Deletion Of Gene Cluster Research Articles

The efficient synthesis strategy of bacillomycin D in Bacillus amyloliquefaciens fmbJ

The cyclic lipopeptide bacillomycin D exhibits potent antifungal activity against a wide range of fungi, particularly filamentous fungi, thereby exhibiting significant potential for application in food preservation. However, the limited yield obtained from wild strains hinders the practical application of bacillomycin D. In this study, we used different strategies to modify the original strain Bacillus amyloliquefaciens fmbJ, including promoter replacement, competitive synthetic gene knockout, and overexpression of the lipopeptide transporter gene. Subsequently, we analyzed changes in the production and expression of bacillomycin D genes in the engineered strain. The results demonstrated that PbacA exhibits the highest promoter activity, leading to a significant increase (2.05-fold) in bacillomycin D production compared with control group. Additionally, deletion of the epsA-O gene cluster also significantly enhances the synthesis of bacillomycin D. However, when amyloid gene cluster tasA-sipW-yqxM was deleted,bacillomycin D accumulation time was shortened, resulting in no significant increase in bacillomycin D production. In addition, lipopeptide transporters SwrC, KrsE and YcxA did not promote bacillomycin D production. Finally, by replacing the promoter PbacA, we successfully disrupted the epsA-O gene cluster, and bacillomycin D production increased by 2.55 times compared to the initial bacillomycin D strain. In conclusion, this study indicated that genetic engineering of regulatory genes was an effective strategy to improve the yields of bacillomycin D and provided promising strains for industrial production of bacillomycin D.

Exploration of Streptomyces fradiae J1-021 as a Potential Host for the Heterologous Production of Spinosad.

Spinosad is a potent insecticide produced by Saccharopolyspora spinosa. However, it harbors certain limitations of a low growing rate and unfeasible genetic manipulation that can be overcome by adopting a superior platform, such as Streptomyces. Herein, we exploited the industrial tylosin-producing Streptomyces fradiae J1-021 for the heterologous production of spinosad. An engineered strain (HW01) with deletion of the tylosin biosynthetic gene cluster (BGC) was constructed and then transformed with the natural spinosad BGC. The distribution and expression levels of the tylosin BGC operons were assessed to construct a natural promoter library. The rate-limiting steps of spinosad biosynthesis were identified by analyzing the transcriptional expression of the spinosad biosynthetic genes. The stepwise engineering work involved the overexpression of the biosynthetic genes participating in rate-limiting pathways using strong promoters, affording an increase in spinosad production to 112.4 μg/L. These results demonstrate that strain HW01 has the potential to be used as a chassis for the heterologous production of polyketides.

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

Modeling of large-scale hoxbb cluster deletions in zebrafish uncovers a role for segmentation pathways in atrioventricular boundary specification.

Hox genes orchestrate the segmental specification of the muscular circulatory system in invertebrates but it has not proven straightforward to decipher segmental parallels in the vertebrate heart. Recently, patients with HOXB gene cluster deletion were found to exhibit abnormalities including atrioventricular canal defects. Using CRISPR, we established a mutant with the orthologous hoxbb cluster deletion in zebrafish. The mutant exhibited heart failure and atrioventricular regurgitation at 5days. Analyzing the four genes in the hoxbb cluster, isolated deletion of hoxb1b-/- recapitulated the cardiac abnormalities, supporting hoxb1b as the causal gene. Both in situ and in vitro data indicated that hoxb1b regulates gata5 to inhibit hand2 expression and ultimately is required to pattern the vertebrate atrioventricular boundary. Together, these data reveal a role for segmental specification in vertebrate cardiac development and highlight the utility of CRISPR techniques for efficiently exploring the function of large structural genomic lesions.

