Polymorphism In Exon Research Articles

Polymorphisms in exon 11 of the mptl-1 gene and monepantel resistance in Haemonchus contortus.

Chemical treatments are the main strategy to control gastrointestinal nematodes in sheep,and the emergence of anthelmintic resistance, as consequence, results in control failures and leads to economic losses. Thus, molecular tests may constitute an excellent tool for the early detection of anthelmintic resistance-related mutations. Thus, a polymerase chain reaction (PCR)-based genotyping assay followed by polyacrylamide gel electrophoresis (PAGE) was developed to detect polymorphisms in exon 11 of the acetylcholine receptor monepantel-1 gene (mptl-1) that were previously associated with monepantel resistance through a genome-wide study in Haemonchus contortus. DNA samples recovered from individual and pooled third-stage larvae from two susceptible field-derived isolates and five (three in vivo-derived and two field-derived) resistant populations were used. New polymorphisms, including a 6-bp deletion and a 3-bp insertion, were detected in resistant individuals. These indels, confirmed using sequencing of cloned PCR products, are predicted to result in amino acid changes in transmembrane domain 2 (TMD2) of the MPTL-1 protein. The two susceptible isolates showed only the presence of the wild-type allele (100%), whereas lower frequencies of the wild-type allele were detected in monepantel-resistant populations (11.1 to 66.7%). These findings report new polymorphisms in the mptl-1 gene, validate the results obtained through genomic mapping for monepantel resistance, and provide a PCR-based assay to genotype indels located in exon 11 of mptl-1 in H. contortus.

P08 - Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene

P08 - Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene

Do follicle-stimulating hormone receptor polymorphisms in infertile men influence intracytoplasmic sperm injection outcomes? A prospective cohort study.

The follicle-stimulating hormone (FSH) and its receptor regulate the quantity and quality of spermatozoa production. Several studies have analyzed the effect of single nucleotide polymorphisms (SNPs) in exon 10 of the FSH receptor (FSHR) on basic semen parameters without yet reaching a firm consensus. The aim of this study was to evaluate the effect of p.Thr307Ala and p.Asn680Ser polymorphisms in exon 10 of the FSHR gene, in infertile men, on intracytoplasmic sperm injection (ICSI) outcomes. This study was conducted between March 2019 and February 2020 on infertile couples undergoing ICSI at Al Hadi Laboratory and Medical Center, Lebanon. Couples with severe infertility factors that may impair gametogenesis/embryogenesis (e.g. advanced maternal age, premature ovarian failure, underwent gonadotoxic treatments, etc.) were excluded from the study. Semen and blood samples were collected from infertile men on the day of oocyte collection. Infertile men (n = 173) were screened for FSHR variants using polymerase chain reaction-restriction fragment length polymorphism. Moreover, fertilization rates, embryo quality, and pregnancy outcomes were evaluated. Higher sperm concentrations were found in the p.Thr307Ala group than the p.Thr307Thr (P < 0.01) and p.Ala307Ala (P < 0.05) groups. Furthermore, fertilization rate was significantly lower in the p.Ala307Ala genotype than in the p.Thr307Thr genotype (P < 0.05). We showed that FSHR variants in infertile men undergoing ICSI could affect sperm concentration, motility, and fertilization rates. Therefore, it will be important to confirm these results in further studies using a larger sample size.

