Beneficial base substitutions in Escherichia coli fucO gene for enhancement of glycolic acid production.

Microbial production of glycolic acid (GA) from ethylene glycol is extensively used in a variety of industries because ethylene glycol is not only an inexpensive raw material but also the main component of industrial wastes. In this study, we produced GA from ethylene glycol using Escherichia coli overexpressing the endogenous 1,2-propanediol oxidoreductase (fucO) and lactaldehyde dehydrogenase (aldA) genes. To increase GA productivity, we screened a random mutant library generated using an error-prone polymerase chain reaction of fucO and obtained FucO mutants MF2-9 and MF6-9 with enhanced GA production in Lysogeny Broth medium containing 800 mM ethylene glycol. MF2-9 contained three amino acid substitutions (D23E, E222K, and G363S) and two synonymous mutations (coding DNA [c.] 93G > A and c.1131T > C) in fucO. MF6-9 contained one amino acid substitution (L377H) in FucO. An amino acid substitution (L377H) and a single synonymous mutation (c.1131T > C) in fucO contributed to the enhancement in GA production. Notably, cell lysates from E. coli harboring a synonymous mutation (c.1131T > C) or amino acid substitution (L377H) in fucO showed that only AldA activity was 1.3-fold higher than that of the cell lysate from E. coli harboring the wild-type fucO. We confirmed that c.1131T > C and L377H mutations increased aldA expression in E. coli. Analysis of mRNA levels and simulation of mRNA stabilization indicated that base substitutions at positions c.1130T, which corresponds to L377H amino acid substitution, and c.1131T increased aldA expression due to partial destabilization of the mRNA. These findings will be useful for the large-scale microbial production of GA from industrial waste.

Novel PAX9 Mutations Causing Isolated Oligodontia.

Hypodontia, i.e., missing one or more teeth, is a relatively common human disease; however, oligodontia, i.e., missing six or more teeth, excluding the third molars, is a rare congenital disorder. Many genes have been shown to cause oligodontia in non-syndromic or syndromic conditions. In this study, we identified two novel PAX9 mutations in two non-syndromic oligodontia families. A mutational analysis identified a silent mutation (NM_006194.4: c.771G>A, p.(Gln257=)) in family 1 and a frameshift mutation caused by a single nucleotide duplication (c.637dup, p.(Asp213Glyfs*104)) in family 2. A minigene splicing assay revealed that the silent mutation resulted in aberrant pre-mRNA splicing instead of normal splicing. The altered splicing products are ones with an exon 4 deletion or using a cryptic 5' splicing site in exon 4. Mutational effects were further investigated using protein expression, luciferase activity assay and immunolocalization. We believe this study will not only expand the mutational spectrum of PAX9 mutations in oligodontia but also strengthen the diagnostic power related to the identified silent mutation.

Molecular Mechanisms and the Significance of Synonymous Mutations.

Synonymous mutations result from the degeneracy of the genetic code. Most amino acids are encoded by two or more codons, and mutations that change a codon to another synonymous codon do not change the amino acid in the gene product. Historically, such mutations have been considered silent because they were assumed to have no to very little impact. However, research in the last few decades has produced several examples where synonymous mutations play important roles. These include optimizing expression by enhancing translation initiation and accelerating or decelerating translation elongation via codon usage and mRNA secondary structures, stabilizing mRNA molecules and preventing their breakdown before translation, and faulty protein folding or increased degradation due to enhanced ubiquitination and suboptimal secretion of proteins into the appropriate cell compartments. Some consequences of synonymous mutations, such as mRNA stability, can lead to different outcomes in prokaryotes and eukaryotes. Despite these examples, the significance of synonymous mutations in evolution and in causing disease in comparison to nonsynonymous mutations that do change amino acid residues in proteins remains controversial. Whether the molecular mechanisms described by which synonymous mutations affect organisms can be generalized remains poorly understood and warrants future research in this area.

Identification of two novel HLA alleles, HLA-A*03:344:02 and -DQB1*04:02:24 in Russian individuals.

The novel HLA alleles HLA-A*03:344:02 and -DQB1*04:02:24 have synonymous mutations.

Identification of polymorphic loci in OSMR and GHR genes and analysis of their association with growth traits in sheep.

The aim of this study was to analyze the effect of OSMR and GHR genes polymorphisms on growth traits in sheep. The single nucleotide polymorphisms of sheep OSMR and GHR genes were identified by DNA sequencing technology. A total of two intronic mutations g.2443 T > C and g.170196 A > G were identified in OSMR and GHR, respectively. Correlation analysis was carried out between the obtained genotypes and the growth traits of sheep. The results showed that at the OSMR g.2443 T > C locus, the body weight, chest circumference and cannon circumference of the TT genotype sheep were significantly higher than those of the CC genotype sheep (p < .05). At the GHR g.170196 A > G locus, the body weight, body length, chest circumference and cannon circumference of the AA genotype sheep were significantly higher than those of the AG genotype and GG genotype sheep (p < .05). Moreover, the body weight of sheep of combination TTOSMR/AAGHR genotype was significantly higher than that of other combination genotypes (p < .05). Therefore, we believe that the polymorphic sites identified in the OSMR and GHR genes can be used as candidate molecular markers for breeding good traits in sheep.

Direct sequencing of ß-globin gene reveals a rare combination of two exonic and two intronic variants in a ß-thalassemia major patient: a case report.

BACKGROUND: Due to indels in the ß-globin gene, patients with ß-thalassemia major exhibit a range of severity, with genotype ß0ß0 > ß0ß+ > ß+ß+, according to the production level of the ß-globin chain. More than 300 mutations have been identified in the ß-globin gene. CASE PRESENTATION: In this case study, we report a compound heterozygous condition with a rare concoction of four different variants (CD 3(T > C), CD41/42 (-CTTT), IVS II-16 (G > C), and IVS II-666 (C > T) in a single ß-globin gene. A regular transfusion-dependent 4-year-old male patient from India was included in the study. Augmented direct sequencing of the ß-globin gene helped reveal the presence of an unusual combination of different variants in a single gene. This patient clinically presented as ß-thalassemia major and was genotypically considered as ß0ß+, although CD41/42(-CTTT) was the only causative/pathogenic mutation in the disease severity. CONCLUSION: Although CD41/42-(CTTT) is the only pathogenic variant among the four variants, the clinical complications of such a combination of variants (pathogenic and benign) is not well understood. Intronic mutations may have the ability to modify clinical characteristics. The variants must therefore be reclassified using additional mRNA splicing and expression-based studies. Additionally, these types of combinations may have significance in studying population migration around the world.

Translated Mutant DSPP mRNA Expression Level Impacts the Severity of Dentin Defects.

Hereditary dentin defects are conventionally classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). Mutations in the dentin sialophosphoprotein (DSPP) gene have been identified to cause DGI type II and III and DD type II; therefore, these are not three different conditions, but rather allelic disorders. In this study, we recruited three families with varying clinical phenotypes from DGI-III to DD-II and performed mutational analysis by candidate gene analysis or whole-exome sequencing. Three novel mutations including a silent mutation (NM_014208.3: c.52-2del, c.135+1G>C, and c.135G>A; p.(Gln45=)) were identified, all of which affected pre-mRNA splicing. Comparison of the splicing assay results revealed that the expression level of the DSPP exon 3 deletion transcript correlated with the severity of the dentin defects. This study did not only expand the mutational spectrum of DSPP gene, but also advanced our understanding of the molecular pathogenesis impacting the severity of hereditary dentin defects.

Evaluation of Suspected Autosomal Alport Syndrome Synonymous Variants.

Background: Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods: We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome (COL4A3 [c.693G>A (p.Val231=)] and COL4A4 [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]). Results: Both in vivo and in vitro splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing. Conclusions: This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.

Recognition of an HLA-DQB1*06:319 variant, HLA-DQB1*06:319:02, in an hematopoietic stem cell donor.

One nucleotide replacement in codon 27 of HLA-DQB1*06:319:01 results in a novel allele, HLA-DQB1*06:319:02.

Mutational cascade of SARS-CoV-2 leading to evolution and emergence of omicron variant.

BACKGROUND: Emergence of new variant of SARS-CoV-2, namely omicron, has posed a global concern because of its high rate of transmissibility and mutations in its genome. Researchers worldwide are trying to understand the evolution and emergence of such variants to understand the mutational cascade events. METHODS: We have considered all omicron genomes (n = 302 genomes) available till 2nd December 2021 in the public repository of GISAID along with representatives of variants of concern (VOC), i.e., alpha, beta, gamma, delta, and omicron; variant of interest (VOI) mu and lambda; and variant under monitoring (VUM). Whole genome-based phylogeny and mutational analysis were performed to understand the evolution of SARS CoV-2 leading to emergence of omicron variant. RESULTS: Whole genome-based phylogeny depicted two phylogroups (PG-I and PG-II) forming variant specific clades except for gamma and VUM GH. Mutational analysis detected 18,261 mutations in the omicron variant, majority of which were non-synonymous mutations in spike (A67, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H), followed by RNA dependent RNA polymerase (rdrp) (A1892T, I189V, P314L, K38R, T492I, V57V), ORF6 (M19M) and nucleocapsid protein (RG203KR). CONCLUSION: Delta and omicron have evolutionary diverged into distinct phylogroups and do not share a common ancestry. While, omicron shares common ancestry with VOI lambda and its evolution is mainly derived by the non-synonymous mutations.

