Search

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. [ABSTRACT FROM AUTHOR]

Cercospora leaf spot (CLS) disease caused by the ascomycete Cercospora beticola is the most widespread fungal leaf disease in sugar beet. Fungicides of two active ingredient classes, quinone-outside inhibitors (QoIs) and demethylation inhibitors (DMIs), were important tools for CLS control. Over the years, C. beticola has become resistant to QoIs and a sensitivity shift has been reported for DMIs. In this study, the mechanisms causing variation in DMI sensitivity in C. beticola isolates from Europe were analyzed. The CYP51 mutations I387M, Y464S, and L144F were detected in many isolates, and most isolates carried the L144F in combination with mutation I309T. Furthermore, single isolates with other mutation combinations have been found. Wildtype isolates were found in low frequency in all European countries. Isolates that contained L144F showed higher EC50 values than those without L144F. Ranges of EC50 values of different CYP51 haplotypes were overlapping, an indication that other resistance mechanisms are present. Mutation L144F is more frequently encoded by codon TTC (96%) than by TTT (4%), and the usage of codon TTC was correlated with increased EC50 values, this being more pronounced for difenoconazole than for mefentrifluconazole. In addition, it could be observed that the usage of codon GAG for E at amino acid position 170, instead of GAA, was more frequently found in isolates with a higher adaptation compared with haplotypes that did not contain L144F. Overall, GAA was present in 67% of all isolates and GAG in 33%, with an unequal distribution within the haplotypes. These data indicate that target site mutations, especially L144F haplotypes, influence DMI sensitivity and that in L144F haplotypes, L144F codon usage might be responsible for variations within L144F haplotypes. The codon usage for E170 may influence sensitivity and increase EC50 variation of wildtype isolates and isolates with "weak" mutations, but not in L144F haplotypes. [ABSTRACT FROM AUTHOR]

Synonymous variants, traditionally regarded as silent mutations due to their lack of impact on protein sequence, structure and function, have been the subject of increasing scrutiny. This commentary explores the emerging evidence challenging the notion of synonymous variants as functionally inert. Analysis of the activity of 70 synonymous variants in the HIV Tat transcription factor revealed that 50% of the variants exhibited significant deviations from wild-type activity. Our analysis supports previous work and raises important questions about the broader impact of non-silent synonymous variants in human genes. Considering the potential functional implications, the authors propose classifying such variants as "synonymous variants of uncertain silence" (sVUS), highlighting the need for cautious interpretation and further investigations in clinical and genetic testing settings.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Data embedding into DNA is a conventional encryption technology since it gives the system security and consumes less energy. This work proposes a model that uses the silent mutation method to apply data embedding into DNA within an encryption model. To produce the key, hash functions are applied first to the original image and its metadata followed by the Arnold Transform technique. The key is then sent to LLCS, which uses it to produce a series of coding rules, and transmitted to HCS, which uses it to generate three chaotic sequences. Five chaotic maps are then used to diffuse the output image of the Arnold Transform. DNA operations are applied to the final image using the chaotic HCS sequences followed by the coding rules sequence. After the image has been decoded and silently mutated into a gene sequence, the altered gene is then obtained. Seven evaluation measures are used on ten grayscale images incorporated with two DNA sequences to assess the proposed model's performance. Results indicate a positive improvement in performance as a result of the model's increased security. [ABSTRACT FROM AUTHOR]

Neural crest cell genes control the migration of neural crest cells to multiple parts of developing vertebrate embryos. A recent hypothesis posits that the "domestication syndrome" characteristic of domesticated animals is driven by selection for tameness acting on neural crest cell genes, particularly those affecting cell migration. This is posited to explain why this syndrome involves many disparate phenotypic effects. These effects can be connected to deficits in neural crest cell migration. This hypothesis predicts that patterns of selection on these neural crest cell genes will differ between domesticated species and related wild species. Specifically, it predicts higher levels of positive selection on these genes in domesticated species, relative to closely related wild species. Here we test this prediction in a comparative framework. We obtained DNA sequences from a public database (NCBI) for eleven key neural crest cell genes from a set of thirty domesticated vertebrates and matched close relatives that remain wild. We used the program Contrast-FEL in the software suite HyPhy to compare the number of sites under positive selection (as measured by non-synonymous to synonymous nucleotide substitution rates across codons) between these two types of taxa in a phylogenetic framework. We found that domesticated lineages showed a consistently higher level of positive selection on these key genes, relative to their closely related wild counterparts. In addition, we found support for relaxation of selection and purifying selection. We argue that this result is consistent with an important role for these genes in the domestication syndrome.