Analysis of Common Mutation of P53 Gene in Male with Lung Cancer in Mosul City

TP53 gene plays a critical role in the follow-up of different cancer cases, including diagnosis and follow-up treatment and the mutation in the P53 gene. It harms the encoded P53 protein and the less function of P53 protein in different types of cancer due to the mutation in the TP53 gene. P53 protein has many mechanisms to eliminate cancer cells, like apoptosis, cell cycle arrest, and DNA repair. This study aims to detect the abnormality change in the sequence of P53 mutation and the correlation with cancer in Mosul city DNA extraction depends on the manual description from blood, the Pro72Arg in exon 4 G\C allele mutation measurement by ARMS-PCR, and the analysis of common mutation in Exon 3,4,5 by DNA sequencing technique. The result of this study shows the observation of different genotypes and allelic frequencies of Pro72Arg polymorphism in exon 4. It was the presence of wild-type genotype CC (pro\pro) 12%, hetero genotype CG (pro\arg) 72% and mutant genotype GG (arg\arg) 16% in patients with Lung cancer. While in healthy people, the wild genotype CC was 26 hetero genotype CG 66 and mutant genotype GG 8%. As for the result of DNA sequencing, this research doesn't find any change in the nucleotide of Exon 5 for the P53 gene of the case study. At the same time, the DNA sequence result of Exon 6 for the P53 gene in patients finds some changes in the nucleotide sequence with sequence. According to this study, the observation of different genotypes and allelic frequency of Pro72Arg polymorphism in exon 4 for the P53 gene present significant variation between patients with lung cancer and healthy group male with Lung cancer Keywords: P53 gene, ARMS-PCR, Mutation, SNP, Lung cancer, Exon.

Identification of a missense variant in the porcine AGPAT gene family associated with intramuscular fat content through whole-genome sequencing.

The 1-acylglycerol-3-phosphate O-acyltransferases (AGPATs) are enzymes that catalyze the conversion of lysophosphatidic acid to phosphatidic acid, which is a precursor of triacylglycerol, the main fat reservoir in mammals. We used whole-genome sequencing of 205 pigs to identify 6639 genetic variants in the porcine AGPAT gene family. Of these, 166 common variants in the AGPAT5 gene had significant associations with fat content and composition traits. We preselected a missense single nucleotide polymorphism in exon 6 of AGPAT5 (rs196952262, A>G) for validation of its associations in 1034 pigs from the same Duroc line. The A allele showed a positive additive effect for intramuscular fat content (+1.12% ± 0.21, p < 0.001, for gluteus medius and +0.89% ± 0.33, p < 0.01, for longissimus). We also observed significant associations with fatty acid composition that were, at least in part, independent of the increased intramuscular fat. The A allele resulted in more monounsaturated fatty acids (+0.34% ± 0.15, p < 0.05, for longissimus) and a greater monounsaturated/polyunsaturated fatty acids ratio (+0.11 ± 0.04, p < 0.01, for gluteus medius and +0.13 ± 0.05, p < 0.05, for longissimus). The effect of the AGPAT5 variant on intramuscular fat was more noticeable in fatter pigs, and AGPAT5 interacts with other genes that affect overall fatness such as LEPR. AGPAT5 was the most expressed gene of the AGPAT family in pig skeletal muscle. This variant can be used as a marker in assisted selection for modulating pig fat deposition and fatty acid content.

Application of HRM Analysis in Detection of PDGFRA Exon 10 Polymorphism in CML Patients with Imatinib Resistance

Introduction: Imatinib mesylate has been widely used as a standard treatment for chronic myeloid leukemia (CML). It acts as a selective competitive inhibitor of the BCR-ABL tyrosine kinase. Despite the excellent efficacy on CML treatment, some patients developed resistance to the treatment. Mutation in the PDGFRA may be one of the factors involved in the mechanism of resistance that affects the response to imatinib. The mutational status of PDGFRA is highly relevant for prognosis and treatment prediction in CML patients. Thus, this study is intended to establish and validate a High Resolution Melting (HRM) analysis for PDGFRA exon 10 c.1432 T&gt;C polymorphism in CML patients. Methods: High resolution melting (HRM) analysis was used to identify the c.1432 T &gt; C polymorphism in PDGFRA exon 10 (n =86; response = 43; resistance = 43). The results from HRM analysis were compared and validated with Sanger sequencing. The association between the polymorphism and treatment response was assessed by statistical analysis using binomial logistic regression analysis. Results: HRM analyses showed two different melt curves. One curve followed the shape of the reference, homozygous wild type (TT) and the other curve showed a different melting profile than the reference with the TC genotype (heterozygous variant). The results revealed that heterozygous variant (TC) genotype showed a high risk of acquiring resistance with an OR of 3.795; 95% CI: 1.502-9.591, with a statistically significant association, p = 0.005. HRM analysis also showed 100% sensitivity and specificity in the detection of PDGFRA exon 10. Conclusion: The HRM analysis of PDGFRA exon 10 c.1432 T&gt;C was successfully established. The exon 10 c.1432 T&gt;C polymorphism shows a higher risk for the development of resistance toward imatinib treatment.

