Stop Mutation Research Articles

Emulsion-based evolution of Escherichia coli for higher growth yield on D-xylose identifies central role of cyclic AMP

D-xylose is an abundant sugar found in plant biomass and can be used as a renewable feedstock for the microbial production of diverse biofuels and bioproducts. However, D-xylose metabolism is slow in many industrial microorganisms, at least as compared to glucose metabolism. Not surprisingly, a number of approaches have been developed for improving D-xylose metabolism in diverse microorganisms. In this work, we applied a previously developed evolution strategy based on media-in-oil emulsions for improving the growth yield of Escherichia coli NCM3722 on D-xylose. After 30 rounds of evolutions, we isolated multiple mutants with increased growth yield on D-xylose. In addition, we also observed similar increases in the growth rate. Three mutants were selected for whole-genome sequencing. Two mutants had an amber stop mutation in adenylate cyclase, which truncates nearly 60% of the enzyme. However, the ability of this mutant to grow on xylose indicated that truncated enzyme, lacking the C-terminal regulatory domain, is still active. The other mutant had a point mutation in the cyclic AMP receptor protein (CRP), near the high affinity binding site for cyclic AMP. Both mutations, when introduced into wild type E. coli, were able to increase the growth yields at levels similar to the isolated mutants. In addition to D-xylose, these mutant strains and their genetic mimics also exhibited higher growth rates and yields on glucose, lactose, and L-arabinose. These results suggest that the improved growth rates and yields are due to changes in the production and sensing of intracellular cyclic AMP concentrations and also suggest native concentrations are suboptimal with respect to the growth rate and yield under the growth conditions tested. Collectively, these results may prove useful for engineering strains of E. coli for high-density fermentations or protein production.

OA28 A rare case of cardiac tumour in infancy mimicking sarcoidosis

Introduction/BackgroundSarcoidosis is a rare multisystemic inflammatory disorder with 2 distinct presentations in childhood. A monogenic form associated with mono-allelic mutation in NOD2 presents with the triad of uveitis, rash and arthritis in children less than 4-years (early-onset sarcoidosis). The other presentation affects older children with similar presentation to adults, with greater pulmonary and lymph node involvement. Histopathological evidence of non-caseating granulomas is essential for the diagnosis but does not exclude alternative diagnoses. We report a 17-month-old infant, who presented with atypical features suggestive of sarcoidosis, but was subsequently found to have a rare cardiac angiosarcoma driving an exaggerated granulomatous reaction.Description/MethodA previously healthy 17-month-old girl born to non-consanguineous Caucasian parents presented with recurrent fever for 3-months, associated with lethargy, weight loss and worsening anemia. Examination revealed an irritable febrile child, weight on 5th centile, with muffled heart sounds; there were no skin lesions, no lymphadenopathy, and no arthritis. Investigations showed severe anemia (Hb 46g/L), leukocytosis (WBC 24/L), thrombocytosis (662/L), high inflammatory markers (CRP >300mg/L, ESR 160mm/hr), high ferritin (600ug/L). CT thorax with cardiac angiogram revealed a large intra-atrial septal mass, extensive pulmonary nodules, bilateral hilar and mediastinal lymphadenopathy and pericardial effusion. She was managed with broad-spectrum antibiotics, blood transfusions and pericardiocentesis with endomyocardial biopsy. Extensive infectious workup, including TB from pericardial exudate, was negative. Serum angiotensin-converting enzyme was normal, with no blasts in blood film; eye examination was unremarkable. The endomyocardial biopsy was inconclusive, so underwent lung and lymph node biopsies, which were initially reported as revealing florid multiple non-necrotising granulomas, but no evidence of malignant cells. Whole-exome sequencing was performed, result pending at that stage. Differential diagnosis included malignancy, infection, immunodeficiency or autoimmune disorder including sarcoidosis, in light of histopathology. With malignancy and infection felt to be excluded, she was managed empirically for atypical sarcoidosis and treated with IV methylprednisolone 30mg/kg followed by tapering steroid and anti-TNF; IL-1 was added later. However, there was no clinical response with ongoing fever, irritability and static inflammatory markers (CRP∼300mg/L). Immunomodulating medications were subsequently stopped. Repeat chest imaging revealed progression of pulmonary findings and increasing cardiac mass. Open-heart bypass surgery with debulking of cardiac mass and further biopsies. Histopathology revealed an extremely rare primary cardiac malignant vascular tumour, angiosarcoma, with loss of SMARCB1 protein staining. Retrospective review and specific staining with SMARCB1 of initial lung biopsy subsequently confirmed that the granulomatous reaction was in response to micro-metastases of tumour throughout the lungs.Discussion/ResultsThe differential diagnosis of granulomatous disease in paediatric patients is very broad. Ultimately, the diagnosis of cancer driving and exaggerated immunological response was provided by a combination of very invasive surgical biopsy of the heart; combined with whole exome sequencing. Whole exome sequencing revealed 2 distinct genetic diagnoses which fully explained the phenotype: heterozygosity for a stop mutation in SMARCB1 (p.R158X) consistent with Rhabdoid Tumour Predisposition Syndrome Type 1; and homozygosity for the hypomorphic PRF1 p.A91V mutation explaining low (but not absent) intracellular perforin levels and exaggerated granulomatous immunological response to tumour. The rare angiosarcoma detected is scarcely reported in the literature, but entirely compatible with the type of cancer associated with RTPS1. Hypomorphic perforin mutation probably explains the exaggerated but frustrated granulomatous immunological response to tumour in this case.Key learning points/ConclusionThis case serves to remind us that granulomas can occur in response to malignancy in children. Lack of response to immunosuppression with ongoing severe systemic inflammation in this case provided an important clue to the need for more invasive diagnostic biopsies. Particular challenges relates to the time it takes to obtain genetic test results; but in this case whole-exome sequencing resolved the phenotype fully. Patients with rare and unusual presentations may have more than one genetic diagnosis, with a blending of phenotypes thereafter. Screening of other family members for the mutation associated with RTPS1 is ongoing.

Abstract EC101: Premature Stop Mutations In Mybphl Share The Common Pathomechanism Of Preventing Protein Incorporation Into The Cardiac Myofilament

Cardiomyopathy and arrhythmia are diseases with significant genetic linkage. Cardiac myosin binding protein-C (cMyBP-C) is a myofilament associated protein with a C-terminal composed of repeating immunoglobulin (Ig) and fibronectin III (FnIII) domains that bind to the myosin thick filaments and titin. Truncating mutations in the C-terminal of MyBP-C result in improper myofilament incorporation and degradation of the truncated protein and is a major cause of hypertrophic cardiomyopathy. Myosin-binding protein-H like ( MyBP-HL ) is a protein that was recently identified that is structurally related to MyBP-C. Both cMyBP-C and MyBP-HL contain repeated C-terminal Ig and FnIII domains that bind to myosin. MyBP-HL is different in that it is primarily expressed in the atria where it interacts with the myofilament. MyBP-HL premature stop mutations are associated with improper myofilament incorporation, and has been linked to ventricular conduction system abnormalities, atria enlargement, and dilated cardiomyopathy (DCM) in humans. The gnomAD database reports 9 stop-gain mutations for MYBPHL in humans including: Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255 and Tyr307. Prior investigation modeled the MyBP-HL Arg255X mutation and showed no MyBP-HL localization into the myofilament. A null mouse model showed that loss of MyBP-HL resulted in arrhythmia and cardiomyopathy, similar to that observed in humans. We hypothesize that all MyBP-HL truncation mutations will share a common mechanism by failing to produce MyBP-HL that integrates into the sarcomere. We test this hypothesis by transfecting neonatal rat cardiomyocytes with MyBP-HL constructs containing these mutations. We tested Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250 and Arg255 MyBP-HL mutations. We measured co-localization events between MyBP-HL mutants and MyBP-C in atrial and ventricular overexpressed cells and observed that stop gained mutations disrupt normal localization as we expected. Moreover, we tested wild-type MyBP-HL overexpression, and both showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. These truncating mutations in MYBP-HL may increase risk for developing arrhythmias, atria enlargement and DCM.

A Novel Homozygous Stop Mutation in IL23R Causes Mendelian Susceptibility to Mycobacterial Disease

PurposeMendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease.MethodsWe performed functional validation of the R381X mutation by evaluating IL23R expression and IL-23 signaling (STAT3 phosphorylation, IFN-γ production) in primary cells (PBMCs, EBV-B cells) and cell lines (HeLa) with or without back-complementation of wild-type IL23R.ResultsWe report on a 48-year-old male with disseminated non-tuberculous mycobacterial disease. We identified and characterized a homozygous loss-of-function stop mutation underlying IL23R deficiency, resulting in near absent expression of membrane bound IL23R. IL23R deficiency was characterized by impaired IL-23-mediated IFN-γ secretion in CD4+, CD8+ T, and mucosal-associated invariant T (MAIT) cells, and low frequencies of circulating Th17 (CD3+CD45RA−CCR4+CXCR3−RORγT+), Th1* (CD45RA−CCR4−CXCR3+RORγT+), and MAIT (CD3+CD8+Vα7.2+CD161+) cells. Although the patient did not have a history of recurrent fungal infections, impaired Th17 differentiation and blunted IL-23-mediated IL-17 secretion in PBMCs were observed.ConclusionWe demonstrate that impaired IL-23 immunity caused by a homozygous R381X mutation in IL23R underlies MSMD, corroborating earlier findings with a homozygous p.C115Y IL23R mutation. Our report further supports a model of redundant contribution of IL-23- to IL-17-mediated anti-fungal immunity.1

A tRNA-Acetylating Toxin and Detoxifying Enzyme in Mycobacterium tuberculosis.

