrenoceptor (β2-AR) gene is due to the fact that a number of studies have proven its role in the development of bronchial asthma (BA), bronchial hyperactivity, the effectiveness of basic treatment. However, these associations show low reproducibility in various studies, so the question of the possibility of clinical application of the results of genetic testing for Arg16Gly polymorphic variant of the β2-AR gene remains unanswered. The main reasons why the clinical significance of this polymorphism is not confirmed in various studies are - population heterogeneity, insufficient sample size, improper characterization of comparison groups.&#x0D; Objective: to study the association of Arg16Gly polymorphism in the β2-adrenoceptor gene with BA clinical course taking into account the age of onset.&#x0D; Materials and methods. We examined 553 BA patients (group I included 282 patients with late-onset asthma and group II included 271 patients with early-onset asthma) and 95 apparently healthy individuals. The study has been approved by the Bioethics Committee of Medical Institute of Sumy State University. Arg16Gly polymorphism in the β2-АR gene (rs1042713) was determined using polymerase chain reaction-restriction fragment length polymorphism analysis. Statistical analysis of obtained results was performed using SPSS–17 program.&#x0D; Results. There was no significant difference in the distribution of genotypes for Arg16Gly polymorphism in the β2-AR gene depending on asthma severity with no regard for the age of onset (χ2 = 5.14; p = 0.27). With regard for the age of onset, we found out that early-onset BA was linked to a difference in genotype distribution for this polymorphic variant in patients with severe and non-severe course (χ2 = 14.76; р = 0.001). The frequency of Gly/Gly genotype was higher in patients with severe course (41.4%) as compared to patients with mild course (16.4%), while the frequency of Arg/Arg (32.9%) and Arg/Gly (50.7%) genotypes was higher in patients with mild asthma as compared to patients with severe course (24.3% and 34.3%). There was no significant difference in the distribution of genotypes in patients with late-onset asthma with regard to course severity (χ2 = 4.94; p = 0.084).&#x0D; The relative risk of severe course for early-onset asthma was 3.84 times higher (95% CI 2.11–7.36; p = 0.001) in the recessive model, 2.58 times higher (95% CI 1.53–4,37, p = 0.001) in the dominant model, and 2.16 times (95% CI 1.56–3.04) higher in the additive model. In patients with late-onset asthma, no association was found in all models.&#x0D; Conclusions. There was no significant difference in the distribution of genotypes for Arg16Gly polymorphism in the β2-AR gene depending on asthma severity with no regard for the age of onset. When adjusted for the age of onset, the analysis revealed a difference in genotype distribution for this polymorphic variant in patients with severe and non-severe course having early-onset BA (р = 0.001). The frequency of Gly/Gly genotype was higher in patients with severe course as compared to patients with mild course. For patients with late-onset asthma, no differences were found (p = 0.084). Heterozygous and homozygous Gly allele carriers have a higher risk of early-onset asthma only.

Necessity of Smc3 in Embryonic and Adult Hematopoiesis with an Emphasis on Myeloid Compartment