Androgen Receptor Gene CAG Repeat Length Varies and Affects Semen Quality in an Ethnic-Specific Fashion in Young Men from Russia.

Male infertility is a multi-factorial and multi-genetic disorder, and the prevalence of male infertility in the world is estimated at 5–35%. The search for the causes of male infertility allowed for identifying a number of genetic factors including a single X-linked gene of the androgen receptor (AR), and some of its alleles are assumed to negatively affect male fertility. Our aim was (1) to study the variability of the length of CAG repeats of the AR gene and possible associations in the AR CAG genetic variants with semen quality and reproductive hormone levels in a population-based cohort of men and (2) to estimate distributions of AR CAG repeat alleles and associations with semen parameters in different ethnic subgroups. The cohort of 1324 young male volunteers of different ethnicities (median age 23.0 years) was recruited from the general population of five cities of the Russian Federation, regardless of their fertility status. Semen quality (sperm concentration, motility and morphology), reproductive hormone levels (testosterone, estradiol, LH, FSH and inhibin B) and trinucleotide (CAG) n repeat polymorphism in exon 1 of the AR gene were evaluated. The semen samples were analyzed according to the WHO laboratory manual (WHO, 2010), serum hormones were measured by enzyme immunoassay, and the AR CAG repeat length was analyzed by direct sequencing of leukocyte DNA. The median AR CAG repeat length in men of our multi-ethnic population was 23 (range 6–39). In the entire study population, a significant difference (p ≤ 0.05) was found in the frequency distribution and the mean values for the CAG repeat length between the groups with normal (23.2 ± 3.3) and impaired semen quality (23.9 ± 3.2). Additionally, we demonstrated that the total sperm count, sperm concentration, progressive motility and normal morphology were lower in the category of long CAG repeats (CAG ≥ 25) compared with those in the category of short CAG repeats (CAG ≤ 19); however, hormonal parameters did not differ between the long and short CAG categories, with the exception of estradiol. Significant differences were observed in the AR CAG repeat length between the most common ethnic cohorts of Slavs (Caucasians), Buryats (Asians), and Yakuts (Asians). The Buryats and Yakuts had a higher number of CAG repeats than the Slavs (medians: Slavs—23; Buryats—24; Yakuts—25). The range of alleles differed among ethnicities, with the Slavs having the largest range (7–36 repeats, 24 alleles total), the Yakuts having the smallest range (18–32 repeats, 14 alleles total) and the Buryats having the middle range (11–39 repeats, 20 alleles total). The longer CAG repeats were associated with an impaired semen quality within the Slavic (CAG ≥ 25) and Buryat (CAG ≥ 28) groups, but this effect was not found in Yakuts. Hormonal parameters did not differ between the three CAG repeat categories in men of all ethnic groups. This is the largest Russian study of the distribution of AR CAG repeats and the search for association between length of AR CAG repeat tract and impaired spermatogenesis in men from the general population. Our results confirmed the association of longer CAG repeats with a risk of impaired semen quality, but this association can be modified by ethnic origin. Identification of the number of AR CAG repeats can be an effective tool to assess the risk of male subfertility and the control of androgen hormone therapy of reproductive diseases.

Aldehyde dehydrogenase 2 and arrhythmogenesis.

Cardiac arrhythmia is a common cardiovascular disease that leads to considerable economic burdens and significant global public health challenges. Despite the remarkable progress made in recent decades, antiarrhythmic therapy remains suboptimal. Aldehyde dehydrogenase 2 (ALDH2), a critical detoxifying enzyme, catalyzes toxic aldehydes and protects individuals from damage caused by oxidative stress. Accumulating evidence has demonstrated that ALDH2 activation has potential antiarrhythmic benefits. The correlation between ALDH2 deficiency and arrhythmogenesis has been widely recognized. In this review, we summarize recent research on the potential role of ALDH2 activation and antiarrhythmic protection, as well as the role played by the ALDH2∗2 polymorphism (rs671) in promoting arrhythmic risk. Additionally, we discuss important new findings illustrating the use of ALDH2 activators, which may prove to be promising antiarrhythmic therapy agents.