Identification of a novel 91.5 kb-deletion (αα)FJ in the α-globin gene cluster using single-molecule real-time (SMRT) sequencing

Objectives To present a novel 91.5-kb deletion of the α-globin gene cluster (αα)FJ identified by genetic assay and prenatal diagnosis in a Chinese family. Subjects and Methods The proband was a 34-year-old G3P1 (Gravida 3, Para 1) female at the gestational age of 21+ weeks with a history of an edematous fetus. A routine genetic assay (reverse dot blot hybridization, RDB) was performed to detect common thalassemia mutations. Multiplex ligation-dependent probe amplification (MLPA) and single-molecule real-time technology (SMRT) were used to detect rare thalassemia mutations. Results The hematological phenotypes of the proband, her mother, elder sister, husband, daughter, and nephew were consistent with the phenotype of α-thalassemia trait. No mutations were found in these family members by RDB, except for the proband’s husband who carried an α-globin gene deletion --SEA/αα. MLPA results showed that the proband and other α-thalassemia-suspected relatives had heterozygous deletions around the POLR3K-3-463nt, HS40-178nt, and HBA-HS40-382nt probes. The 5′-breakpoint was out of probe scope and could not be determined. SMRT was performed and a 91.5-kb deletion (NC_000016.10: g.39268_130758del) in the α-globin gene cluster (αα)FJ was identified in the proband and other suspected relatives, which could explain their phenotypes. At the proband’s gestational age of 22+ weeks, an amniotic fluid sample was collected and analyzed. As only the 91.5-kb deletion (αα)FJ was identified in the fetus with RDB, MLPA, and SMRT. The proband was suggested to continue the pregnancy. Conclusion We first reported a 91.5-kb deletion (NC_000016.10: g.hg38-chr16:39268-_130758del) of the HS-40 region in the α-globin gene cluster (αα)FJ identified in a Chinese family. Since the HS-40 loss of heterozygosity in combination with the heterozygous deletion --SEA might result in Hb Bart’s hydrops fetalis, routine genetic assay, and SMRT were recommended to individuals at risk for prenatal diagnosis.

Regulating Cardiolipin Biosynthesis for Efficient Production of Colanic Acid in Escherichia coli.

Colanic acid has broad application prospects in the food and healthcare market due to its excellent physical properties and biological activities. In this study, we discovered that colonic acid production in Escherichia coli could be enhanced by regulating cardiolipin biosynthesis. Single deletion of clsA, clsB, or clsC related to cardiolipin biosynthesis in E. coli MG1655 only slightly increased colonic acid production, but double or triple deletion of these three genes in E. coli MG1655 increased colonic acid production up to 2.48-fold. Previously, we have discovered that truncating lipopolysaccharide by deletion of the waaLUZYROBSPGQ gene cluster and enhancing RcsA by deletion of genes lon and hns can increase colonic acid production in E. coli. Therefore, these genes together with clsA, clsB, or/and clsC were deleted in E. coli, and all the resulting mutants showed increased colonic acid production. The best colonic acid production was observed in the mutant WWM16, which is 126-fold higher than in the control MG1655. To further improve colonic acid production, the genes rcsA and rcsD1-466 were overexpressed in WWM16, and the resulting recombinant E. coli WWM16/pWADT could produce 44.9 g/L colonic acid, which is the highest titer reported to date.

Preparation of a Klebsiella pneumoniae conjugate nanovaccine using glycol-engineered Escherichia coli

BackgroundEngineered strains of Escherichia coli have been used to produce bioconjugate vaccines using Protein Glycan Coupling Technology (PGCT). Nanovaccines have also entered the vaccine development arena with advances in nanotechnology and have been significantly developed, but chassis cells for conjugate nanovaccines have not been reported.ResultsTo facilitate nanovaccine preparation, a generic recombinant protein (SpyCather4573) was used as the acceptor protein for O-linked glycosyltransferase PglL, and a glycol-engineered Escherichia coli strain with these two key components (SC4573 and PglL) integrated in its genome was developed in this study. The targeted glycoproteins with antigenic polysaccharides produced by our bacterial chassis can be spontaneously bound to proteinous nanocarriers with surface exposed SpyTag in vitro to form conjugate nanovaccines. To improve the yields of the targeted glycoprotein, a series of gene cluster deletion experiments was carried out, and the results showed that the deletion of the yfdGHI gene cluster increased the expression of glycoproteins. Using the updated system, to the best of our knowledge, we report for the first time the successful preparation of an effective Klebsiella pneumoniae O1 conjugate nanovaccine (KPO1-VLP), with antibody titers between 4 and 5 (Log10) after triple immunization and up to 100% protection against virulent strain challenge.ConclusionsOur results define a convenient and reliable framework for bacterial glycoprotein vaccine preparation that is flexible and versatile, and the genomic stability of the engineered chassis cells promises a wide range of applications for biosynthetic glycobiology research.

εγ-Thalassemia, a New Hemoglobinopathy Category.

Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for β-thalassemia. Notably, ϵγδβ-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψβ loci with intact LCR, δ-, and β-regions in 2 women and newborn twins. Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψβ (HBBP1) loci. This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδβ-thalassemia.

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

Metabolic engineering of Corynebacterium glutamicum for l-tyrosine production from glucose and xylose

Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for l-tyrosine (l-Tyr) overproduction was attempted by pushing the carbon flux more towards l-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased l-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase l-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest l-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce l-Tyr from lignocellulosic renewable carbon sources.

Association of DAZL polymorphisms and DAZ deletion with male infertility: a systematic review and meta-analysis.

Various populations have been investigated for the occurrence of two key DAZL polymorphisms, 260A > G (rs11710967) and 386A > G (rs121918346), as well as complete DAZ cluster deletion, with conflicting results. The purpose of the current meta-analysis was to investigate if there is an association between DAZL polymorphisms and complete deletion of the DAZ cluster gene with male infertility. Up until September 2022, a thorough search was conducted in the Pubmed and Google scholar databases. For 260A > G polymorphism, 8 studies with 2077 cases and 1398 controls, 13 studies for 386A > G polymorphism (4343 cases and 3727 controls) and 17 studies of DAZ deletion (2820 cases and 1589 controls) were included in the pooled analysis. All of the studies were statistically analysed by Review Manager 5.4, and publication bias was evaluated with JASP 0.16.2.0 software utilising funnel plots and Egger's linear regression test. The meta analysis result for pooled data indicated no association between 260A > G and 386A > G polymorphisms and male infertility in any of the genetic models or ethnicities. However, there was a definite correlation between complete deletion of the DAZ gene cluster and male infertility, with an OR = 13.23, 95% confidence interval (6.63-26.39), and p < 0.00001. In the stratified analysis by ethnicity, Caucasians and Asian ethnic groups showed the similar relationship. In order to arrive at more definitive conclusions, further study should be conducted, including studies from a larger range of nations and nationalities.

Attenuated Streptococcus agalactiae WC1535 ∆Sia perturbs the gut microbiota of Oreochromis niloticus, massively colonizes the intestine, and induces intestinal mucosal immunity after intraperitoneal inoculation.

We previously developed and assessed the effectiveness of the attenuated Streptococcus agalactiae (Group B Streptococcus, GBS) strain WC1535 ∆Sia (with neuA-D gene cluster deletion) vaccine in tilapia (Oreochromis niloticus). In this study, we characterized the bacterial communities of the tilapia intestines by 16S rRNA high-throughput sequencing and assessed the serum antibody response, expression of immune-related genes, and histological changes following formalin-killed GBS vaccine (FKV) and the live attenuated vaccine ∆Sia (LAV). Results showed that FKV and LAV induced robust systemic and intestinal mucosal immune responses in tilapia without causing obvious pathological changes in the hindgut, spleen, and head kidney but exerted different effects on intestinal bacterial communities. The richness or diversity of the intestinal bacterial community of FKV tilapia showed no significant changes compared with that of the control fish (p > 0.05) at either day 21 post-initial vaccination (21 dpiv) or day 35 (day 14 after the second immunization) (35 dpiv). The community composition of FKV tilapia and controls was significantly similar, although the relative abundance of some genera was significantly altered. Relative to control fish, the gut ecosystem of LAV tilapia was significantly disturbed with a substantial increase in community diversity at 21 dpiv (p < 0.05) and a significant decrease at 35 dpiv in fish with high serum antibody response (ΔSia35H) (p < 0.05). However, there was no significant difference between ΔSia35H and ΔSia35L (low serum antibody response) fish (p > 0.05). Moreover, the community composition of LAV tilapia at 21 dpiv or 35 dpiv was considerably different from that of the controls. Particularly, GBS ∆Sia was found to be abundant in the intestine at 21 and 35 dpiv. This result suggested that the parenteral administration of the LAV (∆Sia) may also have the effect of oral vaccination in addition to the immune effect of injection vaccination. In addition, a significant correlation was found between the expression of immune-related genes and certain bacterial species in the intestinal mucosal flora. Our findings will contribute to a better understanding of the effects of inactivated and attenuated vaccines on gut microbiota and their relationship with the immune response.

