Allelic Variants Research Articles

Compound Heterozygosity Involving Other Dead ( DDX) and Deah ( DHX) Box Helicases Explains Seemingly Monoallelic Somatic and Germline DDX41 Mutations

DDX41 mutations typify a seemingly distinct group of AML/MDS that appear to differ in many features from wild type entities. Classical DDX41 mutant myeloid neoplasia (MN) harbors biallelic mutations whereby, canonical germline (GL), mostly frameshifts (fs, chiefly at amino acid position 140) predispose to the acquisition of somatic canonical R525H and possible other somatic variants in the contralateral allele. Yet, in many instances, only GL or somatic mutations can be found. We stipulated that somatic DDX41 is not present in mature leukocytes but would be detectable in hematopoietic stem cells (HSCs). Indeed, higher VAF is detected in HSCs vs neutrophils likely due to differentiation arrest. However, it is also possible that other somatic mutations ( e.g., in other RNAhelicases) and conversely in cases with R525H without GL DDX41 might be present. In either case, upon our original discovery based on lower ATPase activity of R525H mutants 1 we stipulated that this mutation would be hypomorphic, an indirect evidence that has been, to date, not further substantiated. Here, we propose that R525H may indeed be functional via some unknown mechanisms, such as a GOF variant selected in the mode of somatic gene rescue (SGR) as a maladaptive response to relieve the GL LOF fs alteration. To that end, we studied the molecular makeup of a cohort of 1,893 MN patients (including public datasets 2,3) using NGS. In total, 101 patients with MN harbored DDX41 lesions. Among these, 35 patients had somatic hits other than R525H, detected as a seemingly monoallelic configuration and 15 patients harbored biallelic hits involving D140fs (n=3), M1I/L (n=2), and other pathogenic GL variants (n=10). A total of 51 patients carried GL mutations without matching a second allele hit; including D140 as monoallelic mutation in 4 patients, fs/del/splice sites in 10 patients, and missense variants in 37 patients (among this, M1I/L accounted for 24%). When we studied these monoallelic GL cases, we found a second somatic alteration in other DDX/ DHX helicases in carriers of seemingly monoallelic fs/splice sites. We found somatic hits in DDX10 (n=7), DDX25 (n=2), DDX52 (n=1), DHX16 (n=4) and DHX34 (n=1) suggesting that neoplastic progression occurring in these cases was due to compound heterozygosity involving other helicases substituting for the absence of canonical biallelic configuration. Of note is that no hit in any other helicases was detected in cases with sole GL D140, leaving unexplained the somatic cause driving neoplastic progression in these cases. However, upon manual inspection, R525H was detected in 2 patients with D140 and one patient with M1I albeit at very low VAFs%. Ultra low input NGS of pre-sorted bone marrow cellular fractions showed higher clonal burden of somatic R525H in HSCs, revealing the truly biallelic nature of these cases. 4 We then sought to explain the absence of GL in cases with R525H and other pathogenic lesions. For instance, we found one suspicious GL variant in DDX20 in one patient with R525H and one in DDX31 in one patient with non-R525 hit. These results suggest that monoallelic DDX41 cases may have compound heterozygosity (somatic/GL vs GL/somatic) for DDX41 and other DDX genes. These compound heterozygous cases prompted us to screen other patients with wild type DDX41 for the presence of DDX/DHX mutations. In total, we identified 38 monoallelic cases. Of those, 22 were GL with the most frequent being DDX54 (n=4), DDX11, and DDX20 (n=3; for both), followed by others. We are currently exploring whether these cases might harbor cryptic R525H, which might have been not resolved in bulk sequencing. In summary, we show that the canonical DDX41 biallelic constellation is a more ubiquitous phenomenon in the disease evolution to MN and involves other DDX/DHX helicases. In fact, a proportion of seemingly monoallelic cases might be explained by compound heterozygosity of other RNA helicases. Moreover, most GL DDX41 are cryptic biallelic or compound heterozygous, while cases with somatic canonical DDX41 should be investigated for the presence of GL DDX41 or other RNA helicases using ultra low input NGS. We propose that somatic DDX41 hits correspond to maladaptive SGR of hypomorphic GL mutants in analogy but in reverse fashion compared to GL SAMD9/9L. Studies to be presented at ASH will further substantiate this possibility.

Circulating Tumor DNA Analysis Associates with Progression-Free Survival (PFS) with Odronextamab Monotherapy in Relapsed/Refractory (R/R) Follicular Lymphoma (FL) and Diffuse Large B-Cell Lymphoma (DLBCL): Identification of Minimal Residual Disease Status and High-Risk Subgroups from the Phase 2 ELM-2 Study