Characterization of the novel HLA-DRB1*13:03:12 allele by two next-generation sequencing methods.

The novel HLA-DRB1*13:03:12 allele was characterized using two next-generation sequencing technologies.

Novel homozygous silent mutation of ITGB3 gene caused Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is a rare inherited disease characterized by mucocutaneous bleeding due to the abnormalities in quantity or quality of platelet membrane GP IIb (CD41) or GP IIIa (CD61). GP IIb and GP IIIa are encoded by the ITGA2B and ITGB3 genes, respectively. Herein, we described a 7-year-old Chinese boy of the consanguineous couple who was diagnosed with GT based on the typical clinical manifestations, absence of blood clot retraction and the reduced expression of CD41 and CD61 in platelets. A homozygous silent variant c.1431C > T (p. G477=) of the ITGB3 gene was identified by the Whole-exome sequencing and confirmed by Sanger sequencing. The variant was predicted to affect the splicing. RT-PCR and sequencing revealed that the variant caused a deletion of 95 base pairs and frameshift, and subsequently created a premature stop codon in exon 10 of ITGB3 (p. G477Afs*30). It was indicated that the variant c.1431C > T (p. G477=) of ITGB3 was the cause for Glanzmann thrombasthenia. Our findings expanded the mutation spectrum and provided the information for the genetic counseling, prenatal diagnosis and preimplantation genetic testing (PGT).

Pedigree analysis and prenatal diagnosis of intellectual disability caused by synonymous mutations of p.S434S in DLG3 gene / 中华围产医学杂志

Objective:To analyze the pathogenic gene and prenatal diagnosis of a family with intellectual disability.Methods:Out of this family consisting of 17 members in three generations, four males had intellectual disability. The proband's elder sister (Ⅱ-7) visited Henan Provincial People's Hospital in Oct 2019 for genetic counseling at 8 weeks of gestation. After informed consent was obtained, peripheral blood samples of the family members were collected. The whole exome sequencing was performed on the genome DNA of the proband (Ⅱ-9, male) and his parents to screen the candidate variants for phenotype co-segregated analysis by Sanger sequencing. The expression vectors were constructed by homologous recombination and the splicing experiments were performed in vitro. Reverse transcription polymerase chain reaction, Sanger sequencing, and TA clone sequencing were used to analyze the effect of candidate variants on splicing. After the pathogenic variant was determined the proband's elder sister underwent prenatal diagnosis (Ⅲ-7) using goldeneyeTM20A genotyping system and Sanger sequencing. Results:A hemizygous synonymous variant of c.1302G>A (p. S434S) in DLG3 gene was found in the proband by whole exome sequencing, which was carried by his mother (Ⅰ-1) and co-segregated with the phenotype in other family patients. In vitro splicing experiment showed that c.1302G>A variant led to abnormal splicing of 88.24% transcripts, which further resulted in the reading frame shift and protein function impairment. The mutation was not detected in the fetus (Ⅲ-7), who was born alive later and showed no abnormal mental or behavioral development at the age of one and a half year and is still being followed up. Conclusions:The synonymous mutation c.1302G>A in DLG3 gene was the etiopathogenesis of X-linked intellectual disability in this family.

The HLA-B*58:01:42 allele identified in a volunteer bone marrow donor.

HLA-B*58:01:42 differs from HLA-B*58:01:01:01 in codon 303 in exon 5.

Cross-sectional genomic perspective of epidemic waves of SARS-CoV-2: A pan India study.

BACKGROUND: COVID-19 has posed unforeseen circumstances and throttled major economies worldwide. India has witnessed two waves affecting around 31 million people representing 16% of the cases globally. To date, the epidemic waves have not been comprehensively investigated to understand pandemic progress in India. OBJECTIVE: Here, we aim for pan Indian cross-sectional evolutionary analysis since inception of SARS-CoV-2. METHODS: High quality genomes, along with their collection date till 26th July 2021, were downloaded. Whole genome-based phylogeny was obtained. Further, the mutational analysis was performed using SARS-CoV-2 first reported from Wuhan (NC_045512.2) as reference. RESULTS: Based on reported cases and mutation rates, we could divide the Indian epidemic into seven phases. The average mutation rate for the pre-first wave was <11, which elevated to 17 in the first wave and doubled in the second wave (∼34). In accordance with mutation rate, VOCs and VOIs started appearing in the first wave (1.5%), which dominated the second (∼96%) and post-second wave (100%). Nation-wide mutational analysis depicted >0.5 million mutation events with four major mutations in >19,300 genomes, including two mutations in coding (spike (D614G), and NSP 12b (P314L) of rdrp), one silent mutation (NSP3 F106F) and one extragenic mutation (5' UTR 241). CONCLUSION: Whole genome-based phylogeny could demarcate post-first wave isolates from previous ones by point of diversification leading to incidences of VOCs and VOIs in India. Such analysis is crucial in the timely management of pandemic.