ABSTRACTToxin-antitoxin (TA) systems allow bacteria to adapt to changing environments without altering gene expression. Despite being overrepresented in Mycobacterium tuberculosis, their physiological roles remain elusive. We describe a TA system in M. tuberculosis which we named TacAT due to its homology to previously discovered systems in Salmonella. The toxin, TacT, blocks growth by acetylating glycyl-tRNAs and inhibiting translation. Its effects are reversed by the enzyme peptidyl tRNA hydrolase (Pth), which also cleaves peptidyl tRNAs that are prematurely released from stalled ribosomes. Pth is essential in most bacteria and thereby has been proposed as a promising drug target for complex pathogens like M. tuberculosis. Transposon sequencing data suggest that the tacAT operon is nonessential for M. tuberculosis growth in vitro, and premature stop mutations in this TA system present in some clinical isolates suggest that it is also dispensable in vivo. We assessed whether TacT modulates pth essentiality in M. tuberculosis because drugs targeting Pth might prompt resistance if TacAT is disrupted. We show that pth essentiality is unaffected by the absence of tacAT. These results highlight a fundamental aspect of mycobacterial biology and indicate that Pth’s essential role hinges on its peptidyl-tRNA hydrolase activity. Our work underscores Pth’s potential as a viable target for new antibiotics.IMPORTANCE The global rise in antibiotic-resistant tuberculosis has prompted an urgent search for new drugs. Toxin-antitoxin (TA) systems allow bacteria to adapt rapidly to environmental changes, and Mycobacterium tuberculosis encodes more TA systems than any known pathogen. We have characterized a new TA system in M. tuberculosis: the toxin, TacT, acetylates charged tRNA to block protein synthesis. TacT's effects are reversed by the essential bacterial enzyme peptidyl tRNA hydrolase (Pth), which is currently being explored as an antibiotic target. Pth also cleaves peptidyl tRNAs that are prematurely released from stalled ribosomes. We assessed whether TacT modulates pth essentiality in M. tuberculosis because drugs targeting Pth might prompt resistance if TacT is disrupted. We show that pth essentiality is unaffected by the absence of this TA system, indicating that Pth's essential role hinges on its peptidyl-tRNA hydrolase activity. Our work underscores Pth's potential as a viable target for new antibiotics.

Resistance of Xanthomonas oryzae pv. oryzae to Lytic Phage X2 by Spontaneous Mutation of Lipopolysaccharide Synthesis-Related Glycosyltransferase.

Phage therapy is a promising biocontrol management on plant diseases caused by bacterial pathogens due to its specificity, efficiency and environmental friendliness. The emergence of natural phage-resistant bacteria hinders the application of phage therapy. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of the devastating bacterial leaf blight disease of rice. Here, we obtained a spontaneous mutant C2R of an Xoo strain C2 showing strong resistance to the lytic phage X2. Analysis of the C2R genome found that the CDS2289 gene encoding glycosyltransferase acquired a frameshift mutation at the 180th nucleotide site, which also leads to a premature stop mutation at the 142nd amino acid. This mutation confers the inhibition of phage adsorption through the changes in lipopolysaccharide production and structure and bacterial surface morphology. Interestingly, glycosyltransferase-deficient C2R and an insertional mutant k2289 also showed reduced virulence, suggesting the trade-off costs of phage resistance. In summary, this study highlights the role of glycosyltransferase in interactions among pathogenic bacteria, phages and plant hosts, which provide insights into balanced coevolution from environmental perspectives.

Whole Exome Sequencing Identifies a Rare Mutation in NACAD as a Possible Cause of COVID orchitis in Brothers

IntroductionLittle is known of SARS-CoV2 (COVID-19) pathogenicity, but organ inflammation syndromes have been well observed and documented in the lungs, heart, liver, kidneys, and testes. While the blood-testis barrier is known to protect testes from antigens, current evidence suggests COVID-19 can affect the testis. Reports of bilateral orchitis are growing. Current hypotheses consider causes of (1) cellular induced cytokine storm or (2) IgG deposits in the vascular endothelium to be at blame but causal genes or underlying genetic susceptibility have not yet been identified.ObjectiveThis project sought to uncover genetic explanations as to why certain men face increased susceptibility to developing COVID orchitis.MethodsWe identified and examined six COVID-19 patients who all were confirmed with polymerase chain reaction (PCR), including three COVID-19 (+) men without orchitis (controls) and three COVID (+) men with orchitis (bilateral testicular pain for at least 5 days around the time of testing PCR positive). Of note, among the three men with COVID-19 who had orchitis, two of them were siblings. DNA extraction and whole exome sequencing were performed on blood using the QIAmp blood maxi kit on five of the six patients. Variants were prioritized by being shared between the three patients affected with orchitis, absent in controls, and introducing nonsense, frameshift, splicing or non‐synonymous amino acid changes and less than 10% in population prevalence. Based on WES findings, DuoSet® Human ACE2 reagent kit 2 (catalog number: DY933-05) was purchased from R&D Systems, USA, and used to measure the level of soluble ACE2 in the plasma samples.ResultsThe average age of the men in the study was 25 years old. The average duration of COVID symptoms (fever, sore throat, cough, body aches) were 7 days. Among the men who developed bilateral testis pain, the symptoms lasted for an average of 22 days. A list of 16 variants was generated that found to be shared between the two siblings with COVID orchitis along with the unrelated subject with COVID orchitis, and not present in the two controls. Among the 16 variants, a nonsynonymous non-frameshit deletion in NACAD variant on chromosome 7 with a frequency of 3.9% prevalence in ExAC was prioritized based on known involvement in the ACE2 pathway, read depth, and genotype quality. Phenotypically, we found that circulating levels of soluble ACE2 was 3.72 ng/ml among men who had COVID orchitis and was lower than men who developed COVID without orchitis.ConclusionsWe observed a stop mutation in NACAD in 2 brothers and 1 unrelated man who developed COVID orchitis. Interestingly, we found lower circulating ACE2 serum levels in both brothers with orchitis and the one nonrelated orchitis subject but normal serum levels in all controls. NACAD when involved with cellular ability to shuttle out ACE2 becomes critical for COVID symptomatology. With decreased transcellular and extracellular transport of ACE2 being possible in subjects with the gene mutation, it can be postulated more ACE2 will be found intracellularly leading to increased cellular entry of SARS CoV-2 and possibility of orchitis sequelae.DisclosureNo

Boosting the antioxidant potential of pasta by a premature stop mutation in wheat keto-acythiolase-2

Phenolics are a class of chemical compounds possessing antioxidant activity, which are mainly located in the wheat (Triticum aestivum) bran. Different approaches have been used in food industry to increase the availability of phenolics. Compared to these methods, however, genetic improvement of the wheat antioxidant potential, is a cost-effective, easier and safer approach. Here, we showed a single premature stop mutation in the keto-acythiolase-2 (kat-2b) gene, which significantly improved the antioxidant potential of pasta by a 60 ± 16% increase in its antioxidant potential by increasing the accumulation of ferulic acid. These changes are likely determined by the increased transcription (46% higher) and activity (120% higher) of the phenylalanine lyase genes observed in the mutated line compared to the control. Even if more studies will need to be done, overall, this study suggested that the kat-2b mutant could represent an excellent genetic resource to improve wheat’s antioxidant and health-promoting potential.

Rare PSAP Variants and Possible Interaction with GBA in REM Sleep Behavior Disorder.

PSAP encodes saposin C, the co-activator of glucocerebrosidase, encoded by GBA. GBA mutations are associated with idiopathic/isolated REM sleep behavior disorder (iRBD), a prodromal stage of synucleinopathy. To examine the role of PSAP mutations in iRBD. We fully sequenced PSAP and performed Optimized Sequence Kernel Association Test in 1,113 iRBD patients and 2,324 controls. We identified loss-of-function (LoF) mutations, which are very rare in PSAP, in three iRBD patients and none in controls (uncorrected p = 0.018). Two variants were stop mutations, p.Gln260Ter and p.Glu166Ter, and one was an in-frame deletion, p.332_333del. All three mutations have a deleterious effect on saposin C, based on in silico analysis. In addition, the two carriers of p.Glu166Ter and p.332_333del mutations also carried a GBA variant, p.Arg349Ter and p.Glu326Lys, respectively. The co-occurrence of these extremely rare PSAP LoF mutations in two (0.2%) GBA variant carriers in the iRBD cohort, is unlikely to occur by chance (estimated co-occurrence in the general population based on gnomAD data is 0.00035%). Although none of the three iRBD patients with PSAP LoF mutations have phenoconverted to an overt synucleinopathy at their last follow-up, all manifested initial signs suggestive of motor dysfunction, two were diagnosed with mild cognitive impairment and all showed prodromal clinical markers other than RBD. Their probability of prodromal PD, according to the Movement Disorder Society research criteria, was 98% or more. These results suggest a possible role of PSAP variants in iRBD and potential genetic interaction with GBA, which requires additional studies.

Analysis of entire hepatitis B virus genomes reveals reversion of mutations to wild type in natural infection, a 15year follow-up study.

It has been reported that some mutations in the genome of hepatitis B virus (HBV) may predict the outcome of the virus infection. However, evolutionary data derived from long-term longitudinal analysis of entire HBV genomes using next generation sequencing (NGS) remain rare. In this study, serum samples were collected from asymptomatic hepatitis B surface antigen (HBsAg) carriers from a long-term prospective cohort. The entire HBV genome was amplified by polymerase chain reaction (PCR) and sequenced using NGS. Twenty-eight time series serum samples from nine subjects were successfully analysed. The Shannon entropy (Sn) ranged from 0 to 0.89, with a median value of 0.76, and the genetic diversity (D) ranged from 0 to 0.013, with a median value of 0.004. Intrahost HBV viral evolutionary rates ranged from 2.39E-04 to 3.11E-03. Double mutations at nt1762(A→T) and 1764(G→A) and a stop mutation at nt1896(G→A) were seen in all sequences from subject BO129 in 2007. However, in 2019, most sequences were wild type at these positions. Deletions between nt 2920-3040 were seen in all sequences from subject TS115 in 2007 and 2013 but these were not present in 2004 or 2019. Some sequences from subject CC246 had predicted escape substitutions (T123N, G145R) in the surface protein in 2004, 2013 and 2019 but none of the sequences from 2007 had these changes. In conclusion, HBV mutations may revert to wild type in natural infection. Clinicians should be wary of predicting long-term prognoses on the basis of the presence of mutations.