SMC3 encodes a subunit of cohesin associated with recurrent heterozygous mutations in acute myeloid leukemia (AML) and other myeloid malignancies. About one-third of these mutations are nonsense and splice-site mutations and the rest are missense. To understand whether these missense mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3 deficient and haploinsufficient mouse models.We found that homozygous deletion of Smc3 during embryogenesis (Smc3fl/fl/Vav1-Cre+/-) is embryonic lethal. Grossly, the E13.5 Smc3fl/fl/Vav1-Cre+/- embryos were indistinguishable in size and appearance from other genotypes, except the lack of obvious fetal livers and near complete absence of CD45+Gr1+CD11b+ fetal liver cells, consistent with myeloid biased hematopoietic failure.Likewise, somatic deletion of homozygous Smc3 alleles in adult mice led to complete hematopoietic failure. In Smc3fl/fl/ERT2-Cre+/- mice, tamoxifen-induced Smc3 deletion led to rapid bone marrow (BM) failure and death. In competitive transplantation, the Smc3fl/fl/ERT2-Cre+/- BM cells were quickly outcompeted after tamoxifen treatment, indicating complete loss of hematopoietic stem/progenitor cell (HSPC) functions. Again, the effect was most pronounced within the myeloid compartment. Thus, Smc3 is necessary for both embryonic and adult hematopoiesis.We examined the consequences of Smc3 haploinsufficiency using three mouse models: Smc3fl/+/CMV-Cre+/-, Smc3fl/+/Vav1-Cre+/-, and Smc3fl/+/ERT2-Cre+/- . We found that Smc3 haploinsufficiency led to a normal number of female pups in X-linked CMV-Cre intercrosses, and the female pups had no obvious defects in growth or blood counts, implying tolerance of embryonic heterozygous deletion of Smc3 . In ex vivo methylcellulose assays, Smc3 haploinsufficiency did not result in increased number of colonies, and the Smc3 haploinsufficient BM cells did not replate beyond two weeks. In competitive transplantations, we observed a significant competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells, most pronounced in the Gr1+ myeloid cells.The patterns of missense and nonsense mutations observed in AML patients suggested that SMC3 likely acts as a tumor suppressor, andthat haploinsufficiency would augment pre-leukemic self-renewal. However, we observed a competitive disadvantage across multiple experiments. This contradicts the competitive advantage observed when Smc3 haploinsufficiency was induced using Mx1-Cre (Viny et al . JEM 212 (11): 1819-1832). Therefore, we examined whether Smc3 haploinsufficiency might augment self-renewal under specific conditions of hematopoietic stress, including pIpC. We again observed a marked competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells following tamoxifen induction, although the myeloid competitive disadvantage was partially ameliorated in the pIpC-treated group, whereas 5-FU treatment augmented the myeloid competitive disadvantage.In AML patients, SMC3 mutations frequently co-occurred with DNMT3A mutations. We, therefore, asked whether Smc3 haploinsufficiency might lead to a competitive advantage if it occurred in the background of Dnmt3a haploinsufficiency. With the addition of Dnmt3a haploinsufficiency, the severe myeloid competitive disadvantage was partially abrogated, but the significant competitive disadvantage in other lineages remained intact; even with constitutive Dnmt3a haploinsufficiency, Smc3 haploinsufficiency did not result in competitive growth of BM cells.In summary, our data demonstrate that homozygous Smc3 deletion was incompatible with embryonic or adult hematopoiesis, with the greatest defects noted within myeloid compartment, indicating that AML-associated SMC3 mutations are unlikely to have dominant-negative effects. Smc3 haploinsufficiency resulted in a myeloid competitive disadvantage, which was partially ameliorated by pIpC treatment, and accelerated by the stem cell stress of 5-FU. In the presence of concurrent Dnmt3a haploinsufficiency, the myeloid disadvantage was partially abrogated, while the disadvantage in other lineages remained unperturbed, suggesting interactions between the mutations. DisclosuresNo relevant conflicts of interest to declare.

Variability in cTBS Aftereffects Attributed to the Interaction of Stimulus Intensity With BDNF Val66Met Polymorphism.

Objective: To evaluate whether a common polymorphism (Val66Met) in the gene for brain-derived neurotrophic factor (BDNF)—a gene thought to influence plasticity—contributes to inter-individual variability in responses to continuous theta-burst stimulation (cTBS), and explore whether variability in stimulation-induced plasticity among Val66Met carriers relates to differences in stimulation intensity (SI) used to probe plasticity.Methods: Motor evoked potentials (MEPs) were collected from 33 healthy individuals (11 Val66Met) prior to cTBS (baseline) and in 10 min intervals immediately following cTBS for a total of 30 min post-cTBS (0 min post-cTBS, 10 min post-cTBS, 20 min post cTBS, and 30 min post-cTBS) of the left primary motor cortex. Analyses assessed changes in cortical excitability as a function of BDNF (Val66Val vs. Val66Met) and SI.Results: For both BDNF groups, MEP-suppression from baseline to post-cTBS time points decreased as a function of increasing SI. However, the effect of SI on MEPs was more pronounced for Val66Met vs. Val66Val carriers, whereby individuals probed with higher vs. lower SIs resulted in paradoxical cTBS aftereffects (MEP-facilitation), which persisted at least 30 min post-cTBS administration.Conclusions: cTBS aftereffects among BDNF Met allele carriers are more variable depending on the SI used to probe cortical excitability when compared to homozygous Val allele carriers, which could, to some extent, account for the inconsistency of previously reported cTBS effects.Significance: These data provide insight into the sources of cTBS response variability, which can inform how best to stratify and optimize its use in investigational and clinical contexts.

Influence of Single Nucleotide Polymorphism of ENPP1 and ADIPOQ on Insulin Resistance and Obesity: A Case-Control Study in a Javanese Population.