Frequency of functional exonic single-nucleotide polymorphisms and haplotype distribution in the SLCO1B1 gene across genetic ancestry groups in the Qatari population

Organic anion transporting polypeptides (OATP), which are encoded by SLCO genes, participate in the hepatic elimination of drugs and xenobiotics. SLCO1B1 is an important pharmacogenomic gene (encoding OATP1B1) associated with response to the uptake of endogenous compounds, such as statin and bilirubin. Ethnicity of the patient modulates the response to these drugs; the frequency and haplotype data for SLCO1B1 genetic variants in the Arab population is lacking. Therefore, we determined the frequencies of two well-characterized SLCO1B1 single nucleotide polymorphisms (SNP) and haplotypes that affect the OATP1B1 drugs transportation activity in Qatari population. Genotyping data for two SLCO1B1 SNPs (c.388A > G, c.521 T > C) were extracted from whole exome data of 1050 Qatari individuals, who were divided into three ancestry groups, namely Bedouins, Persians/South Asians, and Africans. By way of using Fisher's exact and Chi-square tests, we evaluated the differences in minor allele frequency (MAF) of the two functional SNPs and haplotype frequencies (HF) among the three ancestry groups. The OATP1B1 phenotypes were assigned according to their function by following the guidelines from the Clinical Pharmacogenetics Implementation Consortium for SLCO1B1 and Simvastatin-Induced Myopathy.The MAF of SLCO1B1:c.388A > G was higher compared to that of SLCO1B1:c.521 T > C in the study cohort. It was significantly high in the African ancestry group compared with the other two groups, whereas SLCO1B1:c.521 T > C was significantly low in the African ancestry group compared with the other two groups. The SLCO1B1 *15 haplotype had the highest HF, followed by *1b, *1a, and *5. Only the SLCO1B1 *5 haplotype showed no significant difference in frequency across the three ancestry groups. Furthermore, we observed that the OATP1B1 normal function phenotype accounted for 58% of the Qatari individuals, the intermediate function phenotype accounted for 35% with significant differences across the ancestry groups, and the low function phenotype accounted for 6% of the total Qatari individuals with a higher trend observed in the Bedouin group.The results indicate that the phenotype frequencies of the OATP1B1 intermediate and low function in the Qatari population appear at the higher end of the frequency range seen worldwide. Thus, a pharmacogenetic screening program for SLCO1B1 variants may be necessary for the Qatari population.

Refining the Camelus dromedarius Myostatin Gene Polymorphism through Worldwide Whole-Genome Sequencing.

