Wt Allele Research Articles

Polymorphism of 8-oxoguanine DNA glycosylase -1(OGG1)in Iraqi Patients with Type2 Diabetes Mellitus

Chronic hyperglycemia in type 2 diabetes mellitus leads to elevated oxidative stress. As a consequence , the accumulation of reactive oxygen species (ROS)may cause additional damage to various biological macromolecules, including DNA. Several studies have demonstrated that oxidative stress plays an important role in the pathogenesis of type2 diabetes mellitus. The aim of this study is to investigate the association of polymorphisms of 8- oxoguanine DNA glycosylase-1(OGG1) repair gene polymorphism to the susceptibility of type 2 DM in an Iraqi population with T2DM patients from Wasit Province. All samples were collected from the local community of Wasit province, Iraq. Forty five type 2 diabetes mellitus patients (22 males and 23 females) and 35 healthy controls (17 males and 18 females) were genotyped for 8-oxoguanine DNA glycosylase-1(OGG1) using PCR- RFLP technique. In both patients and control groups, the distribution frequencies of genotypes and alleles of 8-oxoguanine DNA glycosylase-1(OGG1) A/G was inconsistent with the Hardy-Weinberg equilibrium in T2DM patients(χ2=49.542,P=(0.00001); χ2=82.0185,P=(0.0001) respectively. The Cys/Cys(mt/mt) OGG1 genotype was significantly higher in patients than controls(mt/mt;33(37%)vs,0.00 in controls. The Ser/Cys (wt/mt) was 7(16%) and 6(17%) in patients and controls respectively and Ser/Ser genotypes wt/wt; decreased significantly in patients 5(11%) vs,29(83%) in controls. The wt and mt allele frequencies of OGG1were highly significant between the two groups P=0.00001. The wt allele was the major one in control group with a percent of (90.28) vs. (18.89) in patients group. Whereas the mt the frequent allele in patients with a percent of (81.11) vs. (18.89).This further analysis showed that the individuals carrying the homozygous Cys/Cys(mt/mt) genotype were more likely to have increased the risk of T2DM very significantly with OR= 190.02800 (CI95% 10.8329 to 3342.2603) ,P=0.0003. The genotypes Ser/Ser( wt/wt) and Ser/Cys(wt/mt) decrease the association with T2DM with OR=0.0259 (CI95% 0.0072 to 0.0930), and 0.8904 (CI95% 0.2701 to 2.9347 )respectively, P= 0.0001 and 0.08486 for each genotype respectively, These results suggest that mt allele may be considered as risk allele of T2DM whereas the wt allele is protective agent T2DM.Association analysis showed that the type2 diabetes mellitus risk of females with OGG1 gene Cys/Cys( mt/mt) genotype was highly significant 81.4000 fold higher than that in controls OR= 81.4000 (CI 95% 4.3089 to 1537.7322), P= 0.0033.Ser/Cys( wt/mt) genotype increase the probability of the disease with OR = 1.7647 (CI 95% 0.3745 to 8.3154), P = 0.4727.Similary,Ser/Ser(wt/wt) genotype decrease the association with T2DM with OR= 0.0091 (CI95% 0.0009 to 0.0959), P = 0.0001.Type2 diabetes mellitus patients particularly with Cys/Cys(mt/mt) genotype increases the association about more than 111 times in males patients than that in controls OR= 111.3636 ( CI95% 5.7135 to2170.6226), P = 0.0019 While, genotypes Ser/Cys(wt/mt) and Ser/Ser (wt/wt) reduce the likelihood of T2DM with OR= 0.2222 ( CI95% 0.0209 to 2.3585),P = 0.2120 and 0.0476( CI95% 0.0091 to 0.2484), P= 0.0003 respectively. the genetic model for OGG1 in comparison between T2DM patients and controls . The dominant model indicated that patients and controls of (wt/mt+mt/mt) genotype increased significantly the association with T2DM in patients: (5/7 and 33) comparing with control (29/6 and 0.00) with OR (38.666),P=0.0001.The recessive model revealed that patients carrier the genotype (wt/wt+wt/mt) declined significantly the association with the disease :(33/5 and 7) in patients versus (0.00/29 and 6) in controls,OR=0.0053,P=0.0003.The Over-dominant model showed that patients with the genotype (wt/wt+mt/mt) in creased non-significantly the association with the disease when compare patients (7/5 and 33) with controls(6/29 and 0.00) ,OR=1.123,P=0.848

Allele-specific CRISPR-Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy

The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR/Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at a single nucleotide variant in the COL6A1 gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed spCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep-sequencing for two gRNAs tested showed that single nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele-selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.

Investigating the Correlation between Genotypes and Hepatic Protein Expression of CYP2C9, CYP2C19, CYP2D6, and CYP3A5 Using Postmortem Tissue from a Danish Population.

The cytochrome P450 (CYP) family of enzymes plays a central role in the metabolism of many drugs. CYP genes are highly polymorphic, which is known to affect protein levels, but for some low frequent CYP genotypes the correlation between genotype and CYP protein expression is less established. In this study, we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes of 250 Danish individuals included in a postmortem study. For 116 of the individuals, the hepatic CYP protein levels were investigated by a proteomics approach. Overall, we found the postmortem genetic and proteomic data to be in agreement with those of other studies performed on fresh hepatic tissue, showing the usability of postmortem hepatic tissue for this type of investigation. For less investigated genotypes, we could corroborate previously found results: 1) statistically significantly lower levels of hepatic CYP2C9 protein in individuals carrying the CYP2C9*3 variant compared with individuals with two wild type (wt) alleles; 2) comparable levels of CYP2C19 in CYP2C19*2/*17 and CYP2C19*1/*2 individuals; 3) reduced CYP2D6 protein levels in heterozygous individuals with the CYP2D6*3, CYP2D6*4, and CYP2D6*5 gene deletion variants; and 4) significantly lower levels of CYP3A5 protein in CYP3A5*3 homozygous individuals compared with individuals who were heterozygous for the CYP3A5*3 allele or homozygous individuals for the wt alleles. In conclusion, the use of postmortem tissue significantly increases the access to human specimens for research purposes, and postmortem proteomics can be used to investigate the link between CYP genotypes and hepatic protein expression. SIGNIFICANCE STATEMENT: In tissue samples from a large postmortem cohort (n = 250) we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes. Hepatic CYP protein levels were investigated in 116 individuals using a proteomics approach. For common genotypes, we found results similar to previous knowledge, pointing toward the usability of postmortem tissue. For the less investigated genotypes, we were able to corroborate genotype/protein expression correlations. It is a novel approach to use a large postmortem cohort to investigate genetic/protein expression correlations.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Abstract 3317: KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications

Abstract Alterations in KRAS are a main cancer driver. Amplifications of the KRAS wild-type (wt) allele account for ~7% of all KRAS-driven cancers and are frequent in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). KRASG12V mutations account for ~28% of pancreatic cancers, 9% of colorectal cancers and 6% of lung adenocarcinoma. There is an urgent medical need to identify targeted therapies for these frequent KRAS alterations. Herein, we describe BI 3706674, a novel, potent and orally bioavailable small molecule inhibitor of the KRAS oncogene for clinical testing in patients with tumors harboring KRASG12V mutations and KRAS wild-type amplifications. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Importantly, BI 3706674 is highly selective for KRAS vs HRAS or NRAS, and it is therefore expected to be well-tolerated in normal tissues. In two large cancer cell line panels (PRISM and Horizon), BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wt amplifications and KRAS mutations e.g., G12A/C/D/V, G13D and Q61H). The strongest sensitivity was observed for cell lines with KRAS wt amplifications (relative copy number (CN) of > 10) followed by cell lines with KRASG12V mutations along with significant pharmacodynamic (PD) biomarker modulation (e.g., down-regulation of DUSP6 mRNA). In vivo, BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models of human gastric cancer with KRAS wt CN > 10 and diverse tumor types with KRASG12V mutations. A twice daily oral dose of 30 mg/kg induced significant tumor regression and PD biomarker modulation. Combination of BI 3706674 with standard of care therapies and novel agents are being tested in preclinical models to guide clinical development. Results of the ongoing pre-clinical analysis will be shared. A Phase I clinical trial is in preparation in patients with advanced solid cancers harboring KRASG12V mutations and KRAS wt amplifications to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and efficacy of BI 3706674. Citation Format: Antonio Tedeschi, David H. Peng, Fiorella Schischlik, Lorenz Herdeis, Otmar Schaaf, Valeria Santoro, Daniel Gerlach, Fabio Savarese, Jesse Lipp, Christian Haslinger, Francesca Rocchetti, Johannes Popow, Heinrich J. Huber, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broeker, Tobias Wunber, Michael Gmachl, Klaus Rumpel, Matthew Rees, Melissa Ron, Jennifer Roth, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Scott Kopetz, Michael Kim, Don L. Gibbons, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harbouring KRASG12V mutations and KRAS wild-type amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3317.

Abstract 2906: Inducing and disabling COX-2 S-nitrosylation to investigate its role in breast cancer progression

Abstract Background: We aim to assess the effect of S-nitrosylation of COX-2 on tumor attributes in models of early breast cancer. COX-2 is involved in tumor progression in many solid cancers. It is a known oncogene in murine breast cancer models, and targetable by NSAIDs. In women, its clinical correlation to breast cancer progression as well as its efficacy as a therapeutic target have yielded mixed results. These incongruities may be due, in part, to a post-translational modification, as COX-2 undergoes S-nitrosylation on Cys-526. Commercially-available antibodies selectively bind to either the S-nitrosylated (SNO-COX-2) or non-nitrosylated (nCOX-2) forms, and immunohistochemical staining of human tissue reveals that the two forms of COX-2 localize to different cellular sites in normal breast tissue and have opposite associations with breast cancer progression (PMID 33298921). Hypothesis: S-Nitrosylated and non-nitrosylated COX-2 have distinct roles in cancer progression. Approach: MCF10DCIS.com is a human breast cancer cell line chosen due to its ability to form DCIS-like (“stage 0”) lesions in culture and xenografting, which can progress to invasion when induced by signals from the tumor microenvironment (TME). Approach 1: Detect extracellular matrix (ECM) and ligand factors that upregulate SNO-COX-2, independently or in ECM/ligand pairs. We utilized a 2D microenvironment microarray (MEMA) and tested 357 TME conditions. Approach 2: Validate TME conditions in 3D culture. Approach 3: Develop a mutant MCF10DCIS.com line in which COX-2 cannot be S-nitrosylated by inducing a single disabling missense mutation at Cys-526 via CRISPR. Results: The MEMA revealed several conditions in which SNO-COX-2 was upregulated, including fibular collagens and SSP1. In 3D culture, increased SNO-COX-2 was associated with the presence of markers for EMT and an invasive growth phenotype, whereas nCOX-2 was not. Following CRISPR transformation, 59 individual MCF10DCIS.com clones were isolated and the target site of each sequenced. We identified 10 heterozygotic clones with the desired mutation, including a classical heterozygote that also retained the WT allele. Ongoing: Current studies include a second round of CRISPR to produce a homozygous mutant cell line as well as validation of the mutant protein. The resulting cell lines will model the loss of COX-2 S-nitrosylation in breast cancer cells while retaining otherwise-functional COX-2. Citation Format: Reuben J. Hoffmann, AeSoon Bensen, Elise de Wilde, James E. Korkola, Pepper Schedin. Inducing and disabling COX-2 S-nitrosylation to investigate its role in breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2906.

Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000-8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFβ homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.

Abstract A085: BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in preclinical models and validates wild-type amplified KRAS as a therapeutic target

Abstract Alterations of KRAS are found in approximately one in seven of all human cancers, making KRAS one of the main oncogenic drivers in cancer. Gain-of-function missense mutations in KRAS are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma. In addition, focal high-level amplification of the KRAS wild-type (wt) allele, an alternative means of activating this oncoprotein, is observed in ~7% of cancers and lacks effective targeted therapies. Thus, there is an urgent need to identify effective therapies to address KRAS-driven tumors. Recently, we have reported the identification of compounds active against a broad range of oncogenic KRAS variants (KRASmulti) that selectively inactivate downstream signaling and tumor growth (Kim et al., Nature 2023). Herein we describe BI 3706674, a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including the KRAS wt allele, in the GDP-bound state and thereby disrupts oncogenic signaling. Using several in vitro assays, we show that BI 3706674 is a) highly selective for KRAS vs HRAS or NRAS; b) blocks the interaction between KRAS mutant/wt and its guanidine exchange factor (GEF) SOS1 and c) shows potent antiproliferative activity in isogeneic cell lines that are dependent on various KRAS mutant alleles. In two large cancer cell line panels, BI 3706674 potently and selectively inhibits proliferation of KRAS mutant cancer cell lines as well as cancer cell lines with an amplification of the KRAS wt allele. Significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and DUSP6 mRNA down-regulation) was observed in cell lines with a KRAS wt relative copy number (CN) > 10, treated with BI 3706674. Amplification of the KRAS wt allele is most frequently observed in gastric (~5%), esophageal (~13%) and gastroesophageal junction cancers (~12%). BI 3706674 was well-tolerated and showed dose-dependent efficacy in cell line-derived and patient-derived xenograft models (CDX and PDX) of human gastric cancer with a KRAS wt CN > 10. A twice daily oral dose of 30 mg/kg was sufficient to induce significant tumor regression. BI 3706674 treatment induced PD biomarker modulation (including inhibition of ERK1/2 phosphorylation, DUSP6 down-regulation, repression of the MAPK Pathway Activity Score (MPAS) signature and Ki67 reduction) in two KRAS wt amplified gastric cancer xenograft models. The depth and duration of PD biomarker responses following treatment correlated well with the doses used in the respective efficacy studies. Finally, these results support the clinical use of biomarkers such as the MPAS signature score and Ki67 to investigate treatment effects elicited by BI 3706674 in tumor biopsies. The KRASmulti inhibitor BI 3706674 is undergoing IND enabling studies and is intended to target tumors driven by KRAS wt amplifications as well as oncogenic KRAS missense mutations. Citation Format: Antonio Tedeschi, Lorenz Herdeis, Valeria Santoro, Fabio Savarese, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Bröker, Tobias Wunberg, Michael Gmachl, Rumpel Klaus, Fiorella Schischlik, Jesse Lipp, David H. Peng, Mariah Williams, Charles Deckard, Vandhana Ramamoorthy, Joseph R. Daniele, Jaffer A. Ajani, Funda Meric-Bernstam, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in preclinical models and validates wild-type amplified KRAS as a therapeutic target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A085.

Predicted Functional Consequences of TP53 Alterations and Prognosis in TP53-Mutated Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) cases with TP53 mutations ( TP53mut) are associated with poor responses to chemoimmunotherapy (Eskelund Blood 2017). Across myeloid and solid tumors, predicted functional consequences and pathogenicity of specific TP53 mutations have been linked to differential patient outcomes (Pollock Breast Cancer Res Treat 2022, Yabe Cancers 2021). We aimed to characterize the impact of these features and associated molecular alterations on treatment outcome in TP53mut MCL patients treated with standard frontline regimens and on the ongoing phase 2 BOVEN trial. The BOVEN trial aims to assess the efficacy of frontline treatment with zanubrutinib, obinutuzumab, and venetoclax in TP53mut MCL (Kumar Blood 2021). Methods: The BOVEN trial enrolled 25 patients with TP53mut MCL. Retrospective chart review was performed for 22 patients with TP53mut MCL treated at frontline with standard chemoimmunotherapy regimens (Joffe Blood 2019). Tumor histology and FISH were obtained for each case. Mutational profiling (SNV/indels, CNA) was obtained prior to therapy with various CLIA-certified targeted sequencing platforms (Cheng J Mol Diagn 2015, Dias-Santagata EMBO Mol Med 2010). Mutations in TP53 were annotated using predicted phenotypes and pathogenicity from the TP53 Database (de Andrade Cell Death Differ 2022) and from phenotypic annotation of TP53 mutations (Giacomelli Nat Genet 2018). The primary outcome measures were overall and progression-free survival (OS/PFS). Results: Each patient included in the BOVEN cohort featured variants in TP53 with 26 SNV/indels across all 25 patients. There were 25 SNV/indels across all 22 patients treated with standard regimens. Within the trial cohort 14/25 cases had evidence of biallelic inactivation of TP53 with co-occurring SNV/indels in the gene and 17p loss by FISH or TP53 copy losses by SNParray; the comparator cohort featured 11/22 cases with biallelic losses. Across cohorts, most mutations were missense (>80% in both), with the majority occurring in the DNA binding domain of the p53 protein (76% and 91%, respectively), and with >30% at known hotspot codons in both cohorts. Most mutations (>70% in both cohorts) were predicted to adversely impact WT p53 function, including nonfunctional transactivation activity and dominant negative effect on the WT allele. The majority were also predicted to be highly pathogenic by bioinformatic methods (AGVGD, SIFT, PolyPhen2, PHANTM, REVEL, BayesDel). However, none of these features correlated with outcome. In the BOVEN cohort the variant allele frequency (VAF) of the TP53 SNV/indels approached significant association with poor PFS (HR 33.2, p=0.05) but not OS (HR 28.6, p=0.09). In contrast, in the comparator cohort TP53 VAF appeared to have an opposite effect direction, though this was not significant for PFS (HR 0.98, p=0.2) or OS (HR 0.97, p=0.06). We next investigated the impact of co-occurring molecular alterations on outcome. Besides TP53, the most frequently mutated genes in the trial cohort were KMT2D (24%), ATM (16%), NOTCH2 (12%), and CCND1 (12%). Alterations in these genes did not correlate with outcome. Within the comparator cohort the recurrently mutated genes were SMARCA4 (23%), ARID1A (18%), ARID1B (14%), ATM (14%), and CCND1 (14%). As described previously in this cohort (Joffe Blood 2019), alterations in SMARCA4 were associated with worse PFS (HR 4.2, p=0.02) but not OS (HR 2.2, p=0.27). Mutational burden (SNV and CNA load) was not prognostic in either 12/25 BOVEN samples sequenced with the same panel or the comparator cohort. There was no significant difference in outcome between patients with single mutations in TP53 vs. those with biallelic losses in TP53 in either cohort. Conclusion: Across patients with TP53mut MCL treated either with standard frontline chemoimmunotherapy or on the BOVEN trial, predictions of intrinsic pathogenicity and functional consequences of the TP53 alterations were not prognostic. The impact of TP53 mutation VAF remains unclear, with seemingly opposite effect directions between the two cohorts, though neither finding was significant. As previously described in the comparator cohort, alterations in SMARCA4 are associated with worse PFS in TP53mut MCL, though this was not observed in the BOVEN cohort. There were no significant differences in outcome among patients with biallelic inactivations in TP53 or with higher burdens of somatic alterations.

Rapid and Accurate Remethylation of Dnmt3a Deficient Hematopoietic Cells with Restoration of DNMT3A Activity

Heterozygous loss-of-function mutations in the DNMT3A gene are the most common cause of clonal hematopoiesis, and among the most common initiating events for Acute Myeloid Leukemia (AML). A reduction of DNMT3A activity causes a canonical, focal hypomethylation phenotype in hematopoietic cells, which is associated with immortalization of hematopoietic stem cells, and blockage of myeloid differentiation. The methylation defect can be partially reversed with restoration of physiologic levels of DNMT3A expression over a period of several months (Ketkar et al., PNAS, 2020; PMID 35313694).To identify a more efficient remethylation strategy, we first characterized the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic cell deficiency of Dnmt3a, Dnmt3b (or both enzymes), or expressing the dominant negative Dnmt3aR878H mutation (equivalent to R882H in humans; the most common mutation found in AML patients). Using these different mouse models as substrates, we then defined the patterns and completeness of DNA remethylation after “adding back” supraphysiologic levels of wild type DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive “chaperone” that augments the activity of DNMT3A and DNMT3B in early embryogenesis), with MSCV-based retroviruses transduced into primary mouse hematopoietic stem/progenitor cell s. Overexpression of WT DNMT3A for 2 weeks in vitro (~3 fold) can accurately reverse the differentially methylated regions (DMRs, which are >99% hypomethylated) of Dnmt3a deficient hematopoietic cells, or cells expressing the R878H mutation. The DMRs of Dnmt3b deficient mouse bone marrow cells can be corrected by overexpression of DNMT3A, DNMT3B1, or DNMT3B3; however, DNMT3B3 failed to remethylate the DMRs in Dnmt3a/Dnmt3b double knockout mouse bone marrow cells. Since DNMT3B3 is an inactive enzyme that does not cause remethylation in the absence of DNMT3A, these data suggest that DNMT3B3 functions in a DNMT3A-dependent manner in hematopoietic cells. Importantly, both DNMT3B3 and DNMT3L have been shown to facilitate DNA methylation by acting as a chaperone for DNMT3A (Duymich et al., Nat. Communication, 2016; PMID 27121154). In vitro, DNMT3L copurified with DNMT3A leads to an increase in DNMT3A methyltransferase activity that is ~5 times greater than DNMT3B3 copurified with DNMT3A. Remarkably, overexpression of DNMT3L (which is not expressed in adult hematopoietic cells, or AML cells) can completely correct the hypomethylation phenotype of Dnmt3aR878H/+ bone marrow cells within 2 weeks of in vitro overexpression, or 4 weeks of in vivo overexpression ( Figure 1A and 1B), probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele in these heterozygous mutant cells. Two weeks of overexpression of DNMT3A or DNMT3L in Dnmt3aR878H/+ bone marrow cells (cultured in vitro with IL-3, SCF, FLT3L, and TPO to maintain HSPC populations) can induce a differentiation response, increasing the fraction of mature myeloid cells in the cultures by ~4-fold. This finding was recapitulated in vivo where one month of overexpression of DNMT3A or DNMT3L in transplanted Dnmt3aR878H/+ mouse bone marrow cells increased the fraction of mature myeloid cells by 2-fold by DNMT3L and 4-fold by DNMT3A. Together,these data show that the focal, canonical DNA hypomethylation phenotype of Dnmt3a R878H/+ hematopoietic cells can be accurately and efficiently corrected by overexpressing WT DNMT3A or DNMT3L for several weeks. We have also shown that the restoration of methylation in Dnmt3a R878H/+ mutant cells alters the developmental fate of progenitors, increasing the proportion of mature myeloid cells. These data suggest that DNMT3L expression may represent a novel approach for restoring DNMT3A activity in AMLs initiated by DNMT3A mutations, and could possibly alter AML cell fate. Additional studies are underway to determine whether the correction of DNMT3A activity in Dnmt3aR878H/+ -initiated AML cells with retroviral addback of DNMT3A or DNMT3L will cause a differentiation response that alters AML cell fate.

THU235 Usefulness Of Digital Droplet Polymerase Chain Reaction (ddPCR) For Molecular Diagnosis Of McCune-Albright Syndrome In DNA From Peripheral Blood Leukocytes

Abstract Disclosure: A.G. de Faria: None. L.R. Montenegro: None. A.A. Jorge: None. R.S. Jallad: None. R.M. Martin: None. M.C. Fragoso: None. A.P. Canton: None. C.E. Seraphim: None. F.R. Tinano: None. N.C. Pinto: None. B.B. Mendonca: None. A. Latronico: None. V.N. Brito: None. Background: McCune-Albright Syndrome (MAS) is a rare congenital disorder caused by post-zygotic activating mutations in GNAS gene. Due to the mosaic pattern of this disease, mutation abundance is frequently low in several tissues, including blood cells. The emergence of digital droplet Polymerase Chain Reaction (ddPCR) is a breakthrough technology of quantitative PCR useful for a targeted detection and quantification of rare events. Objective: This study aims to identify the GNAS mutation in peripheral blood leukocytes of patients with MAS using ddPCR. Patients and Methods: Thirty-four patients (25 female and 9 male) with typical (classic triad) or atypical (one or two features) MAS were included. DNA was extracted from peripheral blood and was analyzed through ddPCR method. Two ddPCR mutation detection assays were used, each one targeting the p.R201C or p.R201H mutations. For each assay, the Taqman PCR system was used with two fluorescence probes: the first labeled with HEX, targeting de (WT allele), and the second labeled 6-FAM, targeting the (mutated allele). The mixture (reagents + DNA) was divided into 20,000 nanodrops, which was placed on a PCR plate and amplified by PCR. After amplification by PCR, the fluorescence of each droplet was read on the 200 Droplet Digital PCR system (Bio-Rad), being classified as positive (drop containing the target sequence) or negative. Results were analyzed using Quantasoft software (Bio-Rad) to determine the total amount of positive droplets in the original sample. The results were reported in fractional abundance (FA), corresponding to the percentage of mutated alleles within the total alleles. Results: Among all patients, the most common MAS feature was osteofibrous dysplasia (OFD) (73.5%), followed by peripheral precocious puberty (PPP) (64.7%), and café-au-lait skin spots (52.9%). Acromegaly or hyperthyroidism was diagnosed in 6 and 4 patients, respectively. Classical MAS triad, 2 clinical features, or 1 clinical feature (PPP or OFD) were present in 15, 9, and 10 patients, respectively. GNAS mutations were identified in 12/34 (35.3%) MAS patients: the p.R201C in 5 and the p.R201H in 7. Among them, 8 patients presented typical MAS and 4 patients with two MAS clinical features, respectively. Considering only typical MAS group, the positive molecular test rate was 53.3% (8/15). GNAS mutations were not identified in patients with only one MAS feature. There was a positive correlation between a positive molecular test and the number of MAS clinical features (p = 0.01). Median FA was 1.45%, ranged from 0.25% to 8.4%. There was no association between FA rate and the number of MAS clinical features (p=1.0). Conclusion: ddPCR representeds a more sensitive tool for identifying GNAS mutations in peripheral blood leukocytes, even with low abundance, in patients with classical and atypical MAS. Presentation: Thursday, June 15, 2023

Multimodal single-cell analysis of non-random heteroplasmy distribution in human retinal mitochondrial disease.

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non-random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non-random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

NSCLC presents metabolic heterogeneity, and there is still some leeway for EGF stimuli in EGFR-mutated NSCLC

BackgroundMetabolic remodeling is crucial in carcinogenesis and cancer progression. Oncogenic mutations may promote metabolic reprogramming in cancer cells to support their energy and biomass requirements. EGFR mutations are commonly found in non-small cell lung cancer (NSCLC) and may induce NSCLC metabolic rewiring. Whether EGFR-driven metabolic reprogramming triggers cell vulnerabilities with therapeutic potential remains unknown. MethodsThe role of EGFR signaling activation by EGF was investigated using NSCLC cell lines with different EGFR and KRAS status: A549 (EGFR WT and KRAS c.34G > A), H292 (EGFR WT and KRAS WT) and PC-9 (EGFR exon 19 E746-A750 deletion and KRAS WT). The effect of EGF on NSCLC cell death and cell cycle was evaluated using flow cytometry, and cell migration was assessed through wound healing. EGFR, HER2, MCT1, and MCT4 expression was analyzed through immunofluorescence or western blotting. We explored the impact of glucose and lactate bioavailability on NSCLC cells' metabolic profile using nuclear magnetic resonance (NMR) spectroscopy. Moreover, the expression of several relevant metabolic genes in NSCLC cells or patient samples was determined by RT-qPCR. ResultsWe showed that cell lines presented different metabolic profiles, and PC-9 cells were the most responsive to EGF stimulus, as they showed higher rates of cell proliferation and migration, together with altered metabolic behavior. By inhibiting EGFR with gefitinib, a decrease in glucose consumption was observed, which may be related to the fact that despite PC-9 harbor EGFR mutation, they still express the EGFR WT allele. The analysis of NSCLC patients' RNA showed a correlation between MCT1/MCT4 and GLUT1 expression in most cases, indicating that the metabolic information can serve as a reference in patients' follow-up. ConclusionTogether, this study shows that NSCLC cell lines have heterogeneous metabolic profiles, which may be underlaid by different genetic profiles, revealing an opportunity to identify and stratify patients who can benefit from metabolism-targeted therapies.

HCM-associated ALMS1 variant: Allele drop-out and frequency in Italian Sphynx cats.

Hypertrophic cardiomyopathy (HCM) is the most common cardiomyopathy in domestic cats, and some inherited variants are available for genetic testing. A variant of the Alstrom syndrome protein 1 gene (ALMS1) was recently reported to be associated with HCM in the Sphynx cat breed (A3: g.92439157G>C). Genetic screening of the variant, promoted by the Osservatorio Veterinario Italiano Cardiopatie and Genefast Laboratory, was offered to Sphynx cat owners and breeders in Italy. Genotype data were initially obtained by Sanger sequencing. In one case where the samples of a trio were available, inconsistency in the vertical transmission of the variant suggested an allele dropout (ADO) of the wt allele. A new external primer pair was designed as an alternative to the original. The larger PCR product obtained was sanger sequenced, and five novel single nucleotide variants (SNVs) not yet annotated in open-access databases were detected. Three of these SNVs were within the original primer-binding regions and were assumed to have caused ADO. The haplotype, including the ADO SNVs, was detected in two cats belonging to different lineages. To accurately genotype ALMS1 g.92439157G>C in the samples, we set up a real-time TaqMan MGB assay while avoiding all surrounding SNVs. At g.92439157G>C, for 136 Sphynx cats, g.92439157 C variant was highly widespread (freq. >0.50). The present study reports five new variants surrounding ALMS1 g.92439157G>C that must be considered when designing the test. The study also indicates the need to verify the correspondence between the g.92439157 C variant frequency and the prevalence of HCM by increasing clinical visits and follow-ups and finally to promote genetic counselling for accurate management of mating plans in Italian Sphynx cats.

