Insertion In Exon Research Articles

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinitis pigmentosa, obesity, polydactyly, cognitive impairment and renal failure. Pathogenic variants in 24 genes account for the molecular basis of >80% of cases. Toward saturated discovery of the mutational basis of the disorder, we carefully explored our cohorts and identified a hominid-specific SINE-R/VNTR/Alu type F (SVA-F) insertion in exon 13 of BBS1 in eight families. In six families, the repeat insertion was found in trans with c.1169 T > G, p.Met390Arg and in two families the insertion was found in addition to other recessive BBS loci. Whole genome sequencing, de novo assembly and SNP array analysis were performed to characterize the genomic event. This insertion is extremely rare in the general population (found in 8 alleles of 8 BBS cases but not in >10 800 control individuals from gnomAD-SV) and due to a founder effect. Its 2435 bp sequence contains hallmarks of LINE1 mediated retrotransposition. Functional studies with patient-derived cell lines confirmed that the BBS1 SVA-F is deleterious as evidenced by a significant depletion of both mRNA and protein levels. Such findings highlight the importance of dedicated bioinformatics pipelines to identify all types of variation.

Genetic Basis of Erythrocyte Alloimmunization Among Children with Sickle Cell Anemia in the Dominican Republic

Introduction: In low-resource settings, red blood cell (RBC) transfusions for children with sickle cell anemia (SCA) may be cross-matched for only ABO and RhD antigens, so RBC alloimmunization can develop due to differences in Rh and other antigens between donors and recipients. In the Rh system, these differences can include absence of the antigen or the presence of partial or weak antigens, the latter more common with African ancestry. RBC alloimmunization in SCA has not been described in the Dominican Republic, and the genetic diversity of the RHD and RHCE loci have not been reported in this Hispanic-Afro-Caribbean population. Methods: A pediatric stroke prevention trial in the Dominican Republic (SACRED, NCT02769845) provided serum and dried blood spots from children with SCA, collected at enrollment and stored at -80C. Sera were screened for alloantibodies using a modified test-tube agglutination identification method with commercially available Panocell-20 (Immucor) RBC suspensions of known antigen phenotypes. Reaction patterns identified antigen specificity using standard blood bank methods. Patterns reactive with all cells tested were reported as pan-reactive and reactions without patterns were deemed low frequency antibodies. For all samples with an alloantibody, a matched SACRED control (age, sex, and transfusion history) was identified. Genomic DNA was analyzed by PCR and direct sequencing of each RHD and RHCE exon for single nucleotide polymorphisms (SNPs) that confer weak, partial, or variant phenotypes. Copy number variant PCR techniques with gene-specific primers were used to detect exonic substitutions in the RHD and RHCE gene loci that lead to negative or partial phenotypes. Results: A total of 272 SACRED participants were screened for RBC alloantibodies; 179 (66%) had at least one transfusion prior to enrollment, of whom 37 (21%) had a positive alloantibody screening result. Children with RBC alloantibodies (17 males, 20 females, average age 9.0 ± 2.4 years) had variable transfusion exposure with 23 (62%) having >10 prior transfusions, while 7 (19%) had ≤2 previous transfusions. Among the 37 positive samples, 18 sera had reactivity against Rh antigens including anti-E (12), anti-D (6, including 3 females), and anti-C specificity (5); 8 samples were consistent with a low frequency antibody and 3 were pan-reactive. Other alloantibodies had specificity to K (3, 1 female), Fya (4), Fyb (1), Lea (1), Leb (1), S (1), s (1), Jka (1), and U (1). More than one alloantibody was identified in 7 samples, of which 5 had two Rh antibodies. Genomic DNA analysis revealed numerous RHD variants, including several with one or more exonic SNPs that predict weak or partial RhD phenotypes, as well as predicted D-negative or partial phenotypes with hybrid RHD-CE-D genes or an insert pseudogene (RHDψ). Comparing the alloimmunized patients and controls, there was an equivalent frequency of RHD SNP variants (15/37 = 41%) in both cohorts, but more children with hybrid RHD-CE-D genes affecting multiple RHD exons (10 versus 4, p=0.077). All samples had the homozygous ccee genotype in the RHCE locus. There was a high frequency of RHCE SNP variants in both alloimmunized (31/37 = 84%) and control cohorts (32/37 = 86%), and only one control sample with possible RHD insertion in RHCE exon 5. All six children with anti-D alloantibodies had hybrid RHD-CE-D genes, typically typing as D-negative in tube testing. Among 17 children with anti-C or anti-E alloantibodies, 16 had at least one RHCE SNP, most frequently RHCE*ce.01, but no SNPs were associated with the development of Rh alloantibodies. Conclusions: RBC alloimmunization in children with SCA is common and often unrecognized in low-resource settings. In the Dominican Republic, 21% of children with SCA who entered SACRED with previous transfusions had RBC alloimmunization, including 10% with antibodies against Rh antigens. DNA analysis confirms extensive genetic complexity in both RHD and RHCE genes, with numerous SNPs that alter Rh antigen expression. Large RHD-CE-D insertions may also confer increased risks of anti-D alloimmunization. Lack of typing beyond serologic tube testing of ABO/RhD, and the presence of weak/partial RhD phenotypes in blood donors, may further increase the risk. Extending phenotyping for RhCE, providing RhD-negative blood when available, and performing enhanced screening for RhD-negative donors, will help reduce RBC alloimmunization. Disclosures No relevant conflicts of interest to declare.

Hereditary myotonia in American Bulldog associated with a novel frameshift mutation in the CLCN1 gene

Hereditary myotonia (HM) is a genetic disorder that occurs due to mutations in the chloride channel and results in delayed relaxation of the skeletal muscles. HM has been described in 12 dog breeds, and in five of them, molecular studies of this disorder were performed and mutations in the CLCN1 gene were described. In this study, an affected American Bulldog with HM clinically characterized by muscle hypertrophy, myotonic discharges, and nondystrophic myotonia with a “warm-up” phenomenon was evaluated, and the candidate canine CLCN1 gene was sequenced. The molecular analysis revealed a frameshift mutation NM_001003124.2:c.436_437insCTCT that resulted in a frameshift and a premature stop codon NP_001003124.1:pTyr146SerfsTer49 . Two aberrant alternative CLCN1 transcripts were observed in an affected dog, the expected transcript with the 4 bp insertion, NM_001003124.2:r.436_437insctct, and an unexpected transcript containing parts of intron 6 in addition to the insertion in exon 4, NM_001003124.2:[r.436_437insctct;r.774_775ins79]. In conclusion, the frameshift mutation in the CLCN1 gene is associated with autosomal recessive HM in American Bulldog and this study constitutes the first description of the disease in this breed.

Cadherin repeat 5 mutation associated with Bt resistance in a field-derived strain of pink bollworm

Evolution of resistance by pests reduces the benefits of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance to Bt toxin Cry1Ac in a field-derived strain of pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that the r14 allele of the pink bollworm cadherin gene (PgCad1) has a 234-bp insertion in exon 12 encoding a mutant PgCad1 protein that lacks 36 amino acids in cadherin repeat 5 (CR5). A strain homozygous for this allele had 237-fold resistance to Cry1Ac, 1.8-fold cross-resistance to Cry2Ab, and developed from neonate to adult on Bt cotton producing Cry1Ac. Inheritance of resistance to Cry1Ac was recessive and tightly linked with r14. PgCad1 transcript abundance in midgut tissues did not differ between resistant and susceptible larvae. Toxicity of Cry1Ac to transformed insect cells was lower for cells expressing r14 than for cells expressing wild-type PgCad1. Wild-type PgCad1 was transported to the cell membrane, whereas PgCad1 produced by r14 was not. In larval midgut tissue, PgCad1 protein occurred primarily on the brush border membrane only in susceptible larvae. The results imply r14 mediates pink bollworm resistance to Cry1Ac by reduced translation, increased degradation, and/or mislocalization of cadherin.