Simple SummaryThe dromedary is a multipurpose livestock species. Growing interest in dromedary meat production, increasing intensification of meat production systems and rising business turnover for the dromedary athletic competition sector point to the need for a more effective genetic selection. To this aim, the identification of genetic markers, to be implemented in phenotype–genotype association studies, represents a pre-requisite. The myostatin gene is known to affect, to a various extent, muscularity in several animal species. In this study, we investigated the dromedary myostatin gene sequence variation through the analysis of data from 183 worldwide animals. A total of 99 variants were detected in the target region. Through a bioinformatic approach, we explored the possible functional effects of the detected variants. Several hints emerged, suggesting that natural variants at the dromedary myostatin locus may determine quali-quantitative changes in the myostatin expression, thus likely impacting on dromedary muscularity. Further efforts are needed to collect reliable phenotypic data on dromedary muscularity and racing performances, thus allowing for the unveiling of those genetic markers that are associated with the phenotypic variability in muscularity-related traits. Under intensive rearing systems, large-scale screening of genotypes at those markers in dromedary populations will effectively orient selection decisions and positively impact on rates of genetic gain.Myostatin (MSTN) is a highly conserved negative regulator of skeletal muscle in mammals. Inactivating mutations results in a hyper-muscularity phenotype known as “double muscling” in several livestock and model species. In Camelus dromedarius, the gene structure organization and the sequence polymorphisms have been previously investigated, using Sanger and Next-Generation Sequencing technologies on a limited number of animals. Here, we carried out a follow-up study with the aim to further expand our knowledge about the sequence polymorphisms at the myostatin locus, through the whole-genome sequencing data of 183 samples representative of the geographical distribution range for this species. We focused our polymorphism analysis on the ±5 kb upstream and downstream region of the MSTN gene. A total of 99 variants (77 Single Nucleotide Polymorphisms and 22 indels) were observed. These were mainly located in intergenic and intronic regions, with only six synonymous Single Nucleotide Polymorphisms in exons. A sequence comparative analysis among the three species within the Camelus genus confirmed the expected higher genetic distance of C. dromedarius from the wild and domestic two-humped camels compared to the genetic distance between C. bactrianus and C. ferus. In silico functional prediction highlighted: (i) 213 differential putative transcription factor-binding sites, out of which 41 relative to transcription factors, with known literature evidence supporting their involvement in muscle metabolism and/or muscle development; and (ii) a number of variants potentially disrupting the canonical MSTN splicing elements, out of which two are discussed here for their potential ability to generate a prematurely truncated (inactive) form of the protein. The distribution of the considered variants in the studied cohort is discussed in light of the peculiar evolutionary history of this species and the hypothesis that extremely high muscularity, associated with a homozygous condition for mutated (inactivating) alleles at the myostatin locus, may represent, in arid desert conditions, a clear metabolic disadvantage, emphasizing the thermoregulatory and water availability challenges typical of these habitats.

In vitro evaluation of the impact of Covid-19 therapeutic agents on the hydrolysis of the antiviral prodrug remdesivir

Remdesivir (RDV, Veklury®) is an FDA-approved prodrug for the treatment of hospitalized patients with COVID-19. Recent in vitro studies have indicated that human carboxylesterase 1 (CES1) is the major metabolic enzyme catalyzing RDV activation. COVID-19 treatment for hospitalized patients typically also involves a number of antibiotics and anti-inflammatory drugs. Further, individuals who are carriers of a CES1 variant (polymorphism in exon 4 codon 143 [G143E]) may experience impairment in their ability to metabolize therapeutic agents which are CES1 substrates. The present study assessed the potential influence of nine therapeutic agents (hydroxychloroquine, ivermectin, erythromycin, clarithromycin, roxithromycin, trimethoprim, ciprofloxacin, vancomycin, and dexamethasone) commonly used in treating COVID-19 and 5 known CES1 inhibitors on the metabolism of RDV. Additionally, we further analyzed the mechanism of inhibition of cannabidiol (CBD), as well as the impact of the G143E polymorphism on RDV metabolism. An in vitro S9 fraction incubation method and in vitro to in vivo pharmacokinetic scaling were utilized. None of the nine therapeutic agents evaluated produced significant inhibition of RDV hydrolysis; CBD was found to inhibit RDV hydrolysis by a mixed type of competitive and noncompetitive partial inhibition mechanism. In vitro to in vivo modeling suggested a possible reduction of RDV clearance and increase of AUC when coadministration with CBD. The same scaling method also suggested a potentially lower clearance and higher AUC in the presence of the G143E variant. In conclusion, a potential CES1-mediated DDI between RDV and the nine assessed medications appears unlikely. However, a potential CES1-mediated DDI between RDV and CBD may be possible with sufficient exposure to the cannabinoid. Patients carrying the CES1 G143E variant may exhibit a slower biotransformation and clearance of RDV. Further clinical studies would be required to evaluate and characterize the clinical significance of a CBD-RDV interaction.

A Method to Determine Xenobiotic Acetylation Rate by Taq SNP rs1495741.

Drug acetylation plays an important role in the medical practice. Modern methods of acetylation phenotype prediction are based on genotyping of polymorphisms in the second exon of the gene NAT2. Some disadvantages of these methods limit their application in the clinical practice. We developed a method of human genotyping based on identification of NAT2 gene polymorphism rs1495741 by real-time PCR. This method of genotype determination has a number of advantages: high sensitivity, simplicity, possibility of automated interpretation of the results, and feasibility in clinical laboratories.