1007. Hypermutability in Clinical Strains of Klebsiella pneumoniae: Role of the V76G Mutation in MutH

Abstract Background Hypermutator (HM) bacteria exhibit high spontaneous mutation rates due to DNA mismatch repair (MMR) gene mutations, which may facilitate antibiotic resistance. HM is best described for chronic infections or colonization, in particular with P. aeruginosa. HM K. pneumoniae (KP) and carbapenem resistant Enterobacterales (CRE) are rarely studied. Methods Longitudinal isolates from 5 patients (pts) with long-term ST258 CRKP infections (median: 1.4 yr, 0.5-4.1 yr) underwent Illumina HiSeq whole genome sequencing. Strains from 1 pt were tested for HM and resistance. Mutant strains were created by complementation and CRISPR. Results In each pt, initial and recurrent isolates were genetically related (≤7 core genome (cg) SNP). In 1 pt, infection recurred 3.3 yrs after initial infection; baseline and recurrent (T0) isolates differed by 7-10 cgSNP. 4 ceftazidime-avibactam (CAZ) resistant isolates (T1-T4) were recovered ≥2 wks after treatment of T0 infection. Strains T1-T4 differed from T0 and each other by 109-214 and 58-137 cgSNP (Fig.1), respectively, and carried mutations in MMR genes (mutS -149∆C (∆pmutS); mutH V76G) and blaKPC3 (D179Y). T1-T4 mutations were enriched for genes involved in metabolism (adjusted p=1.46e-10), ABC transport (p=4.1e-7), 2-component systems (p=9.2e-5), signal transduction (p=6e-4), and transcription regulation (p=2.1e-4). mutS and mutH expression was 46-49% lower in T1-T4 than in T0. T1-T4 demonstrated rifampin mutational frequency >10-6.4, compared to < 10-7.3 for earlier strains. Upon passage in meropenem-vaborbactam (MV), colistin and gentamicin, T1-T4 developed resistance faster and higher MICs than T0 (Fig 2). MV resistance was associated with IS5 ompk36 promoter insertions or point, deletion or STOP mutations in ompK36 coding region. Complementation of T1-T4 with wild-type (WT) mutH restored phenotypes. Introduction of V76G to WT mutH in T0 established HM and in vitro passage resistance phenotypes. SNP matrix of 11 clinical isolates from a single patient with recurrent KPC-Kp infections The first 6 isolates were recovered within 6 months of transplant (Tinitial). The later 5 isolates were recovered ~40 months after intial GI colonization. Number of SNPs for each pariwise comparision on isolates are shown. Gray highlighted boxes shown SNP defferences between the 5 later strains. Serial passages of 4 clinical isolates. T1 and T4 harbored ΔpmutS. Ti=Tinitial (baseline) isolates Conclusion MMR mutations emerged in longitudinal CRKP, which conferred HM phenotypes and were associated with CAZ and other anti-CRE antibiotic resistance. mutH V76 is crucial in MMR. Long-term colonization or recurrent infections in face of antibiotic exposure might predispose CRKP strains to HM. Disclosures Cornelius J. Clancy, MD, Merck (Grant/Research Support)

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

Background & AimsSingle immunoglobulin interleukin-1–related receptor (SIGIRR) is a major inhibitor of Toll-like receptor signaling. Our laboratory identified a novel SIGIRR stop mutation (p.Y168X) in an infant who died of severe necrotizing enterocolitis (NEC). Herein, we investigated the mechanisms by which SIGIRR mutations induce Toll-like receptor hyper-responsiveness in the neonatal gut, disrupting postnatal intestinal adaptation.MethodsClustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate transgenic mice encoding the SIGIRR p.Y168X mutation. Ileal lysates, mouse intestinal epithelial cell (IEC) lysates, and intestinal sections were used to assess inflammation, signal transducer and activator of transcription 3 (STAT3) phosphorylation, microRNA (miRNA), and interleukin-1–related–associated kinase 1 (IRAK1) expression. Western blot, quantitative reverse-transcription polymerase chain reaction(qRT-PCR), and luciferase assays were performed to investigate SIGIRR–STAT3 signaling in human intestinal epithelial cells (HIEC) expressing wild-type or SIGIRR (p.Y168X) plasmids.ResultsSigirrTg mice showed increased intestinal inflammation and nuclear factor-κB activation concomitant with decreased IEC expression of miR-146a and miR-155. Mechanistic studies in HIECs showed that although SIGIRR induced STAT3-mediated expression of miR-146a and miR-155, the p.Y168X mutation disrupted SIGIRR-mediated STAT3-dependent miRNA expression. Chromatin immunoprecipitation and luciferase assays showed that SIGIRR activation of STAT3-induced miRNA expression is dependent on IRAK1. Both in HIECs and in the mouse intestine, decreased expression of miR-146a observed with the p.Y168X mutation increased expression of IRAK1, a protein whose down-regulation is important for postnatal gut adaptation.ConclusionsOur results uncover a novel pathway (SIGIRR–STAT3–miRNA–IRAK1 repression) by which SIGIRR regulates postnatal intestine adaptation, which is disrupted by a SIGIRR mutation identified in human NEC. These data provide new insights into how human genetic mutations in SIGIRR identified in NEC result in loss of postnatal intestinal immune tolerance.