Loss-of-function mutations of the X-chromosome gene UPF3B cause male neurodevelopmental disorders (NDDs) via largely unknown mechanisms. We investigated initially by interrogating a novel synonymous UPF3B variant in a male with absent speech. In silico and functional studies using cell lines derived from this individual show altered UPF3B RNA splicing. The resulting mRNA species encodes a frame-shifted protein with a premature termination codon (PTC) predicted to elicit degradation via nonsense-mediated mRNA decay (NMD). UPF3B mRNA was reduced in the cell line, and no UPF3B protein was produced, confirming a loss-of-function allele. UPF3B is itself involved in the NMD mechanism which degrades both PTC-bearing mutant transcripts and also many physiological transcripts. RNAseq analysis showed that ~1.6% of mRNAs exhibited altered expression. These mRNA changes overlapped and correlated with those we identified in additional cell lines obtained from individuals harbouring other UPF3B mutations, permitting us to interrogate pathogenic mechanisms of UPF3B-associated NDDs. We identified 102 genes consistently deregulated across all UPF3B mutant cell lines. Of the 51 upregulated genes, 75% contained an NMD-targeting feature, thus identifying high-confidence direct NMD targets. Intriguingly, 22 of the dysregulated genes encoded known NDD genes, suggesting UPF3B-dependent NMD regulates gene networks critical for cognition and behaviour. Indeed, we show that 78.5% of all NDD genes encode a transcript predicted to be targeted by NMD. These data describe the first synonymous UPF3B mutation in a patient with prominent speech and language disabilities and identify plausible mechanisms of pathology downstream of UPF3B mutations involving the deregulation of NDD-gene networks.

Abstract Background Due to indels in the β-globin gene, patients with β-thalassemia major exhibit a range of severity, with genotype β0β0 > β0β+ > β+β+, according to the production level of the β-globin chain. More than 300 mutations have been identified in the β-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 β-globin gene. A regular transfusion-dependent 4-year-old male patient from India was included in the study. Augmented direct sequencing of the β-globin gene helped reveal the presence of an unusual combination of different variants in a single gene. This patient clinically presented as β-thalassemia major and was genotypically considered as β0β+, 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.

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.

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).

Background: Due to indels in the β-globin gene, patients with β-thalassemia major exhibit a range of severity, with genotype β0β0 > β0β+ > β+β+, according to the production level of the β-globin chain. More than 300 mutations have been identified in the β-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 β-globin gene. A regular transfusion-dependent 4-year-old male patient from India was included in the study. Augmented direct sequencing of the β-globin gene helped reveal the presence of an unusual combination of different variants in a single gene. This patient clinically presented as β-thalassemia major and was genotypically considered as β0β+, 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. [ABSTRACT FROM AUTHOR]

Although synonymous mutations are commonly assumed neutral or nearly so, recent years have seen reports of fitness effects of synonymous mutations detected under laboratory conditions. In a previous study, we used genome editing to construct thousands of yeast mutants each carrying a synonymous or nonsynonymous mutation in one of 21 genes, and discovered that most synonymous and most nonsynonymous mutations are deleterious. A concern was raised that this observation could be caused by the fitness effects of potential CRISPR/Cas9 off-target edits and/or secondary mutations, and an experiment that would be refractory to such effects was proposed. Using genome sequencing, we here show that no CRISPR/Cas9 off-target editing occurred, although some mutants did carry secondary mutations. Analysis of mutants with negligible effects from secondary mutations and new data collected from the proposed experiment confirms the original conclusion. These findings, along with other reports of fitness effects of synonymous mutations from both case and systematic studies, necessitate a paradigm shift from assuming (near) neutrality of synonymous mutations., Competing Interests: Conflict of Interest The authors have no competing interests to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase ( cat ) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR . In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat . Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications., Competing Interests: Competing interests statement:The authors declare no competing interest.

HLA-C*07:02:81 differs from HLA-C*07:02:01:01 by one nucleotide substitution at position 465 (C→A) in exon 3., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Oculocutaneous albinism type 2 (OCA2) is the second most frequent form of albinism and represents about 30% of OCA worldwide. As with all types of OCA, patients present with hypopigmentation of hair and skin, as well as severe visual abnormalities. We focused on a subgroup of 29 patients for whom genetic diagnosis was pending because at least one of their identified variants in or around exon 10 of OCA2 is of uncertain significance (VUS). By minigene assay, we investigated the effect of these VUS on exon 10 skipping and showed that not only intronic but also some synonymous variants can result in enhanced exon skipping. We further found that excessive skipping of exon 10 could be detected directly on blood samples of patients and of their one parent with the causal variant, avoiding invasive skin biopsies. Moreover, we show that variants, which result in lack of detectable OCA2 mRNA can be identified from blood samples as well, as shown for the most common OCA2 pathogenic missense variant c.1327G>A/p.(Val443Ile). In conclusion, blood cell RNA analysis allows testing the potential effect of any OCA2 VUS on transcription products. This should help to elucidate yet unsolved OCA2 patients and improve genetic counseling., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

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. [ABSTRACT FROM AUTHOR]

