Potential Spectrum of Missense and Nonsense Factor (F)VIII Gene (F8) Alleles Based on Single Nucleotide Substitutions - How Many Remain To Be Identified?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4039-4039
Author(s):  
Kevin R. Viel ◽  
Manana Khachidze ◽  
Laura Almasy ◽  
Arthur R. Thompson ◽  
Tom E. Howard
Keyword(s):  
Replacement Therapy ◽  
Hemophilia A ◽  
Protein Domain ◽  
Reference Sequence ◽  
Nucleotide Position ◽  
Loss Of Function ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
The Impact

Abstract Regardless of advances in prenatal diagnosis, carrier detection and gene therapy for hemophilia-A, new patients with bleeding diatheses due to inadequate plasma FVIII activity (FVIII:C) levels will still require specialized management at treatment centers. In the ‘post-genome era’ the possibility exists for personalized medicine, in which an individual’s genetic information will be used to tailor prophylactic and/or treatment regimens that will optimize patient outcomes. As listed in the HAMSTeRS database, ~1,000 distinct loss-of-function F8 variants, representing all mutation types including inversions, insertion/deletions and single nucleotide substitutions (SNSs), have been associated with deficiencies of FVIII. To estimate how soon a complete catalogue of every possible mutation affecting FVIII:C levels may become available, we first determined the theoretical number of potential missense and nonsense F8 alleles, whether loss-of-function or not, based on each possible SNS in the coding region as compared to the reference sequence. While the impact of a missense change on FVIII:C, if any, is not always obvious, in contrast to premature-termination codons (PTCs), which are almost always deleterious, findings from a recent resequencing study raises the possibility that non-hemophilic structural differences between a patient’s endogenous FVIII protein and the infused “wildtype” molecule may increase risk of alloimmunization during replacement therapy. Wildtype FVIII contains 2,351 amino acid (aa) residues: 2,332 in the mature protein and 19 in the signal peptide (SP). Appropriate SNSs within the codons for 793 of these residues would create a PTC (UAA, UAG, UGA). Since three distinct base substitutions are possible at each of the three codon positions, 996 nonsense alleles could theoretically arise naturally. As only 123 distinct nonsense mutations are listed in HAMSTeRS, <15% of the theoretical number, many more likely await discovery. Since suitable SNSs within codons for every residue allow for as many as 15,631 naturally-occurring missense variations, of which 462 are in HAMSTeRS, only 2% of all possible alleles, even more mutations of this type likely remain to be identified. The Table presents the number of possible nonsense and missense F8 alleles and the FVIII protein domain/region affected. Although substantial time and diligent surveillance will be required to document the complete allelic architecture of hemophilia-A, since SNS-mutations can occur at every F8 nucleotide, not just those already identified, doing so could potentially have far reaching implications with respect to personalizing both the current strategy of replacement therapy, based on intravenous infusions and future gene-based methods. Table. Potential nonsense and missense F8 alleles based on the FVIII domain/region and position in codon Nucleotide Position in Codon Domain/Region Amino Acids 1st Codon 2nd Codon 3rd Codon Nonsense (Missense) Nonsense (Missense) Nonsense (Missense) SP 0001–0019 3 (51) 1 (56) 3 (23) A1 0001–0336 61 (911) 27 (981) 38 (317) a1 0337–0372 6 (101) 0 (108) 2 (50) A2 0373–0719 70 (935) 41 (1000) 55 (353) a2 0720–0740 5 (55) 3 (60) 4 (26) B 0741–1648 221 (2400) 139 (2585) 35 (1032) a3 1649–1689 12 (111) 4 (119) 4 (53) A3 1690–2019 71 (887) 15 (975) 45 (364) C1 2020–2172 30 (417) 17 (442) 20 (154) C2 2173–2332 35 (432) 14 (466) 15 (167)

Polymorphisms in the goat β-lactoglobulin gene

2005 ◽  
Vol 72 (3) ◽  
pp. 379-384 ◽  
Author(s):  
Maria Ballester ◽  
Armand Sánchez ◽  
Josep M Folch
Keyword(s):  
Genetic Variants ◽  
Promoter Region ◽  
Point Mutations ◽  
Proximal Promoter ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Β Lactoglobulin ◽  
Pyrosequencing Technology ◽  
Frequent Haplotype

β-lactoglobulin polymorphisms have been reported in the milk of different goat breeds, although no genetic variