Variant cDNA Research Articles

Cellular localization of a variant RAPGEF5 protein associated with idiopathic epilepsy risk in the Belgian shepherd.

The Wnt signaling pathway is critical for normal embryonic development. Disruptions in the Wnt signaling pathway have been linked to neurological disorders. The RAPGEF5 protein is a partner in Wnt signaling and a RAPGEF5 3-bp insertion is associated with increased risk for idiopathic epilepsy in the Belgian shepherd dog. The 3-bp insertion risk variant introduces an alanine residue predicted to disrupt the protein. Wildtype and the risk variant RAPGEF5 cDNAs were cloned into green fluorescent protein (GFP) expression vectors and transfected into canine kidney cells. The cellular localization of each GFP-labeled RAPGEF5 protein was assessed. Variant RAPGEF5 protein was altered in its localization from that of the wildtype protein and rather than localized to the nucleus and cytoplasm as seen for the wildtype, it was predominantly found in the cytoplasm. Belgian shepherds with the risk variant for RAPGEF5 may have altered Wnt signaling due to modified intracellular localization which in turn could thereby contribute to the expression of idiopathic epilepsy.

Effects of HMGCR deficiency on skeletal muscle development.

Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. We knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

Abstract 5584: Experimental ‘loss-of function’ annotation of STK11 mutations with prognostic and therapeutic implications (TNG260mutationfinder.com)

Abstract Loss of function mutations in Serine Threonine Kinase 11 (STK11) occur in 15% of lung adenocarcinoma and have been shown to drive resistance to immune checkpoint blockade clinically, as well as in preclinical models. Though STK11 is commonly inactivated in human cancer with strong implications for treatment outcomes, few STK11 mutations identified from tumor samples have been functionally characterized. TNG260 is an inhibitor of CoREST that is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492). Patients are eligible for enrollment in the TNG260 phase 1/2 trial if their tumor contains a deleterious STK11 mutation. To begin classifying non-annotated variants, over 2,000 distinct mutations in the STK11 gene were identified from STK11 literature or public repositories of tumor sequencing data such as AACR Project GENIE and ClinVar. Where possible, loss-of-function annotations were captured from literature or predictive tools such as PolyPhen-2. However, many STK11 variants, particularly missense mutations, have never been functionally characterized. We developed a functional screening approach to characterize STK11 alterations using the lung adenocarcinoma cell line A549. A549 cells contain homozygous loss of STK11 via a truncation mutation at Q37, and re-expression of wild-type STK11 in these cells strongly impairs their growth in vitro and in vivo. We created a library of STK11 variant cDNAs, each containing a unique barcode. This library was expressed in A549, and cells were maintained in vitro or in vivo to allow for positive selection of STK11 loss-of-function variants and depletion of variants that behave like wild-type STK11. At the end of the screen, variants were quantified by NGS using each mutant cDNA’s unique barcode and compared to well-annotated controls. These data were assembled to generate TNG260mutationfinder.com – the first website to curate STK11 variants with functional annotations. Citation Format: Leanne G. Ahronian, Preksha Shahagadkar, Lauren Flynn, Lauren Grove, Shangtao Liu, Samuel R. Meier, Binzheng Shen, Hannah Stowe, Hsin-Jung Wu, Yi Yu, Andre Mignault, Iga Sienczylo, Heather DiBenedetto, Silvia Fenoglio, Teng. Experimental ‘loss-of function’ annotation of STK11 mutations with prognostic and therapeutic implications (TNG260mutationfinder.com) [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 5584.

Variant functional assessment in Drosophila by overexpression: what can we learn?

The last decade has been highlighted by the increased use of next-generation DNA sequencing technology to identify novel human disease genes. A critical downstream part of this process is assigning function to a candidate gene variant. Functional studies in Drosophila melanogaster, the common fruit fly, have made a prominent contribution in annotating variant impact in an in vivo system. The use of patient-derived knock-in flies or rescue-based, "humanization", approaches are novel and valuable strategies in variant testing but have been recently widely reviewed. An often-overlooked strategy for determining variant impact has been GAL4/upstream activation sequence-mediated tissue-defined overexpression in Drosophila. This mini-review will summarize the recent contribution of ectopic overexpression of human reference and variant cDNA in Drosophila to assess variant function, interpret the consequence of the variant, and in some cases infer biological mechanisms.

Abstract B001: Does deep intronic splicing in breast cancer susceptibility genes contribute to breast cancer?

Abstract Breast cancer (BC) is the most common cancer and leading cause of cancer mortality in women. Average lifetime BC risk for a woman in the United States is 13%. However, pathogenic variants in BC susceptibility genes such as BRCA1/2, ATM, and RAD51C, or high polygenic risk scores substantially increase BC risk. Women with a family history of BC and/or ovarian cancer, BC < age 40, or BC and second primary tumor are considered high-risk with an increased likelihood of having a pathogenic variant in a BC susceptibility gene. Yet, more than 60% of women with high-risk BC test negative for coding variants. In these women, possible causal variation could be non-coding in deep intronic or regulatory regions, or cryptic structural variants (SV). To determine whether deep intronic splicing contributes to BC in high-risk women testing negative for pathogenic coding variants in BC genes, we analyzed the intronic sequences of 649 BRCA1/BRCA2 negative individuals with high-risk BC. The samples had undergone targeted capture sequencing of exons and introns of more than 100 target genes including ATM and RAD51C. From the 649 samples, we identified three intronic splicing variants in ATM and RAD51C in 10 samples (1.5%) that had SpliceAI delta scores >0.5 suggesting changes to splicing. One splicing donor gain variant, RAD51C c.145+246A>T, was found exclusively in women of self-reported African ancestry at a frequency of 0.047. In gnomAD, the variant was found exclusively in individuals of African ancestry at a frequency of 0.012. A 1.3-fold enrichment in variant frequency was observed in AFR women under 65 with breast cancer compared to age-matched cancer free controls in the All of Us dataset. Using lymphoblastoid-cell derived RNA from carriers of RAD51C c.145+246A>T, we performed RT-PCR and cDNA amplification targeting the exons surrounding the variant and observed a lengthened exon. Targeted amplicon sequencing also confirmed the presence of intronic sequence in the variant cDNA. The introduced intronic sequence contains a UGA stop codon 12 bp from the end of the exon which could led to loss of RAD51C function. We have created minigene vectors in the RHCglo background and are performing minigene splicing assays to validate the splicing changes in ATM due to intronic variants. Gene functional assays and association studies will be conducted to determine the functional effects and risk conferred by these variants. Understanding the level of contribution of deep intronic splicing in known BC susceptibility genes is critically important, as results impact sequencing offered to patients, and if positive, their medical management. Citation Format: Mwangala P. Akamandisa, Bradley Wubbenhorst, Kurt D'Andrea, Heather Symecko, Rhonda Gillette, Payal D. Shah, Angela R. Bradbury, Kara N. Maxwell, Susan M. Domchek, Katherine L. Nathanson. Does deep intronic splicing in breast cancer susceptibility genes contribute to breast cancer? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B001.

Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsy

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants invivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Abstract 153: An unbiased in vitro screen for activating ERBB4 mutations

Abstract Introduction: Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites across different samples. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hotspots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and their functional significance remains unknown. Several lines of in vitro and clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations which could serve as predictive markers in oncology. Here, we performed an unbiased functional screen to identify potential activating mutations of ERBB4, a frequently mutated member of the epidermal growth factor receptor family. Method: To identify functional driver mutations of ERBB4, the previously published pipeline, in vitro screen for activating mutations (iSCREAM) [1], was utilized. iSCREAM is a functional genetics screen based on the expression of random cDNA variants and the ability of driver mutations to promote cellular growth in vitro. The expression library encoding randomly mutated ERBB4 variants was retrovirally introduced into murine lymphoid Ba/F3 cells, that normally require interleukin-3 (IL-3) for survival but can exploit ectopic expression of activated variants of oncogenic kinases to compensate for the deficiency of exogenous IL-3. When expressed under an appropriate promoter, the wild-type ERBB4 receptor failed to promote IL-3-independent survival in the presence of the neuregulin-1 ligand, while the cells expressing activating mutations readily proliferated. The identity and frequency of the activating ERBB4 mutations were subsequently determined from the proliferating cell pool using targeted next-generation sequencing. Results: Ten candidate activating mutations were identified out of the over 7000 random ERBB4 missense or nonsense mutations present in the original library. The candidate activating mutations were individually characterized using functional assays both in vitro and in vivo, as well as by structural analyses. The activating mutation were sensitive to clinically used pan-ERBB tyrosine kinase inhibitors afatinib, dacomitinib and neratinib. Conclusions: A subset of ERBB4 missense mutations are activating and sensitive to tyrosine kinase inhibitor drugs. Citation Format: Deepankar Chakroborty, Veera K. Ojala, Anna M. Knittle, Kari J. Kurppa, Klaus Elenius. An unbiased in vitro screen for activating ERBB4 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 153.

Molecular landscape of canine soft tissue sarcoma

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features including hemangiosarcoma, lymphangiosarcoma, histiocytic sarcoma, synovial sarcoma, leiomyosarcoma, and rhabdomyosarcoma. Histologically, canine STS sub‐types are difficult to discern at the light microscopic level due to their overlapping microscopic features. Thus, genomic, and transcriptomic profiling of canine STS may prove more valuable in differentiating the diverse sub‐types of mesenchymal neoplasms within this grouping. To this purpose we sought to characterize the mutation and expression profiles of canine STS. In this study we used whole exome capture (Agilent Sure Select Canine V2) to sequence 29 tumors, grade I‐III, from non‐visceral locations, and matched normal samples, along with RNAseq of tumor samples. For the WES data, 40‐170 million 150 bp paired‐end reads were obtained from Illumina sequencing which were mapped against CanFam3.1 genome via BWA and short variants were called and annotated Mutect2 and VEP tools, respectively. The median depth of sequencing for normals and tumors were 97X (range: 35X–128X) and 110X (range: 22X–133X), respectively. Additionally, RNAseq reads were sequenced from 29 tumors and mapped against CanFam3.1 genome via STAR and gene count data was generated by HTSeq‐count tool. The total number of somatic variants identified across 29 samples ranged from 275 to 4,196, of which, 6% to 31% were annotated as cDNA variants. The protein coding mutations per callable megabase ranged from 0.3 to 37.3. Known cancer genes (COSMIC database) with recurrent mutations were KMT2D(21% of the samples), TP53 (21%), CNTNAP2(14%) genes. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. These gene fusions were confirmed through Sanger sequencing of amplified cDNA. To delineate the transcriptomic sub‐types, we used hierarchical and K‐means clustering methods. This resulted in 4 groups with similar expression profiles comprising of 11, 4, 8 and 6 samples in each. Further, genes set variation analysis (GSVA) was used to profile the distinct pathways across all 4 clusters. Preliminary data indicates that the 11‐sample and 6‐sample clusters had up‐regulated non‐homologous end joining pathway and immune response pathways, respectively. Further study of genes and pathways that are enriched in sample clusters will help in categorizing the canine STS tumors for diagnosis and treatment.

Biologic characterization of ABCA3 variants in lung tissue from infants and children with ABCA3 deficiency.

ABCA3 is a phospholipid transporter protein required for surfactant assembly in lamellar bodies of alveolar type II cells. Biallelic pathogenic ABCA3 variants cause severe neonatal respiratory distress syndrome or childhood interstitial lung disease. However, ABCA3 genotype alone does not explain the diversity in disease presentation, severity, and progression. Additionally, monoallelic ABCA3 variants have been reported in infants and children with ABCA3-deficient phenotypes. The effects of most ABCA3 variants identified in patients have not been characterized at the RNA level. ABCA3 allele-specific expression occurs in some cell types due to epigenetic regulation. We obtained lung tissue at transplant or autopsy from 16 infants and children with ABCA3 deficiency due to compound heterozygous ABCA3 variants for biologic characterization of the predicted effects of ABCA3 variants at the RNA level and determination of ABCA3 allele expression. We extracted DNA and RNA from frozen lung tissue and reverse-transcribed cDNA from mRNA. We performed Sanger sequencing to assess allele-specific expression by comparing the heights of variant nucleotide peaks in amplicons from genomic DNA and cDNA. We found similar genomic and cDNA variant nucleotide peak heights and no evidence of allele-specific expression among explant or autopsy samples with biallelic missense ABCA3 variants (n = 6). We observed allele-specific expression of missense alleles in trans with frameshift (n = 4) or nonsense (n = 1) variants, attributable to nonsense-mediated decay. The missense variant c.53 A > G;p.Gln18Arg, located near an exon-intron junction, encoded abnormal splicing with skipping of exon 4. Biologic characterization of ABCA3 variants can inform discovery of variant-specific disease mechanisms.

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

SUMMARYIndividuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Polymorphic cytochromes P450 in non-human primates.

Cynomolgus macaques (Macaca fascicularis, an Old World monkey) are widely used in drug development because of their genetic and physiological similarities to humans, and this trend has continued with the use of common marmosets (Callithrix jacchus, a New World monkey). Information on the major drug-metabolizing cytochrome P450 (CYP, P450) enzymes of these primate species indicates that multiple forms of their P450 enzymes have generally similar substrate selectivities to those of human P450 enzymes; however, some differences in isoform, activity, and substrate specificity account for limited species differences in drug oxidative metabolism. This review provides information on the P450 enzymes of cynomolgus macaques and marmosets, including cDNA, tissue expression, substrate specificity, and genetic variants, along with age differences and induction. Typical examples of important P450s to be considered in drug metabolism studies include cynomolgus CYP2C19, which is expressed abundantly in liver and metabolizes numerous drugs. Moreover, genetic variants of cynomolgus CYP2C19 affect the individual pharmacokinetic data of drugs such as R-warfarin. These findings provide a foundation for understanding each P450 enzyme and the individual pharmacokinetic and toxicological results in cynomolgus macaques and marmosets as preclinical models. In addition, the effects of induction on some drug clearances mediated by P450 enzymes are also described. In summary, this review describes genetic and acquired individual differences in cynomolgus and marmoset P450 enzymes involved in drug oxidation that may be associated with pharmacological and/or toxicological effects.

Functional characterization and potential therapeutic avenues for variants in the NTRK2 gene causing developmental and epileptic encephalopathies.

Variants within the Neurotrophic Tyrosine Kinase Receptor Type 2 (NTRK2) gene have been discovered to play a role in developmental and epileptic encephalopathies, a group of debilitating conditions for which little is known about cause or treatment. Here, we determine the functional consequences of two variants: p.Tyr434Cys (Y434C) (located in the transmembrane domain) and p.Thr720Ile (T720I) (located in the catalytic domain). Wild-type and variant cDNAs were constructed and transfected into HEK293 cells. In cell culture, variant Y434C exhibited ligand-independent activation of tropomyosin-related kinase B (TRKB) signaling with an associated abnormal response to brain-derived neurotrophic factor (BDNF) stimulation and increased levels of phosphorylated extracellular signal-regulated kinase (ERK) and ETS like-1 protein (ELK1) activity. Expression of variant T720I resulted in decreased TRKB signaling with reduced mTor activity as determined by decreased levels of phosphorylated S6. With the deleterious mechanisms characterized, we utilized mediKanren (a novel artificial intelligence tool) to identify therapeutics to compensate for the pathological effects. Downregulation of TRKB through inhibition with mediKanren-predicted compound 1NM-PP1 led to decreased MEK activity. Upregulation of TRKB signaling by mediKanren-predicted valproic acid led to subsequent increase of mTor activity. Overall, our results provide further characterization of the pathogenicity of these two variants in the NTRK2 gene. Indeed, Y434C is the first patient-specific NTRK2 variant with demonstrated hypermorphic activity. Furthermore, we observed that variants Y434C and T720I result in distinct functional consequences that require distinct therapeutic strategies. These data suggest the possibility that unique mutations within different regions of the NTRK2 gene results in separate clinical presentations, representing distinct genetic disorders requiring unique therapeutics.

The Loss of ALDH9A1 Is a Significant Source of Endogenous DNA Damage Which May be Reversed By the Inhibition of Polyamine Transport System

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome and is caused by impaired DNA interstrand crosslink repair. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage likely play an important role in the pathogenesis of FA.We previously identified loss of ALDH9A1 as a significant source of endogenous DNA damage using a metabolism-focused CRISPR knockout (KO) screen. This finding was validated using Jurkat cells as well as human hematopoietic stem and progenitor cells. Here, we present updates of our project.To determine whether endogenous DNA damage was induced by the combined loss of FANCD2 and ALDH9A1, we investigated markers of DNA damage in bulk-edited cells. We found that the numbers of chromosomal breaks, 53BP1 foci, and gamma-H2AX foci were increased in FANCD2-/-ALDH9A1-/- cells, compared with single KO or wildtype (WT) controls, in the absence of an exogenous DNA damaging agent. These findings are consistent with spontaneously increased basal levels of DNA damage in FANCD2-/-ALDH9A1-/- cells.To study in vivo BMF and tumorigenesis phenotypes of ALDH9A1 deficiency in the setting of FA, we generated a mouse model. Fanca-/-Aldh9a1-/- mice showed the lowest frequency of long-term hematopoietic stem cells and lineage-negative Sca-1-positive cKit-positive cells, however the differences were not significant compared with control groups. While we did not observe aplastic anemia or leukemia, we found a higher incidence of solid tumors, most notably ovarian tumors and hepatocellular carcinoma in aged Fanca-/-Aldh9a1-/- mice. This suggests that the level of endogenous reactive aldehydes created by ALDH9A1 deficiency in mouse is not high enough to cause full-blown hematopoietic phenotypes, most likely due to redundant detoxification pathways. However, in tissues where ALDH9A1 is active and non-redundant, the low level of endogenous DNA damage accumulating over time causes solid tumors.To identify the specific reactive aldehydes responsible for DNA damage in ALDH9A1 deficiency, we performed a growth selection screen using FANCD2-/-ALDH9A1-/- cells. We found that the loss of ATP13A3 conferred survival advantage to FANCD2-/-ALDH9A1-/- cells. ATP13A3 transports endocytosed polyamines into the cytosol where polyamines can be metabolized by serum amine oxidases into 3-aminopropanal, a reactive aminoaldehyde. 3-aminopropanal also undergoes spontaneous decomposition to acrolein, a well-known reactive aldehyde carcinogen. Finding that the loss of ATP13A3 rescues that FANCD2-/-ALDH9A1-/- cells indicates that 3-aminopropanal and/or acrolein induces endogenous DNA damage requiring the Fanconi anemia pathway function for repair.Finally, to determine the contribution of ALDH9A1 variants to clinical manifestations of FA patients, we performed targeted sequencing of DNA from FA patients. We identified five missense variants, four of which had high CADD scores (>20). ALDH9A1 cDNA containing missense variants with high CADD scores expressed in ALDH9A1-/- Jurkat cells resulted in lower protein expression than the WT cDNA. Cell culture supernatant from cells expressing the variant cDNAs also had increased aldehyde levels as assessed by fluorometric assays, suggesting decreased enzymatic activity of the variant proteins. The patients with ALDH9A1 missense variants with high CADD scores had either early hematologic onset of FA (n=3; two patients before age 1 and one patient before age 4) or AML (n=1).In conclusion, we showed that the loss of ALDH9A1 generates endogenous DNA damage necessitating the FA pathway for its repair. Synthetic lethality caused by the combined loss of FANCD2 and ALDH9A1 was rescued by the loss of ATP13A3, which suggests that 3-aminopropanal is the culprit aminoaldehyde that accumulates in ALDH9A1-deficient cells and results in DNA damage. Functionally deleterious ALDH9A1 variants were observed in some FA patients with early onset of disease suggesting that ALDH9A1 could be a modifier of FA in humans. DisclosuresSridhar: Deciphera Pharmaceuticals: Current Employment; CRISPR Therapeutics: Ended employment in the past 24 months. White: Regeneron Pharmaceuticals: Current Employment. Smogorzewska: Rocket Pharmaceuticals: Research Funding.

MRNA Analysis of Frameshift Mutations with Stop Codon in the Last Exon: The Case of Hemoglobins Campania [α1 cod95 (-C)] and Sciacca [α1 cod109 (-C)

An insertion or deletion of a nucleotide (nt) in the penultimate or the last exon can result in a frameshift and premature termination codon (PTC), giving rise to an unstable protein variant, showing a dominant phenotype. We described two α-globin mutants created by the deletion of a nucleotide in the penultimate or the last exon of the α1-globin gene: the Hb Campania or α1 cod95 (−C), causing a frameshift resulting in a PTC at codon 102, and the Hb Sciacca or α1 cod109 (−C), causing a frameshift and formation of a PTC at codon 133. The carriers showed α-thalassemia alterations (mild microcytosis with normal Hb A2) and lacked hemoglobin variants. The 3D model indicated the α-chain variants’ instability, due to the severe structural alterations with impairment of the chaperone alpha-hemoglobin stabilizing protein (AHSP) interaction. The qualitative and semiquantitative analyses of the α1mRNA from the reticulocytes of carriers highlighted a reduction in the variant cDNAs that constituted 34% (Hb Campania) and 15% (Hb Sciacca) of the total α1-globin cDNA, respectively. We developed a workflow for the in silico analysis of mechanisms triggering no-go decay, and its results suggested that the reduction in the variant mRNA was likely due to no-go decay caused by the presence of a rare triplet, and, in the case of Hb Sciacca, also by the mRNA’s secondary structure variation. It would be interesting to correlate the phenotype with the quantity of other frameshift mRNA variants, but very few data concerning α- and β-globin variants are available.

Characterization of the Pathogenicity of Novel Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program for the Molecular and Clinical Biology Study

Von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Von Willebrand factor's (VWF) complex intracellular processing may result in variable VWF expression which requires exploring the underlying mechanism related to individual variants. The mechanisms underlying type 1 VWD are not fully understood, although reduced secretion and increased clearance have been implicated. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program in order to (1) define the underlying mechanism, (2) explore if mutations in a particular domain are mechanistically similar, and (3) correlate expression study data to patient phenotype.Homozygous and heterozygous expression in mammalian cells was used to study the effect of SV on VWF secretion, intracellular retention, function, and multimerization. Novel SV identified throughout the entire VWF protein were introduced into VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Variant VWF cDNA was transfected homozygously and cotransfected heterozygously 1:1 with wild type (WT) VWF pCIneo into Human Embryonic Kidney cells (HEK293T). Supernatants (sup) and cell lysates from 3 separate transfections were collected and analyzed by ELISA for VWF:Ag, VWF binding to platelet glycoprotein 1b (VWF:GPIbM), VWF binding to collagen type III (VWF:CB). Typically VWF:GP1bM/VWF:Ag > 0.7 indicates normal platelet binding and VWF:CB/VWF:Ag > 0.7 indicates normal multimer structure. VWF multimer structure is analyzed by SDS-agarose electrophoresis and western blotting with differential staining of the VWF secreted from variant and WT alleles.V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, and A2569E demonstrated secretion of VWF in sup comparable to that of WT-VWF both when transfected homozygously and hetereozygously, normal VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and normal multimer structure. Two of the stop codon SV W199X and E1660X were not expressed homozgyously (as expected) but demonstrated normal secretion, function, and multimerization when cotransfected 1:1 with WT. Homozygous expression of R960P and C2693F demonstrated reduced VWF secretion, VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and abnormal multimerization. Heterozygous transfection with WT improved VWF secretion, but not multimer defects,reflected by a reduced VWF:CB/VWF:Ag. Homozygous expression of C524Y, G994D, C996W, Q1353X, C2199Y, C2701Y, and C2754Y resulted in absent or greatly reduced secretion with increased intracellular retention. Heterozygous expression resulted in reduced VWF secretion (<50% of WT) for all 7 variants. Q1353X, C2199Y, C2701Y, and C2754Y had normal VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag, and multimer structure, but consisted primarily of secreted WT-VWF. G994D had VWF:CB/VWF:Ag ˂ 0.7 and reduced high molecular weight (HMW) multimers. Co-expression of C524Y and C996W with WT-VWF demonstrated nearly absent VWF secretion, indicating a strong dominant negative effect. The very low VWF:Ag levels precluded assessment of VWF function and multimerization.The VWF:CB/VWF:Ag ratio < 0.7 proved a reliable indicator of multimer structure. No clear domain-specific correlation of VWF secretion or multimer abnormality was observed. Co-expression results of Q1353X, C2199Y, C2701Y, C2754Y, C524Y, G994D, and C996W are consistent with the patient's phenotype, except that patients with C2701Y, and C524Y had slight defect in multimers. In contrast, the in-vitro G994D showed low VWF:CB/VWF:Ag and abnormal multimers but were normal in the patient. C2693F showed consistently abnormal multimer structure in both in vitro and in the patient while R960P had abnormal multimer structure in vitro only.In sum, 52.6% of the 19 SV theoretically are not causative of the VWD phenotype observed in the patients. These subjects had normal VWF propeptide/VWF:Ag suggesting reduced plasma survival is unlikely to be the causative mechanism. The VWD mechanism in these subjects remains undefined. Variants R960P, C2693F, and G994D (15.7%) behaved like Type 2 VWD SV with loss of HMW multimers. We identified 31.6% of the 19 VWF SV as likely to cause the type 1 VWD phenotype with reduced secretion. DisclosuresNo relevant conflicts of interest to declare.