Background Molecular characterization of B-cell non-Hodgkin lymphoma (B-NHL), through circulating tumor DNA (ctDNA) assessment of minimal residual disease (MRD) has been proposed as a tool to predict clinical outcome. Odronextamab, a CD20×CD3 bispecific antibody, demonstrated deep and durable responses and a generally manageable safety profile in patients (pts) with R/R FL or DLBCL in the Phase 2 ELM-2 study (NCT03888105; Kim TM et al. and Kim WS et al. ASH. 2022). In the overall populations, 12-month PFS rates were 64% and 29%, respectively. Using ctDNA from ELM-2, we show that these assessments associate with clinical outcomes following odronextamab treatment. Methods In ELM-2, pts received IV odronextamab in 21-day cycles, with step-up dosing in Cycle (C) 1, 80 mg (FL)/160 mg (DLBCL) QW in C2-4, then 160 mg (FL)/320 mg (DLBCL) Q2W until disease progression or unacceptable toxicity. Baseline (BL) ctDNA and tumor biopsies were used for molecular profiling. BL and on-treatment ctDNA were used for MRD determination in the biomarker population (BP; pts required ≥1 available plasma biomarker sample to be included in the BP). The first post-BL ctDNA sample was collected on C5 Day (D) 1, at the time of positron emission tomography-computed tomography (PET-CT). A modified AVENIO ctDNA analysis workflow (Roche; research only) was used for next-generation sequencing, based on the cancer personalized profiling by deep sequencing technique (Kurtz et al. J Clin Oncol. 2018). Whole blood cell pellets were used to filter out germline allele variants. MRD negativity was reported when the P value for variant allele frequency was >0.005. Results The BP comprised 53 FL pts and 63 DLBCL pts; at BL, all FL pts and all but one DLBCL pt were MRD(+). Pts remaining on study until C5D1 had similar PFS regardless of whether they were in the BP or overall population (FL, n=111; DLBCL, n=83). Pts who were MRD(-) at C5D1 had significantly longer PFS vs. those who remained MRD(+) (FL: HR 0.27 [95% CI 0.09-0.84], P=0.024 [Fig. 1a]; DLBCL: HR 0.29 [95% CI 0.12-0.71], P=0.007). In pts with FL achieving complete response (CR) by PET-CT at C5D1, a trend for prolonged PFS was observed in those who were MRD(-) (n=26) at this timepoint vs. those who were MRD(+) (n=17; HR 0.29 [95% CI 0.07-1.1], P=0.072). Pts with DLBCL who were MRD(-) at C5D1 (n=20) had similar PFS benefit regardless of PET-CT CR status (HR 0.56 [95% CI 0.10-3.11], P=0.511). Four DLBCL pts who were MRD(-) and did not achieve CR by PET-CT at C5D1 (partial response, n=3; stable disease, n=1) went on to achieve PET-CT CR at a later timepoint. Mutational analyses of BL ctDNA identified TP53 as the most frequent mutation (FL, n=34/53 [64%] mutation-evaluable pts; DLBCL, n=44/58 [76%]). In FL pts, TP53 mutations were associated with MRD(+) status at C5D1 (Fisher's exact test, P=0.002) and predicted significantly shorter PFS (HR 3.57 [95% CI 1.01-12.69]; P=0.049); this association was not observed in DLBCL. LymphGen classification (Wright et al. Cancer Cell. 2020) of BL ctDNA in pts with DLBCL identified mostly MCD and EZB subtypes (MCD, n=15; EZB, n=14; ST2, n=1; BN2, n=1; other, n=26). EZB-subtype pts treated with odronextamab had significantly longer PFS compared with MCD-subtype pts (HR 0.23 [95% CI 0.07-0.83]; P=0.025 [Fig. 1b]). Using BL ctDNA, cell of origin was determined in 43/58 pts with DLBCL; odronextamab treatment led to similar PFS in higher-risk non-germinal-center B-cell-like (non-GCB, n=18 [42%]) pts vs. GCB pts (n=25 [58%]; HR 1.62 [95% CI 0.72-3.62]; P=0.244). Further molecular assessment focused on gene fusions in DLBCL pts in the BP (39 with local laboratory gene fusion and ctDNA analyses, 24 with only ctDNA analyses). Odronextamab led to similar PFS in pts with double-hit (n=15) or triple-hit (n=5) fusions compared to those without (n=43; P=0.343 and P=0.140, respectively). Conclusions This study is among the first to analyze ctDNA in pts with R/R FL and DLBCL in a pivotal trial. This non-invasive method allows molecular characterization of pts with no available tissue, enabling identification of high-risk subgroups. CtDNA MRD status at C5D1 of odronextamab treatment was highly associated with PFS in pts with FL and DLBCL and in the future could form the basis of response-directed treatment paradigms. Molecular characterization in tumor biopsies, including CD20, will be presented. Ongoing monitoring of ctDNA may serve as an important early progression marker in R/R FL and DLBCL.

Mutational Landscape of Bone Marrow CD19 and CD138 Cells in Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathy of Undetermined Significance (IgM MGUS)