Diltiazem efficacy and CYP2D6 gene polymorphism in patients with atrial fibrillation with rapid ventricular response

BackgroundDiltiazem stands out as one of the front-line drugs administered in the emergency department to achieve acute rate control in patients suffering from atrial fibrillation with rapid Ventricular Response. One of the cytochrome enzymes involved in the metabolism of diltiazem is cytochrome P450 2D6 (CYP2D6). Interindividual differences can act on drug metabolism and thus drug efficacy due to the genetic polymorphism induced by the CYP2D6 enzyme. This study explores the association between the efficacy of diltiazem and the genetic polymorphism of CYP2D6 in patients with atrial fibrillation with rapid ventricular response.Results87 out of 93 individuals with ventricular rate > 120 beats/min constituted the patient cohort. The patients were administered 0.25 mg/kg diltiazem intravenously. As a second dose, 0.35 mg/kg diltiazem was administered to patients who reportedly did not receive adequate drug efficacy. Heart rate control was considered to be achieved in patients whose heart rate fell below 110 beats/min and did not rise above 110 beats/min for 2 h. CYP2D6 *2, *3, *4 and *10 represent allele variants and *1 represents wild type (wt) allele. Achieving rate control after one or two doses of diltiazem in normal allele (wt/wt) carriers proved significantly higher than wt/*2, wt/*4 and wt/*10 heterozygous variant carriers. No significant difference was noted in wt/*3 heterozygous variant carriers.ConclusionThe presence of *2, *4 and *10 alleles was observed to significantly compromise the drug efficacy. *3 allele was found to bear no relation to the effect of diltiazem on achieving rate control.

Use of single cell DNA sequencing to characterize somatic mosaicism in a patient with two signal transduction and activator of transcription 1 (STAT1) gain-of-function variants

There is increasing recognition that immune defects caused by somatic mosaicism are often clinically indistinguishable from classical germline inborn errors of immunity. Here we demonstrate the utility of single cell DNA sequencing in defining the cellular genotypes and specific cellular populations involved in the pathogenesis of immune dysregulation in a patient with two novel STAT1 variants and a clinical presentation consistent with STAT1 gain-of-function.A 5-month-old male presented with Pneumocystis jiroveci pneumonia, CMV viremia, and immune-mediated hepatitis. Lymphocyte subset analysis was not consistent with a combined immunodeficiency. Sequencing of genomic DNA using targeted panel of 407 inborn error of immunity genes revealed two heterozygous variants in the coiled-coil region of STAT1 (c.496_504del, p.Leu166_Asp168del; c.489_497del, p. Glu164_Leu166del). Functional analysis was consistent with STAT1 gain-of-function – patient peripheral blood mononuclear cells displayed increased IFN-alpha and IFN-beta-mediated STAT1 phosphorylation, and increased expression of downstream IFN-stimulated genes including IFI44, ISG15, and Ly6E.Further examination of the patient’s STAT1 sequencing data revealed that the two in-frame deletion variants were present on distinct alleles, and surprisingly, that a WT allele was also detected. Based on the presence of three distinct alleles, somatic mosaicism was suspected. Parental testing failed to detect either variant, suggesting that the variants were de novo. Sequencing of DNA extracted from a buccal swab of the patient revealed only wild type STAT1 sequence.Single cell DNA sequencing analysis demonstrated that the two in-frame deletions comprised three different genotypes –a compound heterozygous population comprising 9% of leukocytes, a heterozygous p. Leu166_Asp168del/wild type population comprising of 82% of leukocytes, and a WT population comprising 9% of leukocytes. This analysis additionally revealed that all three genotypes were uniformly distributed among both myeloid and lymphoid populations. Functional analysis of the two in-frame deletions demonstrated that both promote increased STAT1 activity.That both STAT1 variants occur at nearly identical locations, and the uniform distribution among leukocytes, suggests a still undefined genetic event occurring prior to the divergence of common myeloid and lymphoid progenitor cells in hematopoietic stem cell ontogeny. This case demonstrates the power of single cell DNA sequencing in precisely defining disease pathogenesis in patients with somatic mosaicism.

BK channels of five different subunit combinations underlie the de novo KCNMA1 G375R channelopathy.

The molecular basis of a severe developmental and neurological disorder associated with a de novo G375R variant of the tetrameric BK channel is unknown. Here, we address this question by recording from single BK channels expressed to mimic a G375R mutation heterozygous with a WT allele. Five different types of functional BK channels were expressed: 3% were consistent with WT, 12% with homotetrameric mutant, and 85% with three different types of hybrid (heterotetrameric) channels assembled from both mutant and WT subunits. All channel types except WT showed a marked gain-of-function in voltage activation and a smaller decrease-of-function in single-channel conductance, with both changes in function becoming more pronounced as the number of mutant subunits per tetrameric channel increased. The net cellular response from the five different types of channels comprising the molecular phenotype was a shift of -120 mV in the voltage required to activate half of the maximal current through BK channels, giving a net gain-of-function. The WT and homotetrameric mutant channels in the molecular phenotype were consistent with genetic codominance as each displayed properties of a channel arising from only one of the two alleles. The three types of hybrid channels in the molecular phenotype were consistent with partial dominance as their properties were intermediate between those of mutant and WT channels. A model in which BK channels randomly assemble from mutant and WT subunits, with each subunit contributing increments of activation and conductance, approximated the molecular phenotype of the heterozygous G375R mutation.

The MC1R r allele does not increase melanoma risk in MITF E318K carriers.