Elevation of propofol sensitivity of cardiac IKs channel by KCNE1 polymorphism D85N.

The slowly activating delayed rectifier K+ channel (IKs ), composed of pore-forming KCNQ1 α-subunits and ancillary KCNE1 β-subunits, regulates ventricular repolarization in human heart. Propofol, at clinically used concentrations, modestly inhibits the intact (wild-type) IKs channels and is therefore unlikely to appreciably prolong QT interval in ECG during anaesthesia. However, little information is available concerning the inhibitory effect of propofol on IKs channel associated with its gene variants implicated in QT prolongation. The KCNE1 single nucleotide polymorphism leading to D85N is associated with drug-induced QT prolongation and therefore regarded as a clinically important genetic variant. This study examined whether KCNE1-D85N affects the sensitivity of IKs to inhibition by propofol. Whole-cell patch-clamp and immunostaining experiments were conducted in HEK293 cells and/or mouse cardiomyocyte-derived HL-1 cells, transfected with wild-type KCNQ1, wild-type or variant KCNE1 cDNAs. Propofol inhibited KCNQ1/KCNE1-D85N current more potently than KCNQ1/KCNE1 current in HEK293 cells and HL-1 cells. Immunostaining experiments in HEK293 cells revealed that pretreatment with propofol (10μM) did not appreciably affect cell membrane expression of KCNQ1 and KCNE1 proteins in KCNQ1/KCNE1 and KCNQ1/KCNE1-D85N channels. The KCNE1 polymorphism D85N significantly elevates the sensitivity of IKs to inhibition by propofol. This study detects a functionally important role of KCNE1-D85N polymorphism in conferring genetic susceptibility to propofol-induced QT prolongation and further suggests the possibility that the inhibitory action of anaesthetics on ionic currents becomes exaggerated in patients carrying variants in genes encoding ion channels.

Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

Functional Genomics of ABCA3 Variants.

Rare or private, biallelic variants in the ABCA3 (ATP-binding cassette transporter A3) gene are the most common monogenic cause of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 disease-associated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPase-mediated phospholipid transport (type II). Therapies remain limited and nonspecific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus in A549 cells with CRISPR/Cas9 genome editing. Next, to generate a parent cell line (A549/ABCA3-/-) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS cassette, FACS, and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural vesicle phenotype) of two individual ABCA3 mutants (type I mutant, p.L101P; type II mutant, p.E292V) in A549/ABCA3-/- cells and in both A549 cells and primary, human alveolar type II cells that transiently express each cDNA after adenoviral-mediated transduction. We also confirmed pharmacologic rescue of ABCA3 variant-encoded mistrafficking and vesicle diameter in A549/ABCA3-/- cells that express p.G1421R (type I mutant). A549/ABCA3-/- cells provide a scalable, genetically versatile, physiologically relevant functional genomics platform for discovery of variant-specific mechanisms that disrupt ABCA3 function and for screening of potential ABCA3 pharmacologic correctors.