The novel HLA alleles HLA‐A*03:344:02 and ‐DQB1*04:02:24 have synonymous mutations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background von Hippel-Lindau (VHL) disease is a familial neoplasia syndrome that results from the germline mutation of VHL. Pathogenic VHL mutations include deletion, frameshift, nonsense and missense mutations. Synonymous mutations are expected to be phenotypically silent and their role in VHL disease remains poorly understood. Case presentation We report a Caucasian male with a family history of pheochromocytoma and the synonymous VHL mutation c.414A > G (p.Pro138Pro). At 47-years, MRI revealed pheochromocytoma in the left adrenal gland and hemangioblastomas in the spine and brain. Pheochromocytoma was treated by adrenalectomy. Radiotherapy, followed by craniotomy and resection were needed to reduce hemangioblastomas to residual lesions. Two of three of the proband’s children inherited the mutation and both presented with retinal hemangioblastomas without pheochromocytoma at age 7: one twin needed four laser treatments. Primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from the family. Mutant fibroblasts downregulated full-length VHL mRNA and protein, and upregulated the short VHL mRNA isoform (a result of exon 2 skipping in splicing) at the mRNA level but not at the protein level. Conclusions Our study shows that the synonymous VHL mutation c.414A > G can within 7 years induce pediatric retinal hemangioblastoma in absence of pheochromocytoma. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is also the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. The mutation c.414A > G translates to p.Pro138Pro, yet it is not functionally silent, because it causes aberrant splicing by skipping exon 2. The reduced but not completely abolished pVHL protein in a loss-of-heterozygosity genetic backdrop may underlie the etiology of VHL disease.

Abstract 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.

Insertion of silent mutations allowing for restriction site modification aids in the screening of successful mutants during site directed mutagenesis. Introducing a new restriction site requires the analysis of degenerate sequences within mutant primer. As the total number of degenerate codons increases, the analysis becomes increasingly laborious, time-consuming and prone to errors. Towards this, a software named as ‘REMP’ (for REstriction site in Mutant Primer) was developed and described here. From the input sequence, REMP instantaneously generates degenerate sequences having restriction sites that are 6–8 base pairs in length. The output sequences are arranged based on the number of bases changed compared to the input sequence. REMP software can be installed and run as a stand-alone program on different operating systems. Any user of REMP can edit the list of restriction sites to be considered by the software, without a need for writing a computer code or knowing a program language.

Background: Determining the impact of somatic mutations requires understanding the functional relationship of genes acquiring mutations; however, it is largely unknown how mutations in functionally related genes influence each other., Methods: We employed non-synonymous-to-synonymous or dNdS ratios to evaluate the evolutionary dependency (ED) of gene pairs, assuming a mutation in one gene of a gene pair can affect the evolutionary fitness of mutations in its partner genes as mutation context. We employed PanCancer- and tumor type-specific mutational profiles to infer the ED of gene pairs and evaluated their biological relevance with respect to gene dependency and drug sensitivity., Results: We propose that dNdS ratios of gene pairs and their derived cdNS (context-dependent dNdS) scores as measure of ED distinguishing gene pairs either as synergistic (SYN) or antagonistic (ANT). Mutation contexts can induce substantial changes in the evolutionary fitness of mutations in the paired genes, e.g., IDH1 and IDH2 mutation contexts lead to substantial increase and decrease of dNdS ratios of ATRX indels and IDH1 missense mutations corresponding to SYN and ANT relationship with positive and negative cdNS scores, respectively. The impact of gene silencing or knock-outs on cell viability (genetic dependencies) often depends on ED, suggesting that ED can guide the selection of candidates for synthetic lethality such as TCF7L2-KRAS mutations. Using cell line-based drug sensitivity data, the effects of targeted agents on cell lines are often associated with mutations of genes exhibiting ED with the target genes, informing drug sensitizing or resistant mutations for targeted inhibitors, e.g., PRSS1 and CTCF mutations as resistant mutations to EGFR and BRAF inhibitors for lung adenocarcinomas and melanomas, respectively., Conclusions: We propose that the ED of gene pairs evaluated by dNdS ratios can advance our understanding of the functional relationship of genes with potential biological and clinical implications., (© 2024. The Author(s).)