Detection of four rare thalassemia variants using Single-molecule realtime sequencing.

Conventional methods for the diagnosis of thalassemia include gap polymerase chain reaction (Gap-PCR), reverse membrane hybridization (RDB), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. In this study, we used single molecule real-time technology (SMRT) sequencing and discovered four rare variants that have not been identified by conventional diagnostic methods for thalassemia. We also performed genotype and phenotype analyses on family members of thalassemia patients. The SMRT technology detected five cases in which the proband had abnormal results by conventional diagnostic methods or inconsistencies between the genotype and phenotype. The variants included two cases of an α-globin gene cluster 27,311 bp deletion, --27.3/αα (hg38 chr16:158664-185974), one case of an HS-40 region 16,079 bp deletion (hg38 chr16:100600-116678), one case of a rearrangement of -α3.7α1α2 on one allele and one case of a ß-globin gene cluster HBG1-HBG2 4,924 bp deletion (hg38 chr11:5249345-5254268). This study clarified the hematological phenotypes of four rare variants and indicated the application value of SMRT in the diagnosis of rare α-globin and ß-globin gene cluster deletions, gene recombination and deletion breakpoints. The SMRT method is a comprehensive one-step technology for the genetic diagnosis of thalassemia and is particularly suitable for the diagnosis of thalassemia with rare deletions or genetic recombination.

Two large novel alpha-globin gene cluster deletions causing alpha(0)-thalassemia in two Chinese families

IntroductionMonosomy of terminal 16p13.3 is a relatively common subtelomeric abnormality, most affected individuals presented α-thalassemia, some also have mental retardation, developmental abnormalities and/or speech delay and facial dysmorphism, which is termed ATR-16 syndrome. Here, we reported two novel 16p13.3 deletions involving the α-globin gene cluster and multispecies conserved sequences (MCSs), causing only a phenotype of α-thalassemia. MethodsSamples were collected from members of the two families and were subjected to haematological and comprehensive genetic analysis. ResultsThe novel 108 Kb deletion in family A extends from the non-protein coding RNA gene (WASIR2) to the NPRL3 gene, removing MCS-R1 to R3. This deletion should arise de novo because it wasn’t detected in both parents. The novel 336 Kb deletion in family B should extend from telomere to ∼ chr16:336000, removing the entire α-globin gene cluster. Carriers of these two deletions presented with microcytosis and hypochromic red cells, in accordance with a phenotype of α0-thalassemia carrier. ConclusionOur study increases the mutation spectrum of α-thalassemia. MCSs deletion should be considered in clinical practice of thalassemia screening and diagnosis.

Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR-AsCpf1 system.

Bacillus subtilis is a useful chassis in the fields of synthetic biology and metabolic engineering for chemical production. Here, we constructed CRISPR-AsCpf1-based expression plasmids with the temperature-sensitive replicon for iterative genome editing in B. subtilis. This method allowed gene insertion and large genomic deletion with an editing efficiency of up 80%-100% and rapid plasmid curing to facilitate the iterative genome editing in B. subtilis 168. Using the customized CRISPR-AsCpf1 system, we successfully and efficiently implemented the related gene editing in B. subtilis 168 for hyaluronic acid (HA) biosynthesis, HA synthase gene (hasA) insertion, UDP-glucose-dehydrogenase gene (tuaD) insertion, and eps gene cluster (epsA-O) deletion. The heterologous production of HA was realized by the engineered strain with a yield of 1.39 g/L. These results support the finding that the CRISPR-AsCpf1 system is highly efficient in bacteria genome editing and provide valuable guidance and essential references for genome engineering in B. subtilis using the CRISPR-AsCpf1 system.

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis.