Single nucleotide polymorphisms (SNPs) in obesity-related genes, such as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and adiponectin (ADIPOQ), potentially increase the risk of insulin resistance, the most common metabolic dysregulation related to obesity. We investigated the association of ENPP1 SNP K121Q (rs1044498) with insulin resistance and ADIPOQ SNP + 267G > T (rs1501299) with circulating adiponectin levels in a case–control study involving 55 obese and 55 lean Javanese people residing in Yogyakarta, Indonesia. Allele frequency was determined by a chi squared test or Fisher’s exact test with an expected value less than 0.05. Odds ratios and 95% confidence intervals were estimated by regression logistic analysis. The presence of the Q121 allele of ENPP1 resulted in significantly higher fasting glucose, fasting insulin levels, and HOMA-IR, as compared to homozygous K121 carriers. The risk of insulin resistance was elevated in obese individuals carrying Q121 instead of homozygous K121. Adiponectin level was significantly lower in the obese group as compared to the lean group. Obese individuals carrying homozygous protective alleles (TT) of ADIPOQ tended to have lower adiponectin levels as compared to GT and GG carriers, however, we did not find statistically significant effects of the +276G > T SNP of the ADIPOQ gene on the plasma adiponectin levels or on the development of obesity.

POLIMORFISME CYP2C19*2 TERHADAP AGREGASI PLATELET DAN KEJADIAN KARDIOVASKULAR PADA PASIEN PENYAKIT JANTUNG KORONER

Coronary Heart Disease (CHD) is a cardiovascular disease that can caused by the presence of coronary artery plaque blockage. Clopidogrel is one of antiplatelet therapy in CHD patients. CYP2C19 *2 polymorphisms can reduce the effectiveness of clopidogrel active metabolites. This research was conducted at RSUD Sidoarjo East Java from November to December 2017continued monitoring of cardiovascular events to March 2018. The research method used is a method Polymerase Chain Reaction (PCR) and the Light Transmittance Aggregometry (LTA) method for platelet aggregation measurement. From 25 sample inclusion,there are wild type alleles (bp values ​​around 120 bp; 1 patient), homozygous alleles (bp values ​​around 169 bp; 18 patients) and allele heterozygotes (bp values ​​around 120bp, 169bp; 6 patients). The most common type of polymorphism is allele homozygotes. There is one patient hyperaggregation and cardiovascular events after being monitored for 3 months. Analysis relationship between CYP2C19*2 genetic polymorphisms with platelet aggregation and cardiovascular events, using the kruskal wallis test. The result is no significant relationship between CYP2C19*2 polymorphisms with platelet aggregation (p=0.512), as well as cardiovascular events did not make any difference which is also significant (p=0.426). The results of the association of platelet aggregation with cardiovascular events showed significant differences (p=0.027).

MO039IDENTIFICATION OF A RECURRENT SYNONYMOUS GENETIC VARIANT IN NPHP3 LEADING TO NEPHRONOPHTHISIS AND CONGENITAL HEPATIC FIBROSIS

Abstract Background and Aims Whole exome sequencing (WES) is becoming part of routine clinical and diagnostic practice and has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants. Filtering of variants and scoring variants in terms of pathogenicity still represents a major challenge and may explain why ∼50% of patients remain without diagnosis after initial assessment. Method In this study, we performed WES to determine the genetic cause of a hepato-renal ciliopathy syndrome in a genetically unsolved consanguineous family from Oman with 2 affected children. For variants filtering and annotation Qiagen Clinical Insight tool was used. Database searches for identical alleles in patients with similar phenotypes were performed using Genomics England, UK Biobank and a Saudi Arabian cohort. RNA studies were used to confirm a splicing defect. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project and from UK Biobank, a major biomedical database. Results Initial bioinformatic analysis of WES data from 2 affected sibs excluded obvious pathogenic variants in known genes associated with primary ciliopathy syndromes with liver and kidney phenotypes. However, by manual curation of variants in candidate genes, a rare homozygous synonymous allele in NPHP3 was identified (c.2805C&amp;gt;T; p.(Gly935Gly)), mid-exon 20 and within a region of shared homozygosity on chromosome 3. Correct segregation was confirmed via Sanger sequencing in the parents and the 2 affected sibs. The variant was rare in gnomAD (2/251374 alleles) and was found heterozygously in just one individual within the UK Biobank cohort of 200,000 exomes. Using various in silico tools, the allele was shown to activate a cryptic splice donor site in the middle of exon 20. RT-PCR with sequencing of parental whole blood RNA confirmed alternative splicing leading to frameshift p.Gly935GlyfsTer47. The identical homozygous allele was identified in 2 additional unsolved families within the Genomics England 100,000 Genomes Project and in 1 Saudi Arabian family with similar hepato-renal phenotypes. Conclusion This study shows that automated filtering of WES data may exclude synonymous variants which are pathogenic, especially if they are mid-exon. Here we identified a recurrent synonymous NPHP3 variant leading to a splice defect as the cause of a hepato-renal ciliopathy phenotype in four families. In unsolved cases, rare synonymous alleles in candidate genes need to be reassessed for impact on RNA splicing and possible pathogenicity.

Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.

Association between MANBA Gene Variants and Chronic Kidney Disease in a Korean Population.

Chronic kidney disease (CKD), a damaged condition of the kidneys, is a global public health problem that can be caused by diabetes, hypertension, and other disorders. Recently, the MANBA gene was identified in CKD by integrating CKD-related variants and kidney expression quantitative trait loci (eQTL) data. This study evaluated the effects of MANBA gene variants on CKD and kidney function-related traits using a Korean cohort. We also analyzed the association of MANBA gene variants with kidney-related traits such as the estimated glomerular filtration rate (eGFR), and blood urea nitrogen (BUN), creatinine, and uric acid levels using linear regression analysis. As a result, 14 single nucleotide polymorphisms (SNPs) were replicated in CKD (p < 0.05), consistent with previous studies. Among them, rs4496586, which was the most significant for CKD and kidney function-related traits, was associated with a decreased CKD risk in participants with the homozygous minor allele (CC), increased eGFR, and decreased creatinine and uric acid concentrations. Furthermore, the association analysis between the rs4496586 genotype and MANBA gene expression in human tubules and glomeruli showed high MANBA gene expression in the minor allele carriers. In conclusion, this study demonstrated that MANBA gene variants were associated with CKD and kidney function-related traits in a Korean cohort.

Overexpression of P-glycoprotein and MRP-1 are pharmacogenomic biomarkers to determine steroid resistant phenotype in childhood idiopathic nephrotic syndrome.

Steroid remains the keystone therapy for Idiopathic Nephrotic Syndrome (NS). Besides genetic factors and histological changes, pharmacogenomic factors also affect the steroid response. The upregulation of P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP-1) modulate the pharmacokinetics of steroids and may contribute to steroid resistance. Flow-cytometric analysis of P-gp, MRP-1 expression and functional activity on peripheral blood mononuclear cells (PBMCs) was carried out in steroid-sensitive nephrotic syndrome (SSNS) (n = 171, male 103, mean age = 8.54 ± 4.3); and steroid-resistant nephrotic syndrome (SRNS) (n = 83, male 43, mean age = 7.43 ± 4.6) patients. The genotypings of MDR-1 gene were carried out using PCR-RFLP. We observed that the percentage expression of P-gp (10.01 ± 2.09 and 3.79 ± 1.13, p < 0.001); and MRP-1 (15.91 ± 3.99 and 7.40 ± 2.33, p < 0.001) on lymphocyte gated population were significantly higher in SRNS than that of SSNS. The functional activity of P-gp and MRP-1 was also significantly escalated in SRNS as compared to SSNS (68.10 ± 13.35 and 28.93 ± 7.57, p < 0.001); (72.13 ± 8.34 and 31.56 ± 8.65, p < 0.001) respectively. AUC-ROC curve analysis revealed that P-gp and MRP-1 expression with a cut-off value of 7.13% and 9.62% predicted SRNS with the sensitivity of 90% and 80.7%; and specificity 90% and 80%, respectively. Moreover, MDR-1 homozygous mutant TT+AA for G2677T/A (rs2032582) was significantly associated with SRNS (p = 0.025, OR = 2.86 CI = 1.14-7.14). The expression of P-gp (9.68 ± 4.99 v/s 5.88 ± 3.38, p = 0.002) was significantly higher in the patients of homozygous mutant alleles compared to wildtype GG. The increased expression and functionality of P-gp and MRP-1 contribute to steroid resistance, and MDR-1 homozygous mutant G2677T/A promotes steroid resistance by inducing P-gp expression in NS.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for β-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in β-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285β+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the β-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74–90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and β-carotene content. The agronomic performance of the four lines was similar (78.83–99.44%) to that of the recurrent parent, and β-carotene content (7.541–8.711 μg/g) was on par with the donor parent. Our study is the first to improve the β-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.