Background: Global developmental delay with speech and behavioral abnormalities (OMIM: 619243) is an autosomal dominant disease caused by variants in TNRC6B gene., Method: We reviewed and summarized clinical manifestations and genotypes in patients previously reported with TNRC6B gene variants. We used several prediction tools to predict pathogenicity and performed minigene assays to verify the function of the synonymous variant affecting RNA splicing., Result: The patient presented with convulsive seizures and developmental delay. WES combined with functional studies diagnosed a child with a synonymous variant in TNRC6B gene. Through minigene assay and Sanger sequencing, we demonstrated that c.3141G > A variant induced exon 7 skipping and the synonymous variant was pathogenic., Conclusion: Synonymous variants do not change the amino acids encoded by the codon, so we usually consider synonymous variants to be benign and ignore their pathogenicity. Minigene assay is a valuable tool to identify the effect of variation on RNA splicing and identify synonymous variants' benign or pathogenic. We showed that the synonymous variant was pathogenic by minigene assay. WES combined with minigene assay establishes a robust basis for genetic counseling and diagnosing diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The Escherichia coli GroEL/ES chaperonin system facilitates protein folding in an ATP-driven manner. There are <100 obligate clients of this system in E. coli although GroEL can interact and assist the folding of a multitude of proteins in vitro. It has remained unclear, however, which features distinguish obligate clients from all the other proteins in an E. coli cell. To address this question, we established a system for selecting mutations in mouse dihydrofolate reductase (mDHFR), a GroEL interactor, that diminish its dependence on GroEL for folding. Strikingly, both synonymous and non-synonymous codon substitutions were found to reduce mDHFR's dependence on GroEL. The non-synonymous substitutions increase the rate of spontaneous folding whereas computational analysis indicates that the synonymous substitutions appear to affect translation rates at specific sites., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Background: Synonymous variants are non-pathogenic due to non-substitution of amino acids. However, synonymous exonic terminal nucleotide substitutions may affect splicing. Splicing variants are easily analyzed at RNA level for genes expressed in blood cells. Minigene analysis provides another method for splicing variant analysis of genes that are poorly or not expressed in peripheral blood., Methods: Whole exome sequencing was performed to screen for potential pathogenic mutations in the proband, which were validated within the family by Sanger sequencing. The pathogenicity of the synonymous mutation was analyzed using the minigene technology., Results: The proband harbored the compound heterogeneous variants c. [291G >A; 572-50C >T] and c.681 + 1G >T in F7, of which the synonymous variant c.291G >A was located at the terminal position of exon 3. Minigene analysis revealed exon3 skipping due to this mutation, which may have subsequently affected protein sequence, structure, and function., Conclusion: Our finding confirmed the pathogenicity of c.291G >A, thus extending the pathogenic mutation spectrum of F7, and providing insights for effective reproductive counseling., (© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Although translational selection to favour codons that match the most abundant tRNAs is not readily observed in humans, there is nonetheless selection in humans on synonymous mutations. We hypothesize that much of this synonymous site selection can be explained in terms of protection against unwanted RNAs - spurious transcripts, mis-spliced forms or RNAs derived from transposable elements or viruses. We propose not only that selection on synonymous sites functions to reduce the rate of creation of unwanted transcripts (for example, through selection on exonic splice enhancers and cryptic splice sites) but also that high-GC content (but low-CpG content), together with intron presence and position, is both particular to functional native mRNAs and used to recognize transcripts as native. In support of this hypothesis, transcription, nuclear export, liquid phase condensation and RNA degradation have all recently been shown to promote GC-rich transcripts and suppress AU/CpG-rich ones. With such 'traps' being set against AU/CpG-rich transcripts, the codon usage of native genes has, in turn, evolved to avoid such suppression. That parallel filters against AU/CpG-rich transcripts also affect the endosomal import of RNAs further supports the unwanted transcript hypothesis of synonymous site selection and explains the similar design rules that have enabled the successful use of transgenes and RNA vaccines., (© 2024. Springer Nature Limited.)

CDA 435C>T is reported as a functional SNP but there is no relevant research on the efficacy/hematological toxicity of gemcitabine-platinum treatment in Chinese non-small-cell lung cancer (NSCLC) patients. In this study, 63 patients who received radical resection (stage IB or IIIA) and 100 advanced NSCLC (stage IIIB or IV) patients have been collected, and all were treated with gemcitabine-platinum regimens from February of 2017 to February 2019. CDA 435C>T polymorphisms have been detected by PCR and direct sequencing. CT scan results and blood routine examinations have been collected to evaluate the efficacy and hematological toxicity. Then the relationships have been analyzed about CDA 435C>T. We found that T allele carriers have better therapy response (p<0.05). Patients carrying CDA 435C/T or T/T genotypes are statistically associated with a better efficacy (p<0.05) but are more prone to leukopenia (p<0.05). Although there is no difference in grade III-IV hematologic toxicity of 163 patients (p>0.05), in the case of 100 stage IIIB-IV patients, the CDA 435C/T and T/T have an increased risk (p<0.05). Regarding the CDA 435C>T polymorphism in the Chinese population, in patients with the mutant T allele, gemcitabine is more effective, but they are more prone to suffering from hematological toxicity, especially the late-stage patients. [ABSTRACT FROM AUTHOR]