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic. The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment. The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

Nitrogen fixation by Paenibacillus polymyxa WLY78 is responsible for cucumber growth promotion

To study nitrogen contribution to cucumber derived from nitrogen fixation of Paenibacillus polymyxa WLY78. The nif gene cluster deletion mutant (ΔnifB-V) of Paenibacillus polymyxa WLY78 was constructed by a homologous recombination method. The effects of plant-growth promotion were investigated by greenhouse experiments. The nitrogen fixation contribution was estimated by 15N isotope dilution method (also being called the 15N natural abundance technique). Deletion of nif gene cluster of P. polymyxa WLY78 resulted in complete loss of nitrogenase activity. Greenhouse experiments showed that inoculation with P. polymyxa WLY78 could significantly enhance the lengths and dry weights of cucumber roots and shoots, but inoculation with ΔnifB-V mutant could not. 15N isotope dilution experiments showed that cucumber plants derive 25.93% nitrogen from nitrogen fixation performed by P. polymyxa WLY78, but the ΔnifB-V mutant nearly could not provide nitrogen for plant growth. This present study demonstrated that nitrogen fixation performed by P. polymyxa WLY78 contributes to plant growth.

Serum resistance factors in avian pathogenic Escherichia coli mediated by ETT2 gene cluster

ABSTRACT Serum resistance is a poorly understood but common trait of some systemic disease pathogenic strains of bacteria. In this study, we analysed the role Escherichia coli type III secretion system 2 (ETT2) of avian pathogenic Escherichia coli (APEC) in serum resistance by bacteria survival number in serum culture, mRNA Seq and Tandem Mass Tag™ (TMT™) detection, lipopolysaccharide (LPS) extraction, and biofilm formation detection. We found that the ETT2 gene cluster deletion strain (ΔETT2) is more resistant to the killing effect of serum than wild-type APEC40. The analysis of ΔETT2 compared to APEC40 in the transcriptomics and proteomics data showed that ETT2 has a negative effect in the ATP-binding cassette (ABC) translator system and quorum sensing system and a positive effect in purine metabolism. ETT2 may affect the LPS, biofilm, flagella, and fimbriae which may affect the serum resistance. These results could lead to effective strategies for managing the infection by APEC. The mRNA Seq data of this study are available in the Sequence Read Archive of the National Center for Biotechnology Information under the BioProject PRJNA757182, and proteomic raw data have been deposited under the accession number IPX0003420000 at iProX.

Genetic research and clinical analysis of β-globin gene cluster deletions in the Chinese population of Fujian province: A 14-year single-center experience.

BackgroundHeterozygotes of HPFH and δβ thalassemia are clinically asymptomatic or have mild hemoglobin (Hb) values. However, when both HPFH and δβ‐thalassemia are coinherited with heterozygous β‐thalassemia, patients may progress to a clinical phenotype of thalassemia intermedia or thalassemia major. The purpose of this study was to characterize the genotypes and analyze the phenotypes of these disorders in Fujian Province, to offer advice for genetic counseling and accurate prenatal diagnosis in this region. A total of 55 001 subjects were participated in thalassemia screening. 142 subjects with HbF levels ≥10%, before the blood transfusion, were selected for further investigation.MethodsMultiplex ligation‐dependent probe amplification (MLPA) and Gap‐PCR were used to screen for three β‐globin gene cluster deletions: Chinese Gγ(Aγδβ)0 thalassemia and Southeast Asia HPFH (SEA‐HPFH) deletion and 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357).ResultsA total of 142 patients with HbF (≥10%) were enrolled to characterize the molecular basis of β‐globin gene cluster deletions in our study; 22 cases 0.04% (22/55 001) were definitively diagnosed with β‐globin gene cluster deletions. Ten cases were heterozygous for the Chinese Gγ(Aγδβ)0‐thal mutations, 10 cases were heterozygous for SEA‐HPFH, and one case was compound heterozygous for SEA‐HPFH and the α‐thal mutation. The 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357) was detected in one case. Moreover, the hemoglobin A2 levels in patients who were heterozygous for Chinese Gγ(Aγδβ)0‐thal were statistically lower than in cases with SEA‐HPFH deletion(p < 0.05).ConclusionIn Fujian Province, the prevalence of common β‐globin gene cluster deletions was 0.04%. What's more, the most common β‐globin cluster deletions are the Chinese Gγ(Aγδβ)0 and SEA‐HPFH.