The mungbean VrP locus encoding MYB90, an R2R3-type MYB protein, regulates anthocyanin biosynthesis.

Anthocyanins are water-soluble pigments present in several tissues/parts of plants. The pigments provide color and are wildly known for health benefits for human, insect attraction for plant pollination, and stress resistance in plants. Anthocyanin content variations in mungbean [Vigna radiata (L.) Wilczek] were first noticed a long time ago, but the genetic mechanism controlling the anthocyanins in mungbean remains unknown. An F2 population derived from the cross between purple-hypocotyl (V2709) and green-hypocotyl (Sulv1) mungbeans was used to map the VrP locus controlling purple hypocotyl. The VrP locus was mapped to a 78.9-kb region on chromosome 4. Sequence comparison and gene expression analysis identified an R2R3-MYB gene VrMYB90 as the candidate gene for the VrP locus. Haplotype analysis using 124 mungbean accessions suggested that 10 single nucleotide polymorphisms (SNPs) in exon 3 may lead to an abolished expression of VrMYB90 and an absence of anthocyanin accumulation in the hypocotyl of Sulv1 and KPS2. The overexpression of VrMYB90 in mungbean hairy root, tobacco leaf, and Arabidopsis resulted in anthocyanin accumulation (purple color). Gene expression analysis demonstrated that VrMYB90 regulated anthocyanin accumulation in the hypocotyl, stem, petiole, and flowers, and the expression was sensitive to light. VrMYB90 protein may upregulate VrDFR encoding dihydroflavonol 4-reductase at the late biosynthesis step of anthocyanins in mungbeans. These results suggest that VrMYB90 is the dominator in the spatiotemporal regulation of anthocyanin biosynthesis. Our results provide insight into the biosynthesis mechanism of anthocyanin and a theoretical basis for breeding mungbeans.

Association of new SNPs at DGAT1 gene with milk quality in Egyptian Zaraibi goat breed

This study aimed to detect putative genomic loci in candidate genes associated with milk composition in Egyptian Zaraibi goats. A total number of 50 samples were tested to detect polymorphism in exons 15 and 16 of the diacylglycerol acyltransferase 1 (DGAT1) gene. The PCR products were sequenced and aligned. Sequence analysis showed three new genotypes in the studied samples: T1C1 (T12C SNP), T2C2 (T84C), and AG (G219A), then three groups were created: the first group was BB with C1T1 and AG genotypes, the second was DD which contains C2T2 and AG genotypes, and the third was AG with only AG genotype. GLM showed that the DD group with C84T and G219A SNPs had significantly the highest fat percent. Meanwhile, the BB group with C84T and G219A SNPs recorded significantly the highest total solids levels. On the other hand, the AG group which has G219A SNP showed a non-significant effect on milk components. Those new SNPs were submitted to GenBank and approved to be published. Moreover, translation of those sequences showed that the G219A SNP causes a substitution of Glycine to Serine in exon 16 at position 106. This SNP (G106S) was predicted to be tolerated by SIFT with a score of 0.48.

Effect of TNF-α gene polymorphism on fertility insufficiency in Holstein cows

The current study was conducted in private station located in the middle of Iraq by using 100 blood samples from 100 Holstein cows to isolate DNA and determine the genotypes of TNF-α gene in tow regions on reproductive performance. Results showed two alleles with co-dominance and three genotypes was detected in 1st position (4th exon), the allele frequency was 0.695 and 0.305 for A and C allele respectively. The allele frequency in 2nd region (Flank region) was 0.64 and 0.36 for A and G allele respectively. Results showed a significant effect of gene polymorphism in 4th exon on retained placenta rate which differed significantly (P≤0.01) among cow groups, the highest rate was recorded in cows with cows with AC genotype about 0.24. A significant effect of gene polymorphism in 4th exon on ovulation weakness rate, the highest rate was recorded in cows with cows with CC genotype about 0.12. Parturition difficulties rate differed significantly (P≤0.01) , the highest rate was recorded in cows with mutant genotype namely,0.18. Results showed a significant effect of gene polymorphism in flank region, retained placenta rate was differed significantly (P≤0.01) among cow groups, the highest rate was recorded in cows with GC genotype about 0.20.