Rosenberg AA, Marx A, Bronstein AM (Codon-specific Ramachandran plots show amino acid backbone conformation depends on identity of the translated codon. Nat Commun. 2022:13:2815) recently found a surprising correlation between synonymous codon usage and the dihedral bond angles of the resulting amino acid. However, their analysis did not account for the strongest known correlate of codon usage: gene expression. We re-examined the relationship between bond angles and codon usage by applying the approach of Rosenberg et al. to simulated protein-coding sequences that (i) have random codon usage, (ii) codon usage determined by mutation biases, and (iii) maintain the general relationship between codon usage and gene expression via the assumption of selection-mutation-drift equilibrium. We observed correlations between dihedral bond angle and codon usage when codon usage is entirely random, indicating possible conflation of noise with differences in bond angle distributions between synonymous codons. More relevant to the general analysis of codon usage patterns, we found surprisingly good agreement between the analysis of the real sequences and the analysis of sequences simulated assuming selection-mutation-drift equilibrium, with 91% of significant synonymous codon pairs detected in the former were also detected in the latter. We believe the correlation between codon usage and dihedral bond angles resulted from the variation in codon usage across genes due to the interplay between mutation bias, natural selection for translation efficiency, and gene expression, further underscoring these factors must be controlled for when looking for novel patterns related to codon usage., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Widespread adoption of DNA sequencing has resulted in large numbers of genetic variants, whose contribution to disease is not easily determined. Although many types of variation are known to disrupt cellular processes in predictable ways, for some categories of variants, the effects may not be directly detectable. A particular example is synonymous variants, that is, those single-nucleotide variants that create a codon substitution, such that the produced amino acid sequence is unaffected. Contrary to the original theory suggesting that synonymous variants are benign, there is a growing volume of research showing that, despite their "silent" mechanism of action, some synonymous variation may be deleterious. Here, we studied the extent of the negative selective pressure acting on different classes of synonymous variants by analyzing the relative enrichment of synonymous singleton variants in the human exomes provided by gnomAD. Using a modification of the mutability-adjusted proportion of singletons (MAPS) metric as a measure of purifying selection, we found that some classes of synonymous variants are subject to stronger negative selection than others. For instance, variants that reduce codon optimality undergo stronger selection than optimality-increasing variants. Besides, selection affects synonymous variants implicated in splice-site-loss or splice-site-gain events. To understand what drives this negative selection, we tested a number of predictors in the aim to explain the variability in the selection scores. Our findings provide insights into the effects of synonymous variants at the population level, highlighting the specifics of the role that these variants play in health and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The interleukin-7 receptor (IL-7R) is primarily expressed on lymphoid cells and plays a crucial role in the development, proliferation, and survival of T cells. Autosomal recessive mutations that disrupt IL-7Rα chain expression give rise to a severe combined immunodeficiency (SCID), which is characterized by lymphopenia and a T - B + NK + phenotype. The objective here was to diagnose two siblings displaying the T - B + NK + SCID phenotype as initial clinical genetic testing did not detect any variants in known SCID genes., Methods: Whole genome sequencing (WGS) was utilized to identify potential variants causing the SCID phenotype. Splicing prediction tools were employed to assess the deleterious impact of the mutation. Polymerase Chain Reaction (PCR), Sanger sequencing, flow cytometry, and ELISA were then used to validate the pathogenicity of the detected mutation., Results: We discovered a novel homozygous synonymous mutation in the IL7R gene. Our functional studies indicate that this variant is pathogenic, causing exon 6, which encodes the transmembrane domain, to be preferentially spliced out., Conclusion: In this study, we identified a novel rare synonymous mutation causing a loss of IL-7Rα expression at the cellular membrane. This case demonstrates the value of reanalyzing genetic data based on the clinical phenotype and highlights the significance of functional studies in determining the pathogenicity of genetic variants., (© 2024. The Author(s).)

Background: Sequence variation produced by mutation provides the ultimate source of natural selection for species adaptation. Unlike nonsynonymous mutation, synonymous mutations are generally considered to be selectively neutral but accumulating evidence suggests they also contribute to species adaptation by regulating the flow of genetic information and the development of functional traits. In this study, we analysed sequence characteristics of ATP6, a housekeeping gene from 139 Phytophthora infestans isolates, and compared the fitness components including metabolic rate, temperature sensitivity, aggressiveness, and fungicide tolerance among synonymous mutations., Results: We found that the housekeeping gene exhibited low genetic variation and was represented by two major synonymous mutants at similar frequency (0.496 and 0.468, respectively). The two synonymous mutants were generated by a single nucleotide substitution but differed significantly in fitness as well as temperature-mediated spatial distribution and expression. The synonymous mutant ending in AT was more common in cold regions and was more expressed at lower experimental temperature than the synonymous mutant ending in GC and vice versa., Conclusion: Our results are consistent with the argument that synonymous mutations can modulate the adaptive evolution of species including pathogens and have important implications for sustainable disease management, especially under climate change., (© 2024. The Author(s).)