Validation of a QTL for Grain Size and Weight Using an Introgression Line from a Cross between Oryza sativa and Oryza minuta

BackgroundGrain size and weight are important target traits determining grain yield and quality in rice. Wild rice species possess substantial elite genes that can be served as an important resource for genetic improvement of rice. In this study, we identify and validate a novel QTL on chromosome 7 affecting the grain size and weight using introgression lines from cross of Oryza sativa and Oryza minuta.ResultsAn introgression line ‘IL188’ has been achieved from a wild species Oryza minuta (2n = 48, BBCC, W303) into O. sativa japonica Nipponbare. The F2 and F2:3 populations derived from a cross between IL188 and Nipponbare were used to map QTLs for five grain size traits, including grain length (GL), grain width (GW), grain length to width ratio (LWR), grain thickness (GT) and thousand grain weight (TGW). A total of 12 QTLs for the five grain traits were identified on chromosomes 1, 2, 3, 6, 7, and 8. The QTL-qGL7 controlling GL on chromosome 7 was detected stably in the F2 and F2:3 populations, and explained 15.09–16.30% of the phenotypic variance. To validate the effect of qGL7, eight residual heterozygous line (RHL) populations were developed through selfing four F2:3 and four F2:4 plants with different heterozygous segments for the target region. By further developing SSR and Indel markers in the target interval, qGL7 was delimited to a ~ 261 kb region between Indel marker Y7–12 and SSR marker Y7–38, which also showed significant effects on grain width and thousand grain weight. Comparing with the reference genome of Nipponbare, stop or frameshift mutations in the exon of the three putative genes LOC_Os07g36830, LOC_Os07g36900 and LOC_Os07g36910 encoding F-box domain-containing proteins may be the candidate genes for qGL7. Scanning electron microscopy analysis of the glume’s epidermal cells showed that the cell length and width of NIL-qGL7IL188 was higher than NIL-qGL7Nip, indicating that qGL7 increases grain size and weight by regulating cell expansion.ConclusionsIn this study, we detected 12 QTLs regulating grain size and weight using an introgression line from a cross between Oryza sativa and Oryza minuta. Of these loci, we confirmed and delimited the qGL7 to a ~ 261 kb region. Three putative genes, LOC_Os07g36830, LOC_Os07g36900 and LOC_Os07g36910 encoding F-box domain-containing proteins may be the candidate genes for qGL7. These results provide a basis for map-based cloning of the qGL7 gene and useful information for marker assisted selection in rice grain quality improvement.

Rare mutations in ATL3, SPTLC2 and SCN9A explaining hereditary sensory neuropathy and congenital insensitivity to pain in a Brazilian cohort

Hereditary sensory neuropathies (HSN) are a group of rare neurological disorders with heterogeneous clinical and genetic characteristics. Although at least 17 different genes have already been associated with HSN, the epidemiology of the disorder in Brazil is still unknown. Performing whole genome sequencing (WGS) in 23 unrelated Brazilian families diagnosed with HSN, we detected pathogenic variants in ATL3, SPTLC2, and SCN9A in 12 patients belonging to five unrelated families. Clinical features associated with heterozygous mutations in ATL3 (c.575A > G; p.(Tyr192Cys)) and SPTLC2 (c.529A > G; p.(Asn177Asp)) were sensory deficits, neuropathic pain, and recurrent ulcerations. Presenting as congenital insensitivity to pain, three unrelated probands carried biallelic loss-of-function mutations in SCN9A. The so far undescribed stop mutation c.2106G > A (p.(Trp702Ter)) and the likewise novel splicing variant c.3319-1G > A were found in compound-heterozygosity with, respectively, the known pathogenic variants c.2908G > T (p.Trp970Ter) and c.2690G > A (p.Glu897Ter). In total, we identified pathogenic mutations in 21.7% of our families, which suggests that most of the cases could be explained by yet to be discovered genes or unusual alleles. Our study represents the first mutational screen in a Brazilian HSN cohort, enabling additional insights for genotype-phenotype correlations, reducing misdiagnoses, and providing early treatment considerations.

Sycp1 Is Not Required for Subtelomeric DNA Double-Strand Breaks but Is Required for Homologous Alignment in Zebrafish Spermatocytes.