Silent variant in F8:c.222G>T (p.Thr74Thr) causes a partial exon skipping in a patient with mild hemophilia A.

One of the challenges of genetic testing in patients with hemophilia A is the interpretation of sequence variants. Here we report a silent variant found in exon 2 in the F8 gene in a 47-year-old patient with a previous von Willebrand disease (VWD) type 1 diagnosis. Clinically he had mild bleeding symptoms restricted to prolonged bleeding from minor wounds. Sanger sequencing of F8 gene using genomic DNA showed a hemizygous silent variant in exon 2: c.222G>T, p.Thr74Thr. When applying ACMG criteria, the variant was predicted to be "likely benign" in the analyzing software or VUS after curating. Sanger sequencing of the patient's cDNA after nested polymerase chain reaction showed that the patient had both a normal transcript containing exons 1-4 and a defect transcript lacking exon 2. These findings explain the patient's low FVIII:C level and led to the diagnosis of mild hemophilia A instead of VWD type 1. This case illustrates that mRNA work-up may be needed to clarify a patient's phenotype-genotype.

Detecting Single Nucleotide Polymorphisms in MEF2B and UCP3 and Elucidating Their Association with Sheep Growth Traits.

Herein we detected single nucleotide polymorphisms in MEF2B and UCP3 by DNA sequencing and the KASPar technology and analyzed their association with sheep growth traits. Two synonymous mutations, g.1826 C > T and g.10266 G > C, were detected, respectively, and they were found to be significantly associated with sheep growth traits (p < 0.05). In case of MEF2B g.1826 C > T, the average body weight and chest and cannon circumference of sheep with the CC genotype were significantly higher than those of sheep with the CT and TT genotypes (p < 0.05). Moreover, in case of UCP3 g.10266 G > C, the average body weight and chest and cannon circumference of sheep with the GG genotype were significantly higher than those of sheep with the GC and CC genotypes (p < 0.05). Moreover, the average body weight of sheep with the CC/GG genotype was higher compared with those of other genotype combinations. We also assessed MEF2B and UCP3 expression in different sheep tissues, confirming their expression in all examined tissues. To summarize, we believe that the polymorphisms identified in MEF2B and UCP3 can serve as molecular markers for sheep growth traits.

A rare gene variation cap +1 (A>C) (HBB: c. -50A>C) associated with codon 5 (-CT) (HBB: c.17_18delCT) mutation in Syrian family.

BACKGROUND: CAP+1 [A>C] (HBB:c.-50A>C) is a rare silent ß-thalassemia (ß-thal) mutation. Carrier individuals of this mutation show borderline hemoglobin (Hb), mean corpuscular volume (MCV) and Hb A2 levels. This mutation was previously reported in combination with different ß-thalassemia mutations, leading to variable phenotypes. CASE PRESENTATION: Here, we describe for the first time the combination of silent CAP+1 [A>C] (HBB:c.-50A>C) mutation with ß0 codon 5 [-CT] (HBB:c.17_18delCT) mutation in a Syrian proband, leading to beta thalassemia intermedia (TI). CONCLUSIONS: The compound heterozygotes of the silent CAP+1 (A>C) together with another severe beta gene mutation, are phenotypically severe enough to present at an early age and require appropriate therapeutic modalities.

Facile method of curing toxicity in large viral genomes by high-throughput identification and removal of cryptic promoters.

Infectious plant virus clones are challenging to construct and manipulate due to the presence of cryptic promoter sequences that induce toxicity in bacteria. Common methods to overcome toxicity include intron insertion to interrupt toxic open reading frames and the use of Rhizobium or yeast species that do not recognize the same cryptic promoters. Unfortunately, intron insertion must be attempted on a trial and error basis within full-length clones and may change the infection characteristics of the virus. We have developed a facile method that can detect multiple cryptic bacterial promoters within large virus genomes. These promoters can then be silenced to obtain infectious clones that can be manipulated in E. coli. Our strategy relies on the generation of a viral library which is cloned upstream of either an eGFP open reading frame for low-throughput analysis or chloramphenicol for next generation sequencing. Pokeweed mosaic virus (PkMV), a 9.5 Kb ssRNA potyvirus, was used as a proof of concept. We found 16 putative promoter regions within 150-250 bp library fragments throughout the PkMV genome. 5'RACE allowed identification of the promoter sequence within each fragment, and subsequent silencing produced infectious clones. Our results indicate that cryptic promoters are ubiquitous within large viral genomes and that promoter screening is a desirable first step when constructing a viral clone. Our method can be applied to large plant and animal viruses as well as any DNA sequence for which low level of background transcriptional activity is required.

Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice.

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.