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chih-Cheng Lai,1 Chi-Chung Chen,2,3 Ying-Chen Lu,3 Yin-Ching Chuang,2,4 Hung-Jen Tang5,6 1Department of Intensive Care Medicine, Chi Mei Medical Center, Liouying, Tainan, Taiwan; 2Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan; 3Department of Food Science, National Chiayi University, Chiayi, Taiwan; 4Department of Internal Medicine, Chi Mei Medical Center, Liouying, Tainan, Taiwan; 5Department of Health and Nutrition, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan; 6Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan Background: The aim of this study was to investigate the genotypic differences between different sequence type MRSA isolates, especially focusing on silent rpoB474 mutations and the relationship between such mutations and ciprofloxacin resistance. Methods: Seventy-nine MRSA isolates were obtained for antibiotic susceptibility tests and molecular study. Results: Among these isolates, we found that the MIC50, MIC90, and minimum inhibitory concentration (MIC) range of ciprofloxacin were much higher for the isolates without the rpoB474 mutation than for isolates with the rpoB474 mutation. A total of 87.5% of the isolates with the rpoB474 mutation were susceptible to ciprofloxacin, but none of the isolates without the rpoB474 mutation were susceptible to ciprofloxacin. For 27 MRSA isolates without rpo474 silent mutation but with gyrA86/126 silent mutation, all of them belonged to SCCmec III, and had high ciprofloxacin MIC levels. For another 44 MRSA isolates with rpo474 silent mutation but without gyrA86/126 silent mutation, all of them showed low ciprofloxacin MIC levels, all of them belonged to either SCCmec IV or V. Furthermore, MRSA ciprofloxacin resistance was found to be associated with the mutations gyrA S84L/parC S80F or gyrA S84L, and S85P/parC S80Y. Conclusion: Most occurrences of this rpoB474 silent mutation were found in community acquired-MRSA (CA-MRSA) isolates with susceptibility to most antibiotics, especially for ciprofloxacin and vice versa. Thus, this mutation may help to differentiate the different microbiologic characteristics of MRSA clinical isolates. Keywords: MRSA, silent mutation, rpoB474, ciprofloxacin, resistance

• Identification of a rare silent pathogenic mutation in the DMD gene. • cDNA investigation helpful in analyzing pathogenicity of silent mutations. • The p.Ser443 = mutation extends the genetic spectrum in Duchenne Muscle dystrophy. Duchenne Muscle dystrophy (DMD) is a X-linked inherited disease predominantly caused by severe mutations in DMD gene leading to absence of dystrophin protein. Here we report a 14-year-old Mongolian boy suffering from proximal muscle weakness, pseudohypertrophic deltoid and gastrocnemius muscles since early childhood. Lactate dehydrogenase (LDH) and creatine kinase (CK) levels were elevated. Mutation analysis including MLPA and sequencing of the DMD gene revealed a hemizygous silent variant, c.1329C>T (p.Ser443=) in exon 11. This silent mutation, listed in the SNP database (rs1060502631), was described as a variant of unknown significance (VUS) in ClinVar database. cDNA analysis demonstrated partial skipping of exon 11 due to this mutation. Although silent mutations are usually considered non-pathogenic, our case emphasizes that silent mutations can be potentially pathogenic. Hence, if silent variants are not annotated in database or not known to be benign, they should be analysed further at cDNA level. [ABSTRACT FROM AUTHOR]

Amelogenesis imperfecta (AI) is a collection of genetic disorders affecting the quality and/or quantity of tooth enamel. More than 20 genes are, so far, known to be responsible for this condition. In this study, we recruited 3 Turkish families with hypomaturation AI. Whole-exome sequence analyses identified disease-causing mutations in each proband, and these mutations cosegregated with the AI phenotype in all recruited members of each family. The AI-causing mutations in family 1 were a novel AMELX mutation [NM_182680.1:c.143T>C, p.(Leu48Ser)] in the proband and a novel homozygous MMP20 mutation [NM_004771.3:c.616G>A, p.(Asp206Asn)] in the mother of the proband. Previously reported compound heterozygous MMP20 mutations [NM_004771.3:c.103A>C, p.(Arg35=) and c.389C>T, p.(Thr130Ile)] caused the AI in family 2 and family 3. Minigene splicing analyses revealed that the AMELX missense mutation increased exonic definition of exon 4 and the MMP20 synonymous mutation decreased exonic definition of exon 1. These mutations would trigger an alteration of exon usage during RNA splicing, causing the enamel malformations. These results broaden our understanding of molecular genetic pathology of tooth enamel formation. [ABSTRACT FROM AUTHOR]