Large β Globin Gene Cluster Deletions and Implications on Transcription Factor Regulation in Hemoglobin Switching - Α Novel ε Gγ Αγ Deletion with Intact LCR

The εγδβ thalassemias (EGDBT) are infrequently occurring deletions of the β globin gene cluster on chromosome 11. EGDBTs can be an unsuspected cause of severe neonatal anemia that resolves during the first year of life into a normal Hb A2 thalassemic phenotype, presumed to be due to an imbalance of β-like chains during development. First described in 1972, they are categorized into two groups, both with loss of the five DNase I hypersensitivity sites upstream of the β gene (HBB) called the locus control region (LCR). In group 1 deletions, all or most of the β globin gene cluster including HBB is deleted; in group 2 deletions, removal of the LCR silences an intact HBB.Large deletions that do not involve LCR but remove the embryonic (HBE) and fetal genes (HBG2/HBG1) [LCR(ε Gγ Aγ) 0δβ] have not been reported. It is not known whether a deletion with intact LCR as well as δ and β genes would similarly manifest as neonatal anemia that resolves into a normocytic phenotype with normal hemoglobin fraction percentages after infancy.Four individuals with an LCR(ε Gγ Aγ) 0δβ deletion have been evaluated. These include monochorionic, diamniotic twin girls whose newborn screen (NBS) returned significant for Hb A percentage greater than Hb F for both twins. NBS was repeated two weeks later with the same result. The newborns were induced late pre-term (36 weeks and 6 days) after an uncomplicated pregnancy, including no features of twin to twin transfusion. Both infants had no anemia and bilirubin levels remained in the low to low-intermediate risk zones. Neither twin received transfusions or phototherapy during admission.At a follow up visit at 20 months of age a hemoglobin electrophoresis was performed which showed borderline Hb A2 and no Hb F in both twins (Table 1). Ferritin levels were low-normal in Twin A (14 µg/L) and low in Twin B (9 µg/L). Both infants were found to have a heterozygous large deletion affecting HBE, HBG2, HBG1, and HBBP1 loci by Multiplex Ligation-dependent Probe Amplification (MLPA), a method that uses multiple DNA probe pairs to determine the size of the DNA segment deleted. MLPA deletion/duplication testing on the α genes was negative. No β globin variants were identified by DNA sequencing. An evaluation was performed on the 25 year-old healthy mother and showed similar findings including the heterozygous large deletion. Mother and infants continue to be asymptomatic with normal red cell indices for age (Table 1).The fourth was an unrelated 40-year-old female with mild anemia screened by hemoglobin electrophoresis for obstetric evaluation who was found to have borderline Hb A2 (see table 1). MLPA showed a heterozygous deletion involving the same loci. Again, no β globin variants were identified by DNA sequencing.All four cases showed the same MLPA pattern except one non-specific probe commonly altered in common gamma gene conversion events. Long range sequencing performed on one case confirmed a single contiguous 32,599 bp deletion that matched the MLPA data (g.5262276-5294875). The detected deletion has not been reported in literature and differs from EGDBT mutations in that the LCR is intact. As the LCR controls expression of the entire gene cluster, this mutation is expected to display different phenotypic features than classic EGDBT. HBD and HBB are not deleted and therefore adequate transcription of Hb A2 and Hb A are expected with no imbalance of α/β chains after the neonatal period. It is postulated this finding explains the decreased Hb F levels in the NBS test result. The Hb A2 increase is likely a product of this deletion and does not indicate β-thalassemia.The heterozygous absence of HBE and HBG2/HBG1 may result in upregulation of HBD and HBB expression through loss of FKLF and FKLF-2 binding. In addition, HBB-EKLF binding would have less competition as the gamma promoter elements have been heterozygously deleted.In summary, in contrast to classic EGDBTs which cause transient severe neonatal anemia and resolve to normal Hb A2 thalassemic indices, this novel LCR(ε Gγ Aγ) 0δβ deletion was associated with normal CBC values with an absence of severe neonatal anemia, inverted Hb F/Hb A percentages at birth and borderline Hb A2 levels. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.