11P Preclinical evaluation of DS-2087b, a novel and selective inhibitor of EGFR/HER2 exon 20 insertions

In-frame insertions in exon 20 of the epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) gene are oncogenic drivers in non-small cell lung cancer (NSCLC), breast cancer, and others. However, no pharmacologic treatments are available for EGFR/HER2 exon 20 insertions (ex20ins), and effective treatment should avoid the inhibition of wild-type (WT) EGFR, which causes dose-limiting toxicities, such as rash and diarrhea. Here we describe DS-2087b, a novel oral, potent, and selective inhibitor of EGFR/HER2 ex20ins. We evaluated the inhibitory effects of DS-2087b and poziotinib, an investigational tyrosine kinase inhibitor against Ba/F3 cells harboring WT EGFR and against EGFR/HER2 ex20ins mutations, including EGFR V769_D770 insertion ASV (EGFR ins.ASV); EGFR D770_N771 insertion SVD (EGFR ins.SVD); and HER2 A775_G776 insertion YVMA (HER2 ins.YVMA). Moreover, we measured the antitumor activity of DS-2087b in an NSCLC patient-derived xenograft (PDX) model with EGFR ins.ASV. DS-2087b inhibited the proliferation of Ba/F3 cells expressing EGFR ins.ASV, EGFR ins.SVD, and HER2 ins.YVMA with 50% maximal inhibition of cell proliferation (GI50) values of 23.5, 21.3, and 29.0 nM, respectively, but the GI50 value for Ba/F3 cells expressing EGFR WT was 368.9 nM. Poziotinib showed similar GI50 values between EGFR/HER2 ex20ins and WT EGFR. In Ba/F3 allograft models with EGFR ins.ASV, EGFR ins.SVD, and HER2 ins.YVMA and the PDX model with EGFR ins.ASV, DS-2087b showed remarkable antitumor effects in a dose-dependent manner without body weight loss. In in vitro and in vivo preclinical assays, DS-2087b is effective against EGFR/HER2 ex20ins and also showed selectivity over WT EGFR. DS-2087b may show clinical utility in patients who harbor EGFR/HER2 ex20ins mutations and reduced toxicities caused by WT EGFR inhibition.

A 63-bp insertion in exon 2 of the porcine KIF21A gene is associated with arthrogryposis multiplex congenita.

A recessive form of arthrogryposis multiplex congenita (AMC) was detected 20years ago in the Swiss Large White (SLW) pig population. A diagnostic marker test enabled the identification of carrier animals, but the underlying causal mutation remains unknown. To identify the mutation underlying AMC, we collected SNP chip genotyping data for 11 affected piglets and 23 healthy pigs. Association testing using 47829 SNPs confirmed that AMC maps to SSC5 (P=9.4×10-13 ). Subsequent autozygosity mapping revealed a common 6.06Mb region (from 66 757970 to 72815151bp) of extended homozygosity in 11 piglets affected by AMC. Using WGS data, we detected a 63-bp insertion compatible with the recessive inheritance of AMC in the second exon of KIF21A gene encoding Kinesin Family Member 21A. The 63-bp insertion is predicted to introduce a premature stop codon in KIF21A gene (p.Val41_Phe42insTer) that truncates 1614 amino acids (~97%) from the protein. We found that this deleterious allele still segregates at a frequency of 0.1% in the SLW pig population. Carrier animals can now be detected unambiguously and excluded from breeding.

Ultrarapid EGFR Mutation Screening Followed by Comprehensive Next-Generation Sequencing: A Feasible, Informative Approach for Lung Carcinoma Cytology Specimens With a High Success Rate

IntroductionFor patients with advanced NSCLC, cytologic samples may be the only diagnostic specimen available for molecular profiling. Although both rapid and comprehensive assessment are essential in this setting, an integrated multitest approach remains an important strategy in many laboratories, despite the risks and challenges when working with scant samples. In this study, we describe our experience and high success rate in using a multitest approach, focusing on the clinical validation and incorporation of ultrarapid EGFR testing using the Idylla system followed by comprehensive next-generation sequencing (NGS). MethodsCytology samples received for routine molecular testing were included in this study. The performance characteristics of the EGFR Idylla assay were assessed; tissue suitability parameters and interpretation criteria to supplement automated mutation calling were established. The assay performance was monitored for 1 year, comparing the results with those of concurrent NGS testing by MSK-IMPACT (primarily) or MSK-AmpliSeq and MSK-Fusion solid panel in a subset of cases. ResultsOverall, 301 samples were studied; 83 samples were included in validation (60.2% [50 of 83] were positive for EGFR mutations). Concordance with the reference method was 96.4% (80 of 83) of the samples with excellent reproducibility. The limit of detection was variable depending on the total tissue input and the specific mutation tested. Unextracted tissue inputs that maintained total EGFR cycle of quantification at less than 23 allowed all mutations to be detected if present at greater than 5% variant allele frequency. Mutations could be detected at 1% variant allele frequency with total EGFR cycle of quantification of 18. During the clinical implementation phase, 218 NSCLC samples were tested by Idylla (24.3% [53 of 218] were EGFR mutation positive). Concurrent NGS testing was requested on 165 samples and successfully performed on 96.4% (159 of 165) of the samples. The Idylla automated results were concordant with those obtained by NGS in 96.2% (153 of 159) of cases and improved to 98.7% (157 of 159) after incorporation of manual review criteria to supplement automated calling, resulting in a diagnostic sensitivity of 95.6% (95% confidence interval: 84.9%–99.5%). In general, 9% (14 of 159) of the cases tested by NGS had EGFR mutations not covered by the Idylla assay, primarily insertions in exon 19 and 20 and minor mutations co-occurring with canonical sensitizing mutations. ConclusionsComprehensive molecular testing is feasible and has a high success rate in NSCLC cytology samples when using a multitest approach. Testing with the Idylla system enables rapid and accurate determination of the EGFR status without compromising subsequent NGS testing.