Background: von Hippel-Lindau (VHL) disease is a familial neoplasia syndrome that results from the germline mutation of VHL. Pathogenic VHL mutations include deletion, frameshift, nonsense and missense mutations. Synonymous mutations are expected to be phenotypically silent and their role in VHL disease remains poorly understood. Case presentation: We report a Caucasian male with a family history of pheochromocytoma and the synonymous VHL mutation c.414A > G (p.Pro138Pro). At 47-years, MRI revealed pheochromocytoma in the left adrenal gland and hemangioblastomas in the spine and brain. Pheochromocytoma was treated by adrenalectomy. Radiotherapy, followed by craniotomy and resection were needed to reduce hemangioblastomas to residual lesions. Two of three of the proband's children inherited the mutation and both presented with retinal hemangioblastomas without pheochromocytoma at age 7: one twin needed four laser treatments. Primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from the family. Mutant fibroblasts downregulated full-length VHL mRNA and protein, and upregulated the short VHL mRNA isoform (a result of exon 2 skipping in splicing) at the mRNA level but not at the protein level. Conclusions: Our study shows that the synonymous VHL mutation c.414A > G can within 7 years induce pediatric retinal hemangioblastoma in absence of pheochromocytoma. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is also the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. The mutation c.414A > G translates to p.Pro138Pro, yet it is not functionally silent, because it causes aberrant splicing by skipping exon 2. The reduced but not completely abolished pVHL protein in a loss-of-heterozygosity genetic backdrop may underlie the etiology of VHL disease. [ABSTRACT FROM AUTHOR]

X-linked hydrocephalus (XLH), a genetic disorder, has an incidence of 1/30,000 male births. The great proportion of XLH is ascribed to loss-of-function mutations of L1 cell adhesion molecule gene (L1CAM), but silent mutations in L1CAM with pathogenic potential were rare and were usually ignored especially in whole-exome sequencing (WES) detection. In the present study, we describe a novel silent L1CAM mutation in a Chinese pregnant woman reporting continuous five times pregnancies with fetal hydrocephalus. After fetal blood sampling, we found c.453G > T (p.Gly151 = ) in the L1CAM gene of the fetus by WES; RT-PCR of the messenger RNA (mRNA) from cord blood mononuclear cells and subsequent sequence analysis identified the mutation created a potential 5′ splice site consensus sequence, which would result in an in-frame deletion of 72 bp from exon 5 and 24 amino acids of the L1CAM protein. Heterozygous mutations were confirmed in analyzing DNA and mRNA from peripheral blood mononuclear cells of the woman, and a severe L1 syndrome was confirmed by fetal ultrasound scan and MRI. Our study first indicated c.453G > T (p.Gly151 = ) in L1CAM could be disease causing for hydrocephalus, which would aid in genetic counseling for the prenatal diagnosis of hydrocephalus. Meanwhile, it suggested some silent mutations detected in WES should not be ignored; splicing predictions of these mutations were necessary.

Accurate identification of driver mutations is crucial in genetic studies of human cancers. While numerous cancer driver missense mutations have been identified, research into potential cancer drivers for synonymous mutations has shown limited success to date. Here, we developed a novel machine learning framework, epSMic, for predicting cancer driver synonymous mutations. epSMic employs an iterative feature representation scheme that facilitates the learning of discriminative features from various sequential models in a supervised iterative mode. We constructed the benchmark datasets and encoded the embedding sequence, physicochemical property, and basic information such as conservation and splicing feature. The evaluation results on benchmark test datasets demonstrate that epSMic outperforms existing methods, making it a valuable tool for researchers in identifying functional synonymous mutations in cancer. We hope epSMic can enable researchers to concentrate on synonymous mutations that have a functional impact on cancer.

Synonymous mutations were considered to lack functional roles in human diseases; however, distinguishing deleterious synonymous mutations from benign ones is still a challenge. In this article, we identified a deleterious synonymous mutation β-codon 16 (C>T). HBB: c.51C>T, in compound heterozygous form with known β-thalassaemia mutation patients who clinically presented as non-transfusion-dependent thalassaemia (NTDT). A total of 9 families with 11 compound heterozygous index cases were reported. In the heterozygous state, codon 16 (C>T) mutation results in borderline HbA 2 (3.18 ± 0.36%) and slightly reduced RBC indices (RBCs: 4.73 ± 0.75 × 10 6 /μL, Hb: 12.26 ± 2.60 g/dL, MCV: 79.48 ± 8.40 fL, MCH: 25.95 ± 4.15 pg). The compound heterozygous patients showed elevated HbA 2 (5.98 ± 1.17%) and HbF (12.75 ± 7.51%) and presented clinically as NTDT with a mean Hb of 6.95 ± 1.29 g/dL. Many of them were dependent on few transfusions and had mild splenomegaly. Of the 11 patients, 5 (45.4%) were treated with hydroxyurea. This study highlights the clinical significance of synonymous mutation, when inherited with other β-thalassaemia mutations leading to the phenotype of NTDT. Thus, the study would help to improve screening protocols for β-thalassaemia carriers, which will ultimately improve the prevention programme., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