Novel allelic variation in the Phospholipase D alpha1 gene (OsPLD\u03b11) of wild Oryza species implies to its low expression in rice bran

Rice bran, a by-product after milling, is a rich source of phytonutrients like oryzanols, tocopherols, tocotrienols, phytosterols, and dietary fibers. Moreover, exceptional properties of the rice bran oil make it unparalleled to other vegetable oils. However, a lipolytic enzyme Phospholipase D alpha1 (OsPLDα1) causes rancidity and ‘stale flavor’ in the oil, and thus limits the rice bran usage for human consumption. To improve the rice bran quality, sequence based allele mining at OsPLDα1 locus (3.6 Kb) was performed across 48 accessions representing 11 wild Oryza species, 8 accessions of African cultivated rice, and 7 Oryza sativa cultivars. From comparative sequence analysis, 216 SNPs and 30 InDels were detected at the OsPLDα1 locus. Phylogenetic analysis revealed 20 OsPLDα1 cDNA variants which further translated into 12 protein variants. The O. officinalis protein variant, when compared to Nipponbare, showed maximum variability comprising 22 amino acid substitutions and absence of two peptides and two β-sheets. Further, expression profiling indicated significant differences in transcript abundance within as well as between the OsPLDα1 variants. Also, a new OsPLDα1 transcript variant having third exon missing in it, Os01t0172400-06, has been revealed. An O. officinalis accession (IRGC101152) had lowest gene expression which suggests the presence of novel allele, named as OsPLDα1-1a (GenBank accession no. MF966931). The identified novel allele could be further deployed in the breeding programs to overcome rice bran rancidity in elite cultivars.

Hexaploid sweetpotato (Ipomoea batatas (L.) Lam.) may not be a true type to either auto- or allopolyploid.

To better define the sweetpotato polyploidy, we sought to reconstruct phylogenies of its subgenomes based on hybridization networks that could trace reticulate lineages of differentiated homoeolog triplets of multiple single-copy genes. In search of such homoeolog triplets, we distinguished cDNA variants of 811 single-copy Conserved Ortholog Set II (COSII) genes from two sweetpotato clones into variation partitions specified by corresponding homologs from two I. trifida lines, I. tenuissima and I. littoralis using a phylogenetic partition method, and amplicon variants of the COSII-marker regions from 729 of these genes from two sweetpotato clones into putative homoeoallele groups using haplotype tree and the partition methods referenced by corresponding homologs from I. tenuissima. These analyses revealed partly or completely differentiated expressed-homoeologs and homoeologs from a majority of these genes with three important features. 1. Two variation types: the predominant interspecific variations (homoeoalleles), which are non-randomly clustered, differentially interspecifically conserved or sweetpotato-specific, and the minor intraspecific ones (alleles), which are randomly distributed mostly at non-interspecifically variable sites, and usually sweetpotato-specific. 2. A clear differentiation of cDNA variants of many COSII genes into the variation partition specified by I. tenuissima or I. littoralis from that by I. trifida. 3. Three species-homolog-specified and one sweetpotato-specific variation partitions among 293 different COSII cDNAs, and two or three out of the four partitions among cDNA variants of 306 COSII genes. We then constructed hybridization networks from two concatenations of 16 and 4 alignments of 8 homologous COSII cDNA regions each, which included three taxa of expressed homoeologs in a triple-partition combination from the 16 or 4 sweetpotato COSII genes and 5 taxa each of respective cDNA homologs from the three sweetpotato relatives and I. nil, and inferred a species tree embodying both networks. The species tree predicted close-relative origins of three partly differentiated sweetpotato subgenomes.