A duplication of the Anti-Müllerian hormone gene is associated with genetic sex determination of different Oreochromis niloticus strains.

Sex determination (SD) mechanisms are ancient and conserved, yet much diversity is exhibited in primary sex-determining signals that trigger male or female development. In O. niloticus, SD is associated with a male-specific locus on linkage group (LG) 23 which harbors the Y-linked Anti-Müllerian hormone (amh) gene, and a truncated duplication, denoted amhΔy. We have evaluated the possible role of identified indels and SNPs in the amh gene on SD, based on conservation in different O. niloticus strains. A fluorescent assay for the detection of a 5 bp insertion in amhΔy exon VI, efficiently discriminated between XX, XY, and YY genotypes. Concordance rate between amhΔy and sex varied in six Oreochromis strains, from 100% (Ghana) through 90% (Swansea) to 85% (Thai-Chitralada). The association of amhΔy with sex was found to be conserved in all tested O. niloticus strains, and thus supports its key role in SD. However, the previously identified missense SNP (C/T) in amh exon II was found only in the Swansea strain, thus excluding its candidacy for the causal variation of SD across all strains. Effects of markers on LGs 1, 3, and 23 (amhΔy) fully explained sex distribution in one Thai-Chitralada family (R2 = 1.0), whereas in another family only the major effect of LG23 (amhΔy) was significant (R2 = 0.37). Thus, amhΔy on LG23 is associated with genetic SD, either as a single causal gene in different O. niloticus strains, or in combination with segregating genes on LGs 1 and 3 in the Thai-Chitralada hybrid strain.

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

BackgroundIn recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants.MethodsWe conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results.ResultsWe successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G).ConclusionWe found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.

A minor QTL, SG3, encoding an R2R3-MYB protein, negatively controls grain length in rice.

SG3, an R2R3 MYB protein coding gene that tightly linked to a major QTLGS3, negatively regulates grain length while dependent on the status ofGS3in rice. It is often very difficult to isolate a minor QTL that is closely linked to a major QTL in rice. In this study, we focused on the isolation of a minor grain length QTL, small grain 3 (SG3), which is closely linked to the major QTL grain size 3 (GS3). The genetic effect of SG3 on grain length was dependent on GS3 status. Its genetic effect was larger in the presence of nonfunctional sg3 than functional SG3. A large number of near-isogenic F2 plants in which GS3 was fixed with nonfunctional alleles were developed to clone SG3. A gene encoding an R2R3 MYB domain transcriptional regulator was identified as the candidate gene for SG3. SG3 overexpression and knockdown plants showed shortened and elongated grains, respectively, which demonstrated that SG3 acts as a negative regulator of grain length. SG3 was preferentially expressed in panicles after flowering, and SG3 acted as a transcription activator. Comparative sequencing analysis identified a 12-bp insertion in the third exon of NYZ that led to a frameshift and resulted in a premature stop codon. The insertion/deletion was associated with grain length in the presence of functional GS3 in the indica subspecies. SG3 and GS3 were frequently in coupling phase in indica rice, making them good targets for the breeding of cultivars with short or long grains. The isolation of the SG3 MYB gene provides new gene resource and contributes to the regulatory network of grain length in rice.

TGF-β1 promotes epithelial-to-mesenchymal transition and stemness of prostate cancer cells by inducing PCBP1 degradation and alternative splicing of CD44.

CD44 is a marker of cancer stem cell (CSC) in many types of tumors. Alternative splicing of its 20 exons generates various CD44 isoforms that have different tissue specific expression and functions, including the CD44 standard isoform (CD44s) encoded by the constant exons and the CD44 variant isoforms (CD44v) with variant exon insertions. Switching between the CD44v and CD44s isoforms plays pivotal roles in tumor progression. Here we reported a novel mechanism of CD44 alternative splicing induced by TGF-β1 and its connection to enhanced epithelial-to-mesenchymal transition (EMT) and stemness in human prostate cancer cells. TGF-β1 treatment increased the expression of CD44s and N-cadherin while decreased the expression of CD44v and E-cadherin in DU-145 prostate cancer cells. Other EMT markers and cancer stem cell markers were also upregulated after TGF-β1 treatment. RNAi knockdown of CD44 reversed the phenotype, which could be rescued by overexpressing CD44s but not CD44v, indicating the alternatively spliced isoform CD44s mediated the activity of TGF-β1 treatment. Mechanistically, TGF-β1 treatment induced the phosphorylation, poly-ubiquitination, and degradation of PCBP1, a well-characterized RNA binding protein known to regulate CD44 splicing. RNAi knockdown of PCBP1 was able to mimic TGF-β1 treatment to increase the expression of CD44s, as well as the EMT and cancer stem cell markers. In vitro and in vivo experiments were performed to show that CD44s promoted prostate cancer cell migration, invasion, and tumor initiation. Taken together, we defined a mechanism by which TGF-β1 induces CD44 alternative splicing and promotes prostate cancer progression.

Immune microenvironment features and efficacy of PD-1/PD-L1 blockade in non-small cell lung cancer (NSCLC) patients with EGFR or HER2 exon 20 insertions.

e21540 Background: This study aimed to investigate the immune microenvironment features and efficacy of PD-1/PD-L1 blockade of NSCLC with insertions in exon 20 (Ex20ins) of EGFR or HER2. Methods: Molecular spectrum, tumor mutational burden (TMB), PD-L1 protein expression, and the abundance of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) were reviewed for NSCLC patients with Ex20ins of EGFR or HER2. Results: Thirty-five patients carrying EGFR Ex20ins and 21 patients harboring HER2 Ex20ins were retrospectively enrolled between April 2016 and September 2018. The average TMB was 3.3 mutations/megabase. PD-L1 expression in patients with EGFR Ex20ins was significantly higher than those with HER2 mutations (48.6% vs. 19.0%, P=0.027). High TMB and PD-L1 expression was independently associated with considerably poor prognosis (P=0.025, P=0.045; respectively). Finally, patients harboring EGFR Ex20ins seemed to be sensitive to PD-1/PD-L1 blockage whereas it showed limited efficacy in patients with HER2 Ex20ins. Conclusions: NSCLC patients with EGFR/ HER2 Ex20ins had distinct immune features. Patients with EGFR Ex20ins had significantly higher PD-L1 expression than those with HER2 mutations, which may be the underlying reason for the different responses to PD-1/PD-L1 blockage.

A key \u2018foxy\u2019 aroma gene is regulated by homology-induced promoter indels in the iconic juice grape \u2018Concord\u2019

‘Concord’, the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special ‘foxy’ aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key ‘foxy’ aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in ‘Concord’ and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the ‘foxy’ aroma trait and makes it genetically trackable and amenable in grapevine breeding.

Corrigendum to: First case of late onset cardiac phenotype Fabry disease due to an AluYb8 insertion in exon 7 of the GLA gene.

Corrigendum to: First case of late onset cardiac phenotype Fabry disease due to an AluYb8 insertion in exon 7 of the GLA gene.

Paraoxonase‐1 regulation of Na/K‐ATPase alpha‐1 Src signaling in Chronic Kidney Disease

BackgroundCardiotonic steroids (CTS) are Na/K‐ATPase alpha‐1 (NKA α‐1) ligands that are increased in volume expanded states such as chronic kidney disease (CKD). We have demonstrated that the NKA α‐1‐Src signaling pathway is a critical mediator of CTS induced renal inflammation and oxidative stress in these settings. Paraoxonase‐1 (PON‐1) is a hydrolytic lactonase enzyme which is synthesized in the liver and circulates bound to high‐density lipoprotein. Clinical data demonstrate an association between diminished lactonase activities of PON‐1 and renal disease severity, yet the underlying mechanism(s) are largely unknown. We have identified novel mechanistic link between PONs and endogenous CTS whereby the lactonase activities of PONs hydrolyze CTS to their inactive open‐ring forms which, unlike native CTS, are incapable of stimulating NKA α‐1 and initiating pro‐inflammatory signaling cascades.AimTo test the hypotheses that PON‐1 can attenuate the progression of renal inflammation in CKD through modulating the pathogenic pathways induced by CTS‐NKA α‐1‐Src signaling.Method and ResultsExperiments were performed on control Dahl salt‐sensitive rats (SSMcwi, hereafter called SS rats) and Pon1 knock‐out rats (designated SS‐Pon1em1Mcwi, hereafter called SS‐Pon1 KO rats) generated by injecting a CRISPR targeting the sequence into SSMcwi rat embryos which resulted in a 7bp frameshift insertion in exon 4 of the Pon1 gene. In order to examine the protective role of the PON‐1 in renal disease progression, age‐matched male SSPon1 KO and SS rats were maintained on 8% high salt diet for up to 5 weeks to initiate the salt‐sensitive hypertensive renal disease characteristic of this model. Upon examination we found that, despite similar blood pressure, SS‐Pon1 KO rats showed significant increase in renal inflammation and fibrosis (both p<0.01), decrease in FITC‐Sinistrin glomerular filtration rate (p<0.05), and increased mortality (p<0.01) compared to SS‐rats. Next, in order to examine involvement of the CTS‐NKA α‐1‐Src kinase signaling axis, SS‐Pon1 KO rats were further divided into two subgroups: one group received a specific NKA α‐1‐Src kinase complex inhibitor, pNaKtide (25 mg/kg i.p.), every week for a total of 4 injections, and the second group, received an anti‐CTS monoclonal antibody (3E9 mAb, 50 μg/kg i.p.) every week for a total of 4 injections, with both sub groups were maintained on the same 8% high salt diet. We found that while SS‐Pon1 KO rats on high salt diet demonstrated significant acceleration in renal disease progression as noted by increased renal inflammation, renal fibrosis and mortality as well as decreased renal function, these effects were all significantly (all p<0.05) attenuated by inhibition of the NKA α‐1‐Src signaling pathway via both pNaKtide and anti‐CTS monoclonal antibody.ConclusionOur data suggest PON‐1 is an endogenous counter‐regulatory mechanism of the CTS‐NKA α‐1‐Src signaling axis which can be therapeutically targeted to attenuate hypertensive renal disease.Support or Funding InformationNIH/NHLBI HL‐137004

Role of Kir4.1 (Kcnj10) in the Regulation of Salt‐Induced Hypertension

In the kidney, Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the aldosterone sensitive distal nephron (ASDN), a major target for multiple hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric Kir4.1/Kir5.1 channels and homomeric Kir4.1 channels. These channels control the transepithelial voltage, fine‐tune renal electrolyte homeostasis and contribute to long‐term blood pressure control, correspondingly. To study the role of Kir4.1 containing channels in the renal control of blood pressure, renin‐angiotensin‐aldosterone system (RAAS) balance, and salt‐sensitive (SS) hypertension, we created a Kcnj10 knockout (SSKcnj10−/−) on the Dahl SS rat background. The SSKcnj10−/− rat was created using CRISPR/Cas9 mutagenesis, resulting in a single base pair insertion in exon 2 of the Kcnj10 gene and producing a frameshift in Kir4.1 protein translation after only 26 amino acids. Homozygous SSKcnj10−/− rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks after birth. Mass spectrometry based quantification of serum RAAS peptides revealed increased Ang I (1631 vs. 560 pM), Ang 1–5 (499 vs. 69 pM), and Ang 1–7 (598 vs. 42 pM) levels in SSKcnj10−/− rats compared to age‐matched SSKcnj10+/+ controls. No differences were observed in serum Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity represented by the Ang II to Ang I ratio was decreased in SSKcnj10−/− rats (0.58 vs. 1.1). The increases in alternative RAS axis peptides (Ang 1–5 and Ang 1–7) and decrease in ACE activity in SSKcnj10−/− rats are suggestive of a low blood pressure phenotype, however, their limited survival precluded blood pressure measurement. Heterozygous (SSKcnj10+/−) developed normally and showed no changes in serum electrolytes. Although survival rate of both male and female SSKcnj10+/− rats was dramatically lower compared to SSKcnj10+/+ rats, they were able to survive much longer than SSKcnj10−/− rats (21 vs. 83 days on average, respectively) with dietary K+ supplementation (HK; 1.41% K+), allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions (LS+HK; 123±2 vs 125±4, mmHg), but salt‐induced (HS, 4% NaCl) elevations in blood pressure were significantly attenuated in SSKcnj10+/− rats fed a HS diet (149±6 vs 183±9 mmHg for SSKcnj10+/− and SSKcnj10+/+ rats, respectively). Furthermore, after 3 weeks on HS, SSKcnj10+/− rats showed decreased diuresis (25±5 vs 38±2, ml/day) and improved albuminuria (6.0±4.0 vs 17.2±7.6, Alb/Cre ratio) compared to SSKcnj10+/+ rats. Our results demonstrate that renal Kir4.1 containing channels mediate salt‐sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.Support or Funding InformationSupported by the National Institutes of Health grants R35 HL135749, R56 DK121750, R24 HL114474, and F31 DK122647