The genetic underpinnings of beta-thalassaemia encompass a myriad of molecular mechanisms. The ability of synonymous mutations, an often-overlooked category of variants, to influence β-globin expression and phenotypic disease is highlighted by this report by Gorivale et al. Commentary on: Gorivale et al. When a synonymous mutation breaks the silence in a thalassaemia patient. Br J Haematol 2024;204:677-682., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Synonymous mutations, which change the DNA sequence but not the encoded protein sequence, can affect protein structure and function, mRNA maturation, and mRNA half-lives. The possibility that synonymous mutations might be enriched in cancer has been explored in several recent studies. However, none of these studies control for all three types of mutational heterogeneity (patient, histology, and gene) that are known to affect the accurate identification of non-synonymous cancer-associated genes. Our goal is to adopt the current standard for non-synonymous mutations in an investigation of synonymous mutations., Results: Here, we create an algorithm, MutSigCVsyn, an adaptation of MutSigCV, to identify cancer-associated genes that are enriched for synonymous mutations based on a non-coding background model that takes into account the mutational heterogeneity across these levels. Using MutSigCVsyn, we first analyzed 2572 cancer whole-genome samples from the Pan-cancer Analysis of Whole Genomes (PCAWG) to identify non-synonymous cancer drivers as a quality control. Indicative of the algorithm accuracy we find that 58.6% of these candidate genes were also found in Cancer Census Gene (CGC) list, and 66.2% were found within the PCAWG cancer driver list. We then applied it to identify 30 putative cancer-associated genes that are enriched for synonymous mutations within the same samples. One of the promising gene candidates is the B cell lymphoma 2 (BCL-2) gene. BCL-2 regulates apoptosis by antagonizing the action of proapoptotic BCL-2 family member proteins. The synonymous mutations in BCL2 are enriched in its anti-apoptotic domain and likely play a role in cancer cell proliferation., Conclusion: Our study introduces MutSigCVsyn, an algorithm that accounts for mutational heterogeneity at patient, histology, and gene levels, to identify cancer-associated genes that are enriched for synonymous mutations using whole genome sequencing data. We identified 30 putative candidate genes that will benefit from future experimental studies on the role of synonymous mutations in cancer biology., (© 2023. The Author(s).)

Background: Single synonymous codon mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~ 200 single synonymous codon mutations in an operonic context by mutating almost all positions of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to most degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed., Results: Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, predicted transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single synonymous codon mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate, but might also alter the folding kinetics of the protein in vivo., Conclusion: In summary, the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations., (© 2023. The Author(s).)

X-linked hydrocephalus (XLH), a genetic disorder, has an incidence of 1/30,000 male births. The great proportion of XLH is ascribed to loss-of-function mutations of L1 cell adhesion molecule gene (L1CAM), but silent mutations in L1CAM with pathogenic potential were rare and were usually ignored especially in whole-exome sequencing (WES) detection. In the present study, we describe a novel silent L1CAM mutation in a Chinese pregnant woman reporting continuous five times pregnancies with fetal hydrocephalus. After fetal blood sampling, we found c.453G > T (p.Gly151 =) in the L1CAM gene of the fetus by WES; RT-PCR of the messenger RNA (mRNA) from cord blood mononuclear cells and subsequent sequence analysis identified the mutation created a potential 5′ splice site consensus sequence, which would result in an in-frame deletion of 72 bp from exon 5 and 24 amino acids of the L1CAM protein. Heterozygous mutations were confirmed in analyzing DNA and mRNA from peripheral blood mononuclear cells of the woman, and a severe L1 syndrome was confirmed by fetal ultrasound scan and MRI. Our study first indicated c.453G > T (p.Gly151 =) in L1CAM could be disease causing for hydrocephalus, which would aid in genetic counseling for the prenatal diagnosis of hydrocephalus. Meanwhile, it suggested some silent mutations detected in WES should not be ignored; splicing predictions of these mutations were necessary. [ABSTRACT FROM AUTHOR]

• FBN1 gene testing in individuals with Marfan syndrome and other fibrillinopathies. • Identification of novel and recurrent causal and potentially causal FBN1 variants. • Summary of molecular and clinical data of Hungarian Marfan syndrome patients. Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder mainly affecting the cardiovascular, ocular and musculo-skeletal systems. FBN1 gene mutations lead to MFS and related connective tissue disorders. In this work we described clinical and molecular data of 26 unrelated individuals with suspected MFS who were referred for FBN1 mutation analysis. FBN1 gene sequencing was performed by next generation sequencing and Sanger sequencing methods. We identified 23 causal or potentially causal (including variants of uncertain significance) FBN1 variants, seven of them was novel (˜30%). About 30% of the cases were sporadic. FBN1 mutations were associated with MFS in the majority of the patients, in two cases with severe and early onset manifestation of the syndrome. Missense mutations were detected in 69.6% (16/23), the majority of them were located in one of the cbEGF motifs and ˜70% of them substituted conserved cystein residues. Small deletions/duplications were identified in 13% of the cases (3/23), while splice site variants were detected in 17.4% (4/23). In three unrelated patients a low frequency recurrent silent variant (c.3294C > T (p.Asp1098=) was identified. FBN1 mRNA analysis showed that the mutation does not lead to aberrant splicing, based on available data the mutation was classified as benign. [ABSTRACT FROM AUTHOR]