Background Waldenström's Macroglobulinemia (WM) is an incurable B-cell neoplasm characterized by serum monoclonal immunoglobulin M (IgM) and clonal lymphoplasmacytic cells infiltrating the bone marrow (BM). Smoldering WM (sWM) is the asymptomatic/indolent form with a high risk of progressing to symptomatic WM requiring treatment whereas IgM monoclonal gammopathy of undetermined significance (IgM MGUS) is an early precursor stage of WM. Despite recurrent and activating mutations, including MYD88, CXCR4, ARID1A, KMT2D, and CD79B have been identified, the genetic basis for WM and the risk of progression of IgM MGUS to WM remain to be fully elucidated. Methods Based on the mutational profiling of WM and our previous wide transcriptome analyses on BM CD19+ and CD138+ cells of patients with WM and IgM MGUS, we decided to investigate the mutation status of these patients as follows: WM (n=10), sWM (n=7), and IgM MGUS (n=5), by performing high throughput targeted AmpliSeq next generation sequencing on 117 target genes potentially involved in B-cell lymphomagenesis and in the risk of progression of IgM MGUS to WM (Trojani A. et al. Cancers, 2021). Results We identified genetic aberrations in multiple genes, including previously described classic mutations in MYD88 (L260P alias L265P) found in 82.3% (14/17) of WM/sWM patients and in 60% (3/5) of IgM MGUS subjects. The CXCR4 mutation (S338Ter) was detected in 23.5% of CD19+ cells of WM patients, whereas it was absent in IgM MGUS subjects. Interestingly, we also identified new mutated genes, including WNK2 somatic mutations affecting 47% of WM patients, for which a recurrent allelic variant (V1635Ter) was observed, and BCL9 with a recurrent frameshift variant (P516Lfs) identified in 29.4% of WM patients exclusively in the CD19 cell fraction. The BCL9 (P516Lfs) variant was also detected in the B cells of one IgM MGUS patient. Moreover, sequencing evaluation revealed recurrently frameshift or missense mutations involving NFKB2 (L473Afs), PTPN13 (P1546Tfs), CARD11 (S622del), KMT2C (I823T), and ATM in WM and IgM MGUS patients (Figure 1). Of interest, KMT2C (also known as MLL3) exhibited a somatic hypermutated phenotype in both CD19+ and CD138+ cells of WM and IgM MGUS samples. The KMT2C mutations were not mutually exclusive with those involving KMT2D in our cohort of patients. Conclusion The present genomic analysis revealed new insights into the mutational landscape of Waldenström Macroglobulinemia, providing evidence of the recurrence of some variants in subjects with IgM MGUS as well. Notably, we uncovered new genetic mutations with somatic variants recurrently observed (n≥3) in KMT2C, WNK2, BCL9, PTPN13, CARD11 and NFKB2 genes. These recurring somatic events provide a genomic basis for future research and a better understanding of the WM pathogenesis as well as the risk of transformation of IgM MGUS to WM.

SLCO1B1 and CYP3A4 allelic variants associated with pharmacokinetic interactions and adverse reactions induced by simvastatin and atorvastatin used in Peru: Clinical implications

Context: Statins reduce the risk of stroke and prevent cardiac events in people with atherosclerosis and diabetes mellitus; and could affect the proliferation, migration, and survival of cancer cells. Aims: To review the most up-to-date and available scientific evidence on the allelic variants of SLCO1B1 and CYP3A4 associated with pharmacokinetic interactions and adverse reactions induced by simvastatin and atorvastatin used in Peru, and their clinical implications. Methods: The bibliographic search was carried out in the PubMed/Medline, Google Scholar and Science Direct databases. The keywords were: “statin”, “atorvastatin”, “simvastatin” in combination with “pharmacokinetics”, “pharmacogenetics”, “CYP3A4”, “SLCO1B1” or “drug interactions” considering the eligibility criteria defined by the PRISMA-2020 international statement. Results: Scientific evidence indicates a significant association between SLCO1B1 rs4149056 c.521T>C (521CC and 521TC) and increased plasma levels, area under the plasma concentration curve (AUC) and maximum plasma concentration (Cmax) of simvastatin, compared to wild-type SLCO1B1*1/*1 521TT (p<0.05). SLCO1B1 521C is not associated with atorvastatin (p>0.05). Patients with SLCO1B1 521CC had a significantly higher risk of myopathy and rhabdomyolysis induced by simvastatin compared to TT (p<0.05). An association was also found between CYP3A4*1/*22/CYP3A4*3/*22 and increased pharmacokinetic parameters of simvastatin compared to CYP3A4*1/*1 (p< 0.05). Conclusions: Based on the review of the published scientific evidence, it is concluded that individuals carrying the allelic variants SLCO1B1 (c.521T>C), CYP3A4*1/*22 and CYP3A4*3/*22 could be associated with an increase in the pharmacokinetic parameters and with an increased risk of myopathy and rhabdomyolysis induced by simvastatin, and not by atorvastatin.

Implementing a high-reliability, system-wide program for pre-emptive DPD deficiency testing for patients planned for a systemic fluoropyrimidine.

396 Background: Systemic fluoropyrimidine indications span several solid tumor types including gastrointestinal, breast, and head and neck malignancies. Dihydropyrimidine dehydrogenase (DPD) is the primary enzyme involved in fluoropyrimidine metabolism. Pathogenic polymorphisms of the gene encoding DPD (DPYD), occur at a rate of 4-7% and correlate with DPD enzyme deficiency. Failure to detect patients with DPD deficiency, and adjust the dosage preemptively, may result in severe toxicity, including death. Methods: Between December 2022 and May 2023, Dana-Farber Cancer Institute (DFCI) implemented a program across all sites of care to simplify and standardize preemptive DPYD testing for patient planned for a systemic fluoropyrimidine. The opt-out program includes an automated alert recommending DPYD testing when an oncologist orders a new regimen containing either 5FU or capecitabine, education for providers, pharmacists and nurses, patient education materials, standardized tracking of pending results, closed loop notification of providers of abnormal test results, dose adjustment guidelines, and patient tracking. DPYD allelic variants tested: *2A, *7, *8, *10, *13, rs67376798, rs75017182, rs115232898. Results: DPYD testing has increased from a baseline of 14% to 89%. Abnormal DPYD activity (score <2) has been detected in 5.1% of cases (28 of 553). 23 tests were performed preemptively and 5 reactively (all 5 after > grade 3 toxicity events). 27 cases had a single abnormal allele (activity score 1.5 or 1.0); a single case had 2 abnormal alleles (homozygous for rs75017182). Among 11 of the original 23 preemptively tested patients who continued fluoropyrimidine treatment at our facility, standardized dosage reductions were successfully applied for 10 (90.9%). None have experienced > grade 3 toxicity and subsequent dose escalation was possible for 5 (45.5%) patients. Conclusions: We have implemented a voluntary approach to facilitate routine preemptive DPYD testing for patients planned for a systemic fluoropyrimidine achieving an 89% usage rate. 28 of 553 (5.1%) patients were found to have at least a single allelic variant. Preemptive dose reductions were successfully applied for 90.9% of cases. The results provide proof of principle that pharmacogenomic testing can be applied in routine clinical practice and sets the stage for testing additional genes.