An exonic insertion in the NAGLU gene causing Mucopolysaccharidosis IIIB in Schipperke dogs

Mucopolysaccharidosis (MPS) IIIB (Sanfilippo syndrome B; OMIM 252920), is a lysosomal storage disease with progressive neurological signs caused by deficient activity of alpha-N-acetylglucosaminidase (NAGLU, EC 3.2.1.50). Herein we report the causative variant in the NAGLU gene in Schipperke dogs and a genotyping survey in the breed. All six exons and adjacent regions of the NAGLU gene were sequenced from six healthy appearing and three affected Schipperkes. DNA fragment length and TaqMan assays were used to genotype privately owned Schipperkes. A single variant was found in exon 6 of MPS IIIB affected Schipperkes: an insertion consisting of a 40–70 bp poly-A and an 11 bp duplication of the exonic region preceding the poly-A (XM_548088.6:c.2110_2111ins[A(40_70);2100_2110]) is predicted to insert a stretch of 13 or more lysines followed by either an in-frame insertion of a repeat of the four amino acids preceding the lysines, or a frameshift. The clinically affected Schipperkes were homozygous for this insertion, and the sequenced healthy dogs were either heterozygous or homozygous for the wild-type allele. From 2003–2019, 3219 Schipperkes were genotyped. Of these, 1.5% were homozygous for this insertion and found to be clinically affected, and 23.6% were heterozygous for the insertion and were clinically healthy, the remaining 74.9% were homozygous for the wild-type and were also clinically healthy. The number of dogs homozygous and heterozygous for the insertion declined rapidly after the initial years of genotyping, documenting the benefit of a DNA screening program in a breed with a small gene pool. In conclusion, a causative NAGLU variant in Schipperke dogs with MPS IIIB was identified and was found at high frequency in the breed. Through genotyping and informed breeding practices, the prevalence of canine MPS IIIB has been drastically reduced in the Schipperke population worldwide.

A 14-bp insertion in endothelin receptor B-like (EDNRB2) is associated with white plumage in Chinese geese

BackgroundGang goose is a native species with gray plumage in Sichuan, China. As a result of overhunting, the number of gray Gang geese has decreased dramatically. To keep the species from extinction, conservation work for Gang geese was undertaken. In the process of pure breeding of gray Gang geese, approximately 2% of the offspring of each generation were white. This study aims to explain the genetic mechanism of this phenomenon and provide reliable molecular markers for goose-related plumage color breeding.ResultsWe used the method of pooled whole genome sequencing and Fst (fixation statistics) to identify the differentiation degree of alleles between gray Gang geese and white Gang geese from their offspring. In this way, EDNRB2, a key gene that affects the migration of melanoblasts, was identified. Then, the transcriptome was sequenced for the two geese plumage color populations, and the DEGs (differentially expressed genes) were analyzed. The results indicated that EDNRB2, as a possible candidate gene, had a significantly differential mRNA expression. In addition, a 14-bp insertion (NW_013185915.1: g. 750,748–750,735 insertion. CACAGGTGAGCTCT) in exon 3 of EDNRB2 was analyzed and found to have a significant association between gray geese and Chinese white breeds (P = 0.00), while this mutation was not found in European geese. Meanwhile, the insertion was homozygous in all the white geese we detected and heterozygous in gray geese, indicating that this mutation is recessive. Furthermore, this 14-bp insertion leads to a frameshift mutation in the EDNRB2 coding region and nonsense-mediated mRNA decay.ConclusionOur study strongly suggests that the 14-bp insertion in exon 3 of the EDNRB2 gene is associated with the white plumage phenotype in Chinese geese. This study is the first to investigate the relationship between EDNRB2 and white plumage in geese.