Abstract 2692: Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene

Abstract Castration resistant prostate cancer (CRPC) involves the upregulation of androgen receptor variants (AR-Vs), of which AR-V7 is clinically the most relevant. By lacking the ligand-binding domain, AR-V7 is constitutively active and may therefore bypass AR signaling inhibitors. Treating CRPC involves improved AR signaling inhibitors or taxane-based chemotherapy. However, the optimal treating sequence may differ from patient to patient. The presence of AR-V7 in the nucleus of circulating PCa cells, which is associated with AR targeted therapy resistance, is used as a therapy-guiding biomarker. This stratification has a high predictive power and specificity, but its sensitivity is unsatisfying and can only be done by highly specialized laboratories. A PCR/sequencing based test would improve the sensitivity of this biomarker, but it requires a better understanding of AR-V7’s functional regulation. Therefore, we aim here to analyze single nucleotide polymorphisms (SNPs) in the 3’ untranslated region (UTR) of AR-V7 that might have an impact on AR-V7’s expression and/or functional activity. The LD trait tool of NIH was used to search for trait association with AR cryptic exon (CE3) SNPs. Four RNA binding motif databases (oRNAment, ATtRACT, RBP map, and RBPDB) and SpliceAid v2 were used for evaluating SNPs in their capacity to alter motifs. By CRISPR/CAS9 mediated non-homologous end joining (NHEJ) these predictions were assessed experimentally using qPCR and Westen Blot. SNP rs5918762 was selected as it is in linkage disequilibrium with several PCa tagSNPs (rs5919393, rs5919432). rs5918762T/C is characterized by an unequal minor allele frequency (MAF) distribution among different populations. Using an in silico approach, this SNP is predicted to destroy/create a binding site for the splicing machinery involved protein SRSF9. Disrupting this SNP region via NHEJ resulted respectively in an increased and decreased expression of AR-FL and AR-V7 mRNA in 22RV1 cells (C allele). Additionally, analysis of the SU2C dataset (CRPC patients) correlated SRSF9 expression with AR-V7 expression. Subsequently, SRSF9 knock out (KO) resulted in reduced AR-V7 mRNA expression. Our data suggests that SNP rs5918762T/C is located in a region important for CE3 inclusion during AR splicing, which might involve SRSF9. Further validation of these results is in need and encompasses splicing assays by an AR-V7 mini-gene assay, protein-RNA interaction assays and an analysis of a lack of effect in cell lines expressing the T allele. Citation Format: Jasper Van Goubergen, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2692.

Evaluation of the relationships between HLA-G 14 bp polymorphism and two acute leukemia in a Saudi population

The non-classical Human Leukocyte Antigen G (HLA-G) is an immunomodulatory molecule, with low polymorphism frequency and restricted tissue distribution. Its higher expression was associated with immunoinhibitory properties causing tolerance for cancer progression. The rs371194629 is a 14 bp insertion/deletion polymorphism (Ins/Del) in exon 8 of the 3′ untranslated region (3′-UTR) of the HLA-G gene, has been associated with the stability and the expression level of HLA-G mRNA. In this study, we evaluated the relationship between the HLA-G 14-bp Ins/Del polymorphism and two common blood cancers including acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) in a population from Riyadh in Saudi Arabia. The study groups include 145 patients diagnosed for ALL, 98 patients diagnosed for AML and 115 healthy individuals. For all these individuals, the 14 bp Ins/Del polymorphism was genotyped using PCR methodology. mRNA gene expression of the HLA-G was assessed using quantitative RT-PCR. Logistic regression model analysis for HLA-G 14 bp Ins/Del polymorphism shows no statistically significant correlation with AML and ALL. For the HLA-G mRNA expression, while slightly increased level among healthy individuals compared to patients was observed, no statistically significant differences were obtained. Although our results did not show association between HLA-G with the two leukemia cancer diseases, the role of HLA-G gene awaits further investigations including large number of subjects with more clinical data combinations.