In meiotic prophase I, homologous chromosomes are bound together by the synaptonemal complex, in which two axial elements are connected by transverse filaments and central element proteins. In human and zebrafish spermatocytes, homologous recombination and assembly of the synaptonemal complex initiate predominantly near telomeres. In mice, synapsis is not required for meiotic double-strand breaks (DSBs) and homolog alignment but is required for DSB repair; however, the interplay of these meiotic events in the context of peritelomeric bias remains unclear. In this study, we identified a premature stop mutation in the zebrafish gene encoding the transverse filament protein Sycp1. In sycp1 mutant zebrafish spermatocytes, axial elements were formed and paired at chromosome ends between homologs during early to mid-zygonema. However, they did not synapse, and their associations were mostly lost in late zygotene- or pachytene-like stages. In sycp1 mutant spermatocytes, γH2AX signals were observed, and Dmc1/Rad51 and RPA signals appeared predominantly near telomeres, resembling wild-type phenotypes. We observed persistent localization of Hormad1 along the axis in sycp1 mutant spermatocytes, while the majority of Iho1 signals appeared and disappeared with kinetics similar to those in wild-type spermatocytes. Notably, persistent Iho1 foci were observed in spo11 mutant spermatocytes, suggesting that Iho1 dissociation from axes occurs in a DSB-dependent manner. Our results demonstrated that Sycp1 is not required for peritelomeric DSB formation but is necessary for complete pairing of homologs in zebrafish meiosis.

Investigation of Staphylococcus aureus positive for Staphylococcal enterotoxin S and T genes.

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.

High frequency of biotinidase deficiency in Italian population identified by newborn screening

The biotinidase (BTD) enzyme is essential for recycling biotin, a water-soluble B-complex vitamin that is the coenzyme of four carboxylases involved in fatty acid synthesis, amino acid catabolism and gluconeogenesis. If untreated, total or partial BTD deficiencies lead to an autosomal recessive inherited organic aciduria whose clinical features, mainly presenting in the first years of life, include, seizures, skin rash, and alopecia. Based on residual BTD enzyme activity it is possible to identify partial or total biotinidase deficiency. The incidence of profound and partial biotinidase deficiency worldwide is estimated to be about 1 in 60.000.We report twelve years of experience in the newborn screening of biotinidase deficiency on 466.182 neonates. When a positive screening result occurred, a clinical evaluation was made of the patient and genetic counselling was offered to the family. Molecular analysis the BTD gene was carried out in all recalled neonates.Newborn screening lead to the identification of 75 BTD deficiencies with an incidence of about 1:6.300 births, ten times higher than the reported worldwide incidence. BTD deficiency was confirmed at a genomic level in all patients, demonstrating a high frequency of the p.(Asp444His) amino acid substitution and the complex allele p.(Ala171Thr)/p.(Asp444His) in the analyzed Italian newborns. Four new mutations (two small deletions, one stop mutation and one missense mutation) and a new combined allelic alteration were identified.Our data suggests that there is a high incidence of the biotinidase defect in the Italian population, most likely due to the high frequency of certain mutations.

Case report: Pitt-Hopkins like syndrome with CNTNAP2 mutation

BackgroundPitt-Hopkins syndrome (PHS) is a rare cause of severe intellectual disability, seizures, language impairment, and peculiar facial dysmorphism. It is caused by a mutation in transcription factor 4 (TCF4). Through molecular karyotyping and mutational analysis, a study identified recessive defects in two genes, contactin associated protein like 2 (CNTNAP2) and Neurexin I (NRXN1), in patients with similar presentations of Pitt-Hopkins syndrome and called Pitt-Hopkins-like syndrome (Zweier et al., J Med Genet 80: 994-1001, 2007).We present the first case report of a child in Iraq with Pitt-Hopkins-like syndrome that was referred to the Welfare Children’s Hospital/Medical City of Baghdad because of her intellectual disability.Case presentationThe patient was 4-year-old female child who presented with psychomotor delay and language impairment. She had frequent attacks of the respiratory tract and eye infections. Ophthalmologic examination revealed left-sided esotropia and severe myopia. Routine hematologic, serologic, and chemistry tests were within normal ranges. EEG revealed diffuse theta slowing and diffuse beta activity. The audiological test was normal. NCS and EMG showed normal results. Echo study, chest X-ray, and abdominal/pelvic ultrasound revealed normal findings. Brain MRI showed mild bilateral frontal-temporal atrophy. Whole-exome sequencing (WES) revealed a homozygous stop mutation in CNTNAP2 with a heterozygous state in both parents.ConclusionIntellectual disability may result from different types of abnormal cellular processes and with widening the use of molecular gene analysis in cases of intellectual disability, underdiagnosed cases of Pitt-Hopkins and Pitt-Hopkins-like syndromes may be uncovered.