Endoplasmic Reticulum Stress Activation in Alport Syndrome Varies Between Genotype and Cell Type.

Alport syndrome is a hereditary progressive chronic kidney disease caused by mutations in type IV collagen genes COL4A3/4/5. X-linked Alport syndrome (XLAS) is caused by mutations in the COL4A5 gene and is the most common form of Alport syndrome. A strong correlation between the type of COL4A5 mutation and the age developing end-stage renal disease in male patients has been found. Mutation to the α (IV) chain causes retention of the protein to the endoplasmic reticulum lumen, which causes endoplasmic reticulum stress (ERS) and subsequent exertion of deleterious intracellular effects through the activation of ERS. The exact time point that mutant type IV collagen α chain exerts its deleterious effects remains elusive. In this study, we explored the relationship between the COL4A5 genotype and cell type in ERS activation. We obtained skin fibroblasts from Alport syndrome patients with different COL4A5 mutation categories [i.e., a missense mutation (c.4298G > T, p.Gly1433Val) in exon 47, a splicing mutation (c.1949–1G > A) in intron 25 and an insertion (c.573_c.574insG, p. Pro193Alafs*23) in exon 10], and then reprogrammed these fibroblasts into induced pluripotent stem cells (iPSCs). Interestingly, no significant dysregulation of ERS pathway markers was observed for the three COL4A5 mutant iPSCs; however, significant activation of ERS in COL4A5 mutant fibroblasts was observed. In addition, we found that the activation levels of some ERS markers in fibroblasts varied among the three COL4A5 mutation types. Mutant COL4A5 proteins were demonstrated to have different effects on cells at different stages of ontogenesis, providing a theoretical basis for choosing the timing of intervention. The observed differences in activation of ERS by the COL4A5 mutant fibroblasts may contribute to the intracellular molecular mechanisms that describe the correlation between genotype and clinical features in XLAS.

In silico analysis of the fragrance gene (badh2) in Asian rice (Oryza sativa L.) germplasm and validation of allele specific markers

AbstractBadh2 of rice is considered to be the major gene responsible for the fragrance in rice. The wild type badh2 allele encodes betaine aldehyde dehydrogenase 2 (BADH2) enzyme while the mutated version of badh2 gene encodes non-functional BADH2 enzyme that leads to the accumulation of 2-acetyl-1-pyrroline (2AP), the principal fragrant compound in rice. There are many mutated recessive alleles causing fragrance in global rice germplasm, although the badh2.1 allele present in Basmati type rice is the most well-known among breeders. In this study, we attempted to reveal potential fragrance causing mutations, and the respective varieties carrying them, through in silico analysis based on the sequences available in the Rice SNP-Seek-Database of International Rice Research Institute. The sequences of 1878 rice accessions from 22 countries were analysed to identify mutations in each exon of badh2 comparatively with the non-fragrant ‘wildtype’ GenBank sequence in Nanjing11, Oryza sativa indica (EU770319.1). Results revealed that 63 varieties from 12 countries possessed the most prevalent allele, badh2.1 having an 8 bp deletion and three single nucleotide polymorphisms in the 7th exon. The second most prevalent allele in genotypes from Asia was badh2.7 having a ‘G’ insertion in the 14th exon. A novel allele with a T deletion in 9th exon was detected in a Thai rice accession. Rice varieties containing either badh2.1 or badh2.7 alleles could be identified with DNA markers for badh2.1 (frg) and badh2.7 (Bad2.7CAPS). The marker, Bad2.7CAPS, co-segregated with the fragrance phenotype in two crosses, confirming the possibility of employing it in marker assisted breeding.