Skewed X-Chromosome Inactivation as a Possible Marker of X-Linked CNV in Women with Pregnancy Loss

Skewed X-chromosome inactivation (sXCI) can be a marker of lethal genetic variants on the X chromosome in a woman since sXCI modifies the pathological phenotype. The aim of this study was to search for CNVs in women with miscarriages and sXCI. XCI was assayed using the classical method based on the amplification of highly polymorphic exon 1 of the androgen receptor (AR) gene. The XCI status was analysed in 313 women with pregnancy loss and in 87 spontaneously aborted embryos with 46,XX karyotype, as well as in control groups of 135 women without pregnancy loss and 64 embryos with 46,XX karyotype from induced abortions in women who terminated a normal pregnancy. The frequency of sXCI differed significantly between women with miscarriages and women without pregnancy losses (6.3% and 2.2%, respectively; p = 0.019). To exclude primary causes of sXCI, sequencing of the XIST and XACT genes was performed. The XIST and XACT gene sequencing revealed no known pathogenic variants that could lead to sXCI. Molecular karyotyping was performed using aCGH, followed by verification of X-linked CNVs by RT-PCR and MLPA. Microdeletions at Xp11.23 and Xq24 as well as gains of Xq28 were detected in women with sXCI and pregnancy loss.

Survey of the association between polymorphisms of CTLA-4 exon 1 49 A/G genes with rheumatoid arthritis in Iran

ABSTRACT Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which suppresses T cell proliferation, is a promising candidate for the susceptibility genes to rheumatic arthritis diseases (RA). This study aims to examine the association between the polymorphisms of the CTLA-4 exon 1(+ 49) genes with RA in the Qazvin city of Iran population. The polymerase chain reaction of genomic DNA-restriction fragment length polymorphism (PCR-RFLP) was applied to genotype the CTLA-4 exon 1(+ 49) polymorphisms in 105 RA patients and 90 control subjects. Laboratory diagnostic tests were also measured for RA and control groups. Our results did not demonstrate a significant difference in allele and genotype frequencies of the CTLA-4 exon 1(+ 49) between RA patients and the control group (p < .0001). There was no significant difference in age at onset, CRP, RF value in patients with RA according to the CTLA-4 polymorphisms; just anti-CCP showed a significant difference. Our data declared that polymorphisms of CTLA-4 exon 1(+ 49) genes are not correlated with RA susceptibility and its clinical and paraclinical manifestations.

Association Study between Mucin 4 (MUC4) Polymorphisms and Idiopathic Recurrent Pregnancy Loss in a Korean Population.

Recurrent pregnancy loss (RPL) is the loss of two or more consecutive pregnancies before 20 weeks of gestational age. Our study investigated whether mucin 4 (MUC4) polymorphisms are associated with RPL. MUC polymorphisms (rs882605 C>A, rs1104760 A>G, rs2688513 A>G, rs2258447 C>T, and rs2291652 A>G) were genotyped in 374 women with RPL and 239 controls of Korean ethnicity using polymerase chain reaction-restriction fragment length polymorphism analysis and the TaqMan probe SNP genotyping assay. Differences in genotype frequencies between cases of RPL and the controls were compared. MUC4 rs882605 C>A and rs1104760 A>G polymorphisms were associated with increased incidence of RPL in three and four or more pregnancy loss patients. The haplotype analyses showed a tendency for the allelic effect including the association of MUC4 rs882605 A and rs1104760 G alleles with increased incidence of RPL. In addition, the MUC4 rs882605 CA/MUC4 rs2258447 CC genotype combination was associated with increased RPL prevalence. The two exonic polymorphisms lead to amino acid changes of protein and may act as pathogenic variants for RPL. In conclusion, the MUC4 rs882605 C>A and MUC4 rs1104760 A>G polymorphisms were associated with the susceptibility of RPL and we considered them as potential biomarkers for RPL.