Cloning of wheat keto-acyl thiolase 2B reveals a role of jasmonic acid in grain weight determination

Grain weight (GW) is one of the component traits of wheat yield. Existing reports have shown that multiple phytohormones are involved in the regulation of GW in different crops. However, the potential role of jasmonic acid (JA) remains unclear. Here, we report that triticale grain weight 1 (tgw1) mutant, with marked reductions in both GW and JA content, is caused by a premature stop mutation in keto-acyl thiolase 2B (KAT-2B) involved in β-oxidation during JA synthesis. KAT-2B overexpression increases GW in wild type and boosts yield. Additionally, KAT-2B compliments the grain defect in tgw1 and rescues the lethal phenotype of the Arabidopsis kat2 mutant in a sucrose-free medium. Despite the suppression of JA synthesis in tgw1 mutant, ABA synthesis is upregulated, which is accompanied by enhanced expression of SAG3 and reduction of chlorophyll content in leaves. Together, these results demonstrate a role of the JA synthetic gene KAT-2B in controlling GW and its potential application value for wheat improvement.

Recurrent Crebbp Mutations in Follicular Lymphoma Appear Localized to the Committed B-Cell Lineage

Background: Follicular lymphoma (FL) is genetically characterized by translocations involving the BCL2 locus on chromosome 18q21. However, up to 70% of healthy individuals also carry detectable t(14;18)-positive cells, suggesting BCL2 translocation is critical but not sufficient for FL development. Chromatin modifying genes (CMGs) including KMT2D, CREBBP, EZH2, and EP300 are almost ubiquitously mutated in FL. We previously reported the direct characterization by ultra-deep sequencing of pre-diagnostic blood and tissue specimens from 19 subjects who ultimately developed FL. CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, detected in blood a median of 7.5 years before diagnosis in patients developing FL (8/19, 42%) but not in healthy adults with or without detected BCL2 translocations (0/13, p=0.01 and 0/20, p<0.001, respectively) (Schroers-Martin et al, ASH Annual Meeting 2017). While the BCL2 translocation is thought to occur in pre-B-cell precursors in the bone marrow, the high prevalence of CMG mutations raises the possibility that, analogous to myeloid age-related clonal hematopoiesis, such early lesions could occur in hematopoietic stem cells (HSCs) rather than in the committed B-cell lineage. Methods: To address this question of mutational hierarchy, we studied blood, bone marrow aspirate, and lymph node samples from 6 patients with CREBBP or KMT2D mutations in their FL tumor. We analyzed flow-sorted purified hematopoietic cell populations by deep sequencing, including isotype-specific mature B-cell, mature T-cell, and precursor populations encompassing HSCs and common lymphoid progenitors (CLPs). Patients additionally underwent routine clinical sequencing of tumor biopsy and marrow specimens. Results: Bulk sequencing of a diagnostic bone marrow aspirate from patient FL002 revealed a CREBBP mutation concordant with FL tumor biopsy. To ascertain the population bearing this mutation, we sorted to high purity viable marrow aspirate cells (Fig. A). The CREBBP mutation was confirmed in the mature B-cell compartment at an AF of 40.3% but was not detected in other cell populations. To validate this finding, a similar sorting strategy was employed on viable bone marrow aspirate or peripheral blood samples from another 4 FL patients bearing CREBBP mutations (Fig B). In each case, CREBBP was absent from the CD34+ precursor population. In patient CIML004 a characteristic KMT2D stop mutation was likewise absent in precursors. An atypical case sheds additional light on the localization of early FL mutations. Patient LYM267 was diagnosed with Grade 1-2 FL bearing CREBBP and NRAS mutations. Eight years into a prolonged remission after chemoimmunotherapy, he developed cutaneous and gingival myeloid sarcoma without radiographic or histopathological evidence of FL recurrence. While the CREBBP mutation was not detected in myeloid sarcoma or bone marrow biopsies, concordant NRAS mutations and clonal VDJ rearrangements were seen in all 3 compartments (Fig. C). This unusual clonal lineage favors the occurrence of the CREBBP mutation later than the branch point between morphologically distinct lymphoid and myeloid tumors, likely in the committed B-cell lineage after pre-BCR rearrangements (Fig D). Conclusions: HSCs are believed to be the cell of origin in several lymphoid leukemias, and mouse models have demonstrated lymphoma development with induced CREBBP lesions in HSCs (Horton et al Nat Cell Bio 2017). However, in sorted hematopoietic cell populations from marrow and peripheral blood, we observed CREBBP mutations in B-cell lineages but never in CD34+/CD20- precursor populations or paired lymphoid/myeloid disease. Our data therefore are not in support of HSCs as a precursor reservoir in FL. Given that cells harboring the t(14;18) translocation in healthy individuals appear derived from the germinal center, recurrent mutations in CREBBP are likely to occur after the pre-B-cell stage. Figure Disclosures Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Khodadoust:Kyowa Kirin: Consultancy; Seattle Genetics: Consultancy. Nadel:Innate Pharma: Research Funding; Institut Roche: Research Funding. Diehn:RefleXion: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; BioNTech: Consultancy; AstraZeneca: Consultancy. Roulland:Celgene/BMS: Research Funding; Roche: Honoraria. Alizadeh:Pharmacyclics: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Gilead: Consultancy; Pfizer: Research Funding; Roche: Consultancy.