Detected Genetic Markers for Three Varieties of Rice (Oryza sativa L.) under Nano- Particles

Despite the enormous advancements in breeding, fresh and accessible methods of plant development are preferred. Many research have been done to determine the impact of zinc oxide nanoparticles (ZnO NPs) and magnesium nanoparticles (MgONPs) on plant growth. ZnO NPs and MgONPs have been used extensively in agriculture. Few research have looked into the potential impact of ZnO NPs on grain Zn content or cereal yield formation to date. The goal of the study was to demonstrate how varied concentrations of nanoparticles affected root germination in three different rice types (Giza 177, Sakha super 300, and Sakha 108) and the fungus Fusariummoniliforme, which causes foot rot in rice. Here, we performed a pot experiment to assess the effects that ZnO nanoparticles and MgONPs had on the shoot length, root length, and germination of rice. The experiment involved three dosages of Zn and Mg nanoparticle solution at varying concentrations of 10, 20, and 100 ppm. The outcomes showed that, when compared to the control treatment, ZnO NPs and MgONPs increased the length of the shoot, the length of the roots, and the rate of germination. Using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers, the genetic makeup of plants treated with 10, 20, and 100 ppm AgNPs was also examined. In vitro rooted shoots were acclimated in a greenhouse before being tested for biochemical stability and phenotypic stability. In vitro, callogenesis and caulogenesis were both reduced by AgNPs at the greatest dose (100 ppm). In order to maximise genome coverage, three rice cultivars from distinct origins were chosen, and they were tested using three RAPD markers and seven selected ISSR markers. AgNPs-treated plants using ISSR markers produced 87 amplified bands in total, 45 polymorphic allelic variants, and 36 monomorphic allelic variations. The Pearson correlation coefficient was used in the Mantel test to determine whether there was a significant association between the Jaccard's dissimilarity matrices based on ISSR and RAPD markers (r = 0.69; P 0.05). Morphological characteristics, ISSR, and RAPD analysis were used to partition the UPGMA Dendrogram into two groupings. Jaccard's coefficient was used to analyse the genetic similarity matrix. Sakha Super 300 and Giza 177 had the most genetic similarity (95%) while Sakha 108 and Giza 177 had the lowest genetic similarity (90%) according to ISSR and RAPD study. The classification of rice germplasm, breeding initiatives, and conservation efforts all heavily rely on the determination of genetic diversity within the rice species. To find genetic variations, morphological characteristics, ISSR, and RAPD analysis are useful techniques. The findings demonstrated the large ratio of variation in rice. This work successfully established the possibility of using MgONPs and ZnO NPs as high-performing fertilisers to improve rice output and quality.This study could serve as a guide for future research on rice and could support efforts to understand the species and improve breeding stock.

Investigation of the Differences in the Pharmacokinetics of CYP2D6 Substrates, Desipramine, and Dextromethorphan in Healthy African Subjects Carrying the Allelic Variants CYP2D6*17 and CYP2D6*29, When Compared with Normal Metabolizers.

This study investigated the differences in the pharmacokinetics (PK) of dextromethorphan and desipramine in healthy African volunteers to understand the effect of allelic variants of the human cytochrome P450 2D6 (CYP2D6) enzyme, namely the diplotypes of CYP2D6*1/*2 (*1*1, *1*2, *2*2) and the genotypes of CYP2D6*17*17 and CYP2D6*29*29. Overall, 28 adults were included and split into 3 cohorts after genotype screening: CYP2D6*1/*2 (n=12), CYP2D6*17*17 (n=12), and CYP2D6*29*29 (n=4). Each subject received a single oral dose of dextromethorphan 30mg syrup on day1 and desipramine 50mg tablet on day8. The PK parameters of area under the plasma concentration-time curve from time of dosing to time of last quantifiable concentration (AUClast), and extrapolated to infinity (AUCinf), and the maximum plasma concentration (Cmax) were determined. For both dextromethorphan and desipramine, AUCinf and Cmax were higher in subjects of the CYP2D6*29*29 and CYP2D6*17*17 cohorts, as compared with subjects in the CYP2D6*1/*2 diplotype cohort and with normal metabolizers from the literature. All PK parameters, including AUCinf, Cmax, and the elimination half-life, followed a similar trend: CYP2D6*17*17>CYP2D6*29*29>CYP2D6*1/*2. The plasma and urinary drug/metabolite exposure ratios of both drugs were higher in subjects of the CYP2D6*17*17 and CYP2D6*29*29 cohorts, when compared with subjects in the CYP2D6*1/*2 diplotype cohort. All adverse events were mild, except in 1 subject with CYP2D6*17*17 who had moderately severe headache with desipramine. These results indicate that subjects with CYP2D6*17*17 and CYP2D6*29*29 genotypes were 5-10 times slower metabolizers than those with CYP2D6*1/*2 diplotypes. These findings suggest that dose optimization may be required when administering CYP2D6 substrate drugs in African patients. Larger studies can further validate these findings.

Polymorphism of allelic variants of milk protein genes

The polymorphism of allelic variants of milk productivity genes (alpha-lactoglobulin, beta-casein, kappa-casein, prolactin, somatotropin) in cattle of the yaroslavl breed was studied in conjunction with an assessment of the genetic diversity of the studied population. The distribution of frequencies of occurrence of genotypes in the genes of the studied herds (0.1...0.716049) was determined. Based on the calculations of the average values ??of the genetic diversity indices, the effective number of alleles (1.679...1.994), the Shannon information index (0.594...0.692), the observed heterozygosity (0.087...0.716), the expected heterozygosity (0.404...0.498), fixation index (-0.498...0.823).The coefficient of inbreeding of subpopulations in the studied genes relative to the entire population indicates a weak differentiation of different herds (-0.023 ... -0.002). According to the analysis of the equilibrium distribution of genotypes according to the Hardy-Weinberg law, it is noted that the largest part of the herds has an equilibrium distribution of genotypes. The exception is yaroslavka breeding Plant JSC for the A1A2 beta-casein gene (20.095) and Agromir LLC (Mikhailovskoe branch) for the beta-casein gene (15.581).

Genetic structure of the collection of ryegrass (Lolium) cultivars: a study based on SSR and SCoT markers

Background. Current molecular and genetic approaches make it possible to accelerate ryegrass breeding, simplify source material evaluation, and increase its accuracy. The efficiency of PCR-based SSR and SCoT marker techniques was studied in the context of evaluating the genetic structure of annual and perennial ryegrass accessions and defining DNA-identifying markers.Materials and methods. Genomic DNA was isolated from the aggregate sample of 30 seedlings from each of the 15 analyzed cultivars according to the modified SDS DNA extraction protocol. In total, 20 SSR and 22 SCoT markers were used to assess genetic polymorphism. Basic parameters of the markers’ informative efficiency were identified. Genetic relationships among the studied cultivars were analyzed on the basis of the Neighbor-Joining dendrogram and Bayesian model.Results. To assess the genetic polymorphism of ryegrass species and varieties, 7 SSR loci were selected, for which 110 allelic variants were identified (34 alleles were unique for individual cultivars), and 9 SCoT loci, for which 78 polymorphic amplification fragments were identified, with 28 being cultivar-specific. The dendrogram of genetic similarity and modeling in the Structure v2.3.4 program according to the results of SSR and SCoT analyses distributed the cultivars by their traits according to their species, ploidy level, origin, and similarity. Based on PCoA, carried out using summarized data of SSR and SCoT analyses, a multidimensional diagram of genetic relationships among ryegrass cultivars was constructed.Conclusions. The systems of SSR and SCoT markers appeared to be an efficient tool to reveal genetic polymorphism and identify differences among ryegrass species and cultivars. We found unique DNA profiles that can be used for genetic identification. The results of the study have practical significance in cultivar-specific identification and selection of cultivars for various purposes.

Marker-assisted selection in the development of advanced apple-tree forms and donors combining scab resistance with increased fruit storability

Background. Cultivation of scab-resistant apple cultivars with better fruit storability increases the economic efficiency and environmental friendliness of horticulture. Hence, the development of such cultivars is an important task in modern apple-tree breeding.Materials and methods. A set of 646 hybrid plants obtained in six cross combinations (Renet Simirenko/Modi, Renet Simirenko/Smeralda, Renet Simirenko/Renoir, Renet Simirenko/Fujion, Renoir/Granny Smith, and Modi/Granny Smith) was studied. Their scab resistance was assessed under natural infection pressure for two years using a quantitative scale. The Rvi6 gene was identified using the PCR analysis with the primer pair VfC1+VfC. The Md-ACS1 gene alleles were detected with a known SCAR DNA marker. The chi-square test was applied for statistical confirmation of the data.Results. Based on the DNA marker analysis, 328 plants carrying the dominant allele of the Rvi6 gene were identified. The results of the phenotypic resistance assessment confirmed the correspondence of the resistant phenotype / dominant allele of the gene for most plants. Molecular genetics analysis of the Md-ACS1 gene disclosed its allelic combinations. A total of 190, 322, and 126 plants carrying allelic variants of Md-ACS1-2/2, Md-ACS1-1/2, and Md-ACS1-1/1, respectively, were identified. Among the plants with the dominant allele of the scab resistance Rvi6 gene, 92 plant forms were identified with Md-ACS1-2/2, and 143 heterozygous ones (Md-ACS1-1/2), also valuable for breeders.Conclusion. A wide range of apple breeding forms carrying valuable alleles of the Rvi6 and Md-ACS1 genes were selected. Hybrid forms with target alleles of both genes responsible for important agronomic traits would contribute to the intensification of breeding efforts aimed at producing scab-resistant apple cultivars with increased fruit storability. They can also be used for breeding purposes as complex donors.

Pharmacogenetics May Prevent Psychotropic Adverse Events in Autism Spectrum Disorder: An Observational Pilot Study.

Up to 73% of individuals with autism spectrum disorder (ASD) and intellectual disability (ID) currently have prescriptions for psychotropic drugs. This is explained by a higher prevalence of medical and psychiatric chronic comorbidities, which favors polypharmacy, increasing the probability of the appearance of adverse events (AEs). These could be a preventable cause of harm to patients with ASD and an unnecessary waste of healthcare resources. To study the impact of pharmacogenetic markers on the prevention of AE appearance in a population with ASD and ID. This is a cross-sectional, observational study (n = 118, 72 participants completed all information) in the ASD population. Sociodemographic and pharmacological data were gathered. The Udvalg for Kliniske Undersøgelser Scale (UKU Scale) was used to identify AEs related to the use of psychotropic medication. Polymorphisms of DOP2, ABCB1, and COMT were genotyped and correlated with the AE to find candidate genes. Furthermore, a review of all medications assessed in a clinical trial for adults with autism was performed to enrich the search for potential pharmacogenetic markers, keeping in mind the usual medications. The majority of the study population were men (75%) with multiple comorbidities and polypharmacy, the most frequently prescribed drugs were antipsychotics (69%); 21% of the participants had four or more AEs related to psychotropic drugs. The most common were "Neurological" and" Psychiatric" (both 41%). Statistical analysis results suggested a significant correlation between the neurological symptoms and the DOP2 genotype, given that they are not equally distributed among its allelic variants. The final review considered 19 manuscripts of medications for adults with ASD, and the confirmed genetic markers for those medications were consulted in databases. A possible correlation between neurologic AEs and polymorphisms of DOP2 was observed; therefore, studying this gene could contribute to the safety of this population's prescriptions. The following studies are underway to maximize statistical power and have a better representation of the population.

Fifty novel HLA-DPB1 alleles identified in a UK cohort of unrelated hematopoietic cell donors and recipients.

HLA-DPB1 is the classical HLA class II genes with the least recorded variation on the IPD-IMGT/HLA Database, suggesting the full extent of its diversity is perhaps yet to be characterized. Here, a full-gene typing strategy was employed to genotype a UK cohort of 1470 HCT recipients (n = 744) and donors (n = 726). In total, 2940 full-length HLA-DPB1 sequences were generated, comprising 193 distinct alleles. Of these, 107 sequences contained novel variation, totaling 49 unique intronic HLA-DPB1 alleles, and one coding variant (HLA-DPB1*1188:01). Full-gene sequencing resulted in zygosity changes for 129 individuals by identifying two distinct intronic variants of the same coding allele. We verified the existence of nine unconfirmed alleles and extended the sequence of two existing alleles on the IPD-IMGT/HLA Database.

Interleukin-37: associations of plasma levels and genetic variants in gout

ObjectivesIL-37 is an anti-inflammatory cytokine involved in inflammatory and autoimmune diseases. We aimed to investigate the association between IL-37 genetic variants, IL-37 plasma levels, and various clinical phases of gout.MethodsThe study included a control group with no history of primary hyperuricemia/gout, (n = 50), asymptomatic hyperuricemia (n = 74), intercritical gout (n = 200), acute gouty flare (n = 18), and chronic tophaceous gout (n = 30). Plasma IL-37 was analysed using enzyme-linked immunosorbent assay. All coding regions and intron–exon boundaries of IL-37 and exons 1–5 were amplified and sequenced.ResultsPlasma levels of IL-37 were significantly higher in asymptomatic hyperuricemic (p = 0.045), intercritical gout (p = 0.001), and chronic tophaceous gout (p = 0.021) cohorts when compared to control group. The levels of IL-37 in patients with acute gouty flare were comparable to control group (p = 0.061). We identified 15 genetic variants of IL-37: eight intron (rs2708959, rs2723170, rs2708958, rs2723169 rs2466448, rs3811045, rs3811048, rs2708944) and seven non-synonymous allelic variants (rs3811046, rs3811047, rs2708943, rs2723183, rs2723187, rs2708947, rs27231927), of which rs2708959 showed an over-presentation in gouty and acute flare cohorts (p = 0.003 and 0.033, respectively) compared to European population (minor allelic frequency MAF = 0.05) but not in control and hyperuricemic cohorts (p/MAF = 0.17/0.08 and 0.71/0.05, respectively).. On the contrary, rs3811045, rs3811046, rs3811047, and rs3811048 were underrepresented among individuals with tophaceous gout (MAF = 0.57) compared to European MAF 0.70–0.71, but not compared to the control cohort (MAF = 0.67).ConclusionsWe demonstrated the up-regulation of IL-37 levels across the clinical phases of gout: asymptomatic hyperuricemia, intercritical, and chronic tophaceous gout compared to control. Moreover, 15 genetic variants of IL-37 were identified and their associations with the clinical variants of gout were evaluated.

Analysis of the Structural Organization and Expression of the Vrn-D1 Gene Controlling Growth Habit (Spring vs. Winter) in Aegilops tauschii Coss.

The duration of the vegetative period is an important agronomic characteristic of cereal crops. It is mainly influenced by the Vrn (response to vernalization) and Ppd (response to photoperiod) genes. In this work, we searched for alleles of several known genes of these two systems of response to external conditions in 15 accessions of Aegilops tauschii Coss. (syn. Ae. squarrosa L.), with the aim of studying the impact these alleles have on the vegetative period duration and growth habit. As a result, three allelic variants have been found for the Vrn-D1 gene: (i) one intact (winter type), (ii) one with a 5437 bp deletion in the first intron and (iii) one previously undescribed allele with a 3273 bp deletion in the first intron. It has been shown that the spring growth habit of Ae. tauschii can be developed due to the presence of a new allele of the Vrn-D1 gene. Significant differences in expression levels between the new allelic variant of the Vrn-D1 gene and the intact allele vrn-D1 were confirmed by qPCR. The new allele can be introgressed into common wheat to enhance the biodiversity of the spring growth habit and vegetative period duration of plants.

Allelic variant 6A (rs35068180) of the MMP-3 promoter region as a predictor of cardiotoxicity after the end of adjuvant chemotherapy with doxorubicin in patients with breast cancer

Aim. To evaluate the relationship of polymorphic variants rs2232228 of the HAS3 gene, rs8187710 of the ABCC2 gene, rs35068180 of the MMP-3 gene with cardiotoxicity after the end of adjuvant chemotherapy in patients with breast cancer.Material and methods. The study included 100 patients (women, mean age 52,5±9,4 years) diagnosed with breast cancer who received anthracycline antibiotics (doxorubicin, total dose 240 mg/m2 or 360 mg/m2). Echocardiography was performed before and after the end of chemotherapy. Polymorphic status of selected targets was determined using the real-time polymerase chain reaction.Results. After the end of chemotherapy, based on the changes of left ventricular ejection fraction and global longitudinal strain, cardiotoxicity (CT) was detected in 20 patients. There were following significant differences between subgroups: rs8187710 of the ABCC2 gene — not identified; rs2232228 of the HAS3 gene — genotype AA, odds ratio (OR) 3,37 (95% confidence interval (CI) 1,14; 9,97) and allelic variant A, OR 2,17 (95% CI 0,98; 4,80) are significantly more common (p<0,05) in the cardiotoxicity+ subgroup; rs35068180 of the MMP-3 gene — genotype 6A/6A, OR 2,53 (95% CI 0,93; 6,88) and allelic variant 6A, OR 2,19 (95% CI 1,08; 4,44) are significantly more often (p<0,05) in the cardiotoxicity+ subgroup.Conclusion. Genotype 6A/6A, allelic variant 6A rs35068180 of the MMP-3 gene, genotype AA and allelic variant A rs2232228 of the HAS3 gene can be considered as predictors of early cardiotoxicity after the end of chemotherapy in patients with breast cancer receiving doxorubicin.

The Influence of Human IgG Subclass and Allotype on Complement Activation.

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating that a long-standing conundrum in the literature depends on cellular context. Furthermore, we compared IgG1 and IgG3 under different circumstances, showing that Ag density and Ab hinge length, but not complement regulators, define the context dependency of Ab-mediated CDC activity. Our results point toward a variation in the capacity of IgG subclasses to activate complement due to single amino acid changes and hinge length differences of allotypes to activate complement, which might give new insights on susceptibility to infectious, alloimmune, or autoimmune diseases and aid the design of Ab-based therapeutics.

Metabolism of testosterone and progesterone by cytochrome P450 2C19 allelic variants.

CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450nm) typical of P450 in the reduced CO-difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α-/17- and progesterone 6β-,21-,16α-/17α-hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone.

Genetic risk variants for childhood nephrotic syndrome and corticosteroid response.

The etiology of most cases of nephrotic syndrome (NS) remains unknown, therefore patients are phenotypically categorized based on response to corticosteroid therapy as steroid sensitive NS (SSNS), or steroid resistant NS (SRNS). Genetic risk factors have been identified for SSNS from unbiased genome-wide association studies (GWAS), however it is unclear if these loci are disease risk loci in other forms of NS such as SRNS. Additionally, it remains unknown if these risk loci are associated with response to therapy. Thus, we investigated the association between SSNS risk loci and therapy response in a large, multi-race cohort of children along the entire spectrum of childhood-onset NS. We enrolled 1,000 patients with childhood-onset NS comprised of SSNS and SRNS. Genotyping was done using TaqMan and Direct Sanger Sequencing for 9 previously reported childhood SSNS risk loci. We compared the allele frequencies (AF) and variant burden between NS vs. controls and SRNS vs. SSNS. All 9 risk loci were associated with NS compared with healthy controls (p = 3.5 × 10-3-<2.2 × 10-16). Variant burden greater than 7 was associated with risk of SRNS (OR 7.4, 95% CI 4.6-12.0, p = 8.2 × 10-16). Our study showed that genetic risk loci for childhood SSNS are associated with pattern of therapy response, may help predict disease outcome, and set the stage for individualized treatment of NS.

FRI267 Carney Complex From A Single Familycarney Complex From A Single Family Followed Up Over 20 Years With Different Clinical Pattern Evolution

Abstract Disclosure: A.W. Kuhn: None. D.S. de Melo: None. H.L. Charchar: None. B.M. Mariani: None. M.Y. Nishi: None. F.L. Ledesma: None. F.Y. Tanno: None. V. Srougi: None. J.L. Chambo: None. A. Latronico: None. B.B. Mendonca: None. M.Q. Almeida: None. M.C. Fragoso: None. Background: Carney complex (CNC) is a hereditary syndrome with autosomal dominant inheritance, composed of multiple neoplasms, such as benign and malignant, endocrine and nonendocrine tumors (cardiac, cutaneous and neural). The primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of Cushing syndrome is the most common endocrine neoplasia in CNC. The most frequent genetic cause is related with the presence of germline pathogenic allelic variants on PRKAR1A. Overall CNC penetrance among carriers of the PRKAR1A allelic variant is approximately 97.5%, however, families with long-term follow up is rarely described in literature. Clinical Case: A 17-year-old male, was referred to our Hospital in 2003 due to weight gain (≈10 Kg), BMI 27.27 Kg/m2, acne, occurrence of abdomen purplish striae (&amp;gt;1cm), facial plethora and arrest of pubertal development for last 2 years. He has had pigmented facial lesions since 9 years old. Laboratory revealed suppressed ACTH &amp;lt;18.0 pg/mL, failure to suppress cortisol levels after low dose dexamethasone test (DST-1mg): cortisol 29 µg/dL (normal range: &amp;lt;1.8 µg/dL), and total urinary cortisol (TUC), 2809 µg/24 h (normal range: 30-300 µg/24h). Abdominal CT scan showed normal right adrenal gland and a micronodule (0.8 cm) in the left adrenal gland. Bone densitometry showed an important loss of density. Owing to ACTH-independent Cushing's syndrome and adrenal with micronodule, the most plausible diagnostic hypothesis was PPNAD associated to CNC. He underwent bilateral laparoscopic adrenalectomy and the histological data confirmed PPNAD on both adrenal glands. He developed primary adrenal insufficiency and he maintains continuous replacement with gluco and mineralocorticoids. The genetic analysis identified a heterozygous germline nonsense pathogenic variant on PRKAR1A (p.Tyr21X) in exon 2. His relatives, such as father, two younger sisters, one paternal aunt and a female cousin carried the same nonsense variant. All of them also presented facial lentiginous but without Cushing features. In the first investigation, all his relatives showed possible autonomous cortisol secretion. In follow-up of almost 20 years, the index case and his family members did not develop other manifestations of CNC. Only his father developed metabolic syndrome, but all relatives maintained subclinical hypercortisolism due to possible autonomous cortisol secretion without any clinical repercussions. Clinical Lessons: Carney complex is a rare disease and the follow-up of familial cases over the years can show different clinical pattern despite the same germline pathogenic variant. Probably somatic molecular mechanisms as modulators could trigger specifically clinical manifestations. Presentation: Friday, June 16, 2023

THU522 Symptomatic Hypoglycemia As The First Manifestation Of Multiple Endocrine Neoplasia Type 1 (MEN-1): Case Report

Abstract Disclosure: V.D. Oliveira: None. S.C. Tormin: None. B.V. Dias: None. N. Scalissi: None. R.D. Scalco: None. C. Olivati: None. A. de Moricz: None. L.A. Szutan: None. J.V. Lima: None. Introduction: MEN-1 is a rare, autosomal dominant syndrome that occurs due to a pathogenic variant of MEN1. The incidence of insulinoma in MEN-1 is relatively rare. 4-6% of patients with insulinoma will develop MEN-1. Unlike sporadic insulinomas that usually develop after the age of 40, MEN-associated insulinoma usually occur before 40 years of age and even sometimes before 20. CASE REPORT: A 26-year-old male patient had a 3-year history of Whipple's triad associated with significant weight gain with a body mass index (BMI) of 33 kg/m² to 58 kg/m² during this period. His father has a metastatic carcinoid tumor, being treated with palliative chemotherapy. Laboratory tests at the time of hypoglycemia were: blood glucose 42mg/dL (RV 75-99), insulin 39 mU/L (RV &amp;lt; 3) and C-peptide 11.6 ng/dL (RV &amp;lt; 0.2). Magnetic resonance imaging (MRI) of pancreas showed 3 hypervascular nodules, the largest in the tail, measuring 3.3 cm, with neuroendocrine characteristics. Distal pancreatectomy, splenectomy and lymphadenectomy were indicated. After surgery, the episodes of hypoglycemia disappeared, with a weight loss of 25 kg. He required postoperative metformin, with satisfactory glycemic control. During the investigation, a prolactinoma was diagnosed (pituitary MRI, which showed a 4 mm adenoma, prolactin 56ng/mL (LR &amp;lt; 25) and hypogonadotrophic hypogonadism), and cabergoline 0.25 mg/week was started. In addition, diagnosis of mild primary hyperparathyroidism (PTH 188 pg/mL and calcium 10.4mg/dL). Parathyroid scintigraphy with 99mTc-MIBI showed increased uptake in the right lower parathyroid. Genetic testing was performed by Next Generation Sequence (NGS) of the MEN1 gene, which was positive for the pathogenic variant c.825-2A&amp;gt;G in heterozygosis in intron 5. A molecular study was performed on the patient's father and sister, which identified the same pathogenic allelic variant in both. Conclusion: We described a rare case of MEN-1 whose first manifestation was insulinoma. Presentation: Thursday, June 15, 2023