Population-wide screening for melanoma is not cost-effective, but genetic characterization could facilitate risk stratification and targeted screening. Common Melanocortin-1 receptor (MC1R) red hair colour (RHC) variants and Microphthalmia-associated transcription factor (MITF) E318K separately confer moderate melanoma susceptibility, but their interactive effects are relatively unexplored. To evaluate whether MC1R genotypes differentially affect melanoma risk in MITF E318K+ vs. E318K- individuals. Melanoma status (affected or unaffected) and genotype data (MC1R and MITF E318K) were collated from research cohorts (five Australian and two European). In addition, RHC genotypes from E318K+ individuals with and without melanoma were extracted from databases (The Cancer Genome Atlas and Medical Genome Research Bank, respectively). χ2 and logistic regression were used to evaluate RHC allele and genotype frequencies within E318K+/- cohorts depending on melanoma status. Replication analysis was conducted on 200 000 general-population exomes (UK Biobank). The cohort comprised 1165 MITF E318K- and 322 E318K+ individuals. In E318K- cases MC1R R and r alleles increased melanoma risk relative to wild type (wt), P < 0.001 for both. Similarly, each MC1R RHC genotype (R/R, R/r, R/wt, r/r and r/wt) increased melanoma risk relative to wt/wt (P < 0.001 for all). In E318K+ cases, R alleles increased melanoma risk relative to the wt allele [odds ratio (OR) 2.04 (95% confidence interval 1.67-2.49); P = 0.01], while the r allele risk was comparable with the wt allele [OR 0.78 (0.54-1.14) vs. 1.00, respectively]. E318K+ cases with the r/r genotype had a lower but not significant melanoma risk relative to wt/wt [OR 0.52 (0.20-1.38)]. Within the E318K+ cohort, R genotypes (R/R, R/r and R/wt) conferred a significantly higher risk compared with non-R genotypes (r/r, r/wt and wt/wt) (P < 0.001). UK Biobank data supported our findings that r did not increase melanoma risk in E318K+ individuals. RHC alleles/genotypes modify melanoma risk differently in MITF E318K- and E318K+ individuals. Specifically, although all RHC alleles increase risk relative to wt in E318K- individuals, only MC1R R increases melanoma risk in E318K+ individuals. Importantly, in the E318K+ cohort the MC1R r allele risk is comparable with wt. These findings could inform counselling and management for MITF E318K+ individuals.

Mutation-agnostic RNA interference with engineered replacement rescues Tmc1-related hearing loss.

Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations in TMC1, a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approaches is limited by the infeasibility of development and validation of novel constructs for each variant. We developed an allele-non-specific approach consisting of mutation-agnostic RNAi suppression of both mutant and WT alleles, co-delivered with a knockdown-resistant engineered WT allele with or without the use of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to augment transgene expression. This therapeutic construct was delivered into the mature murine model of DFNA36 with an AAV vector and achieved robust hair cell and auditory brainstem response preservation. However, WPRE-enhanced Tmc1 expression resulted in inferior outcomes, suggesting a role for gene dosage optimization in future TMC1 gene therapy development.

The Piwil1 N domain is required for germ cell survival in Atlantic salmon.

Genetic introgression of farmed salmon into wild populations can damage the genetic integrity of wild stocks and is therefore considered as an environmental threat. One possible solution is to induce sterility in farmed salmon. We have searched for proteins potentially essential for germline survival in Atlantic salmon. One of these is the argonaute protein Piwil1, known to be required for germ cell survival. To examine Piwil1 function in salmon, we induced indels in the N domain by CRISPR-Cas9. The encoded domain is present in all vertebrate Piwi proteins and has been linked to Tdrd1 protein interaction and PAZ lobe structure. The F0 founder generation of piwil1 crispant males and females displayed a mosaic pattern of piwil1 mutations, exhibiting highly mutated alleles (53%–97%) in their fin gDNA samples. In general, piwil1 crispants carried germ cells, went through puberty and became fertile, although a transient and partial germ cell loss and delays during the spermatogenic process were observed in many male crispants, suggesting that Piwil1 functions during salmon spermatogenesis. By crossing highly mutated F0 founders, we produced F1 fish with a mixture of: loss-of-function alleles (−); functional in frame mutated alleles ( + ) and wt alleles (+). In F1, all piwil1 −/− fish lacked germ cells, while piwil1 +/+ siblings showed normal ovaries and testes. Yet, most juvenile F1 piwil1 +/−males and females displayed an intermediate phenotype with a higher somatic/germ cell ratio without an increase in germ cell apoptosis, suggestive of a gene dose effect on the number of germ cells and/or insufficient replacement of lost germ cells in heterozygous fish. Interestingly, the two longest in-frame indels in the N domain also ensured germ cell loss. Hence, the loss of 4–6 aa in this region Phe130-Ser136 may result in crucial changes of the protein structure, potentially affecting piRNA binding of the PAZ lobe, and/or affecting the binding of Piwil1 interacting proteins such as Tdrd protein, with critical consequences for the survival of primordial germ cells. In conclusion, we show that loss of piwil1 leads to loss of germ cells in salmon and that part of the N domain of Piwil1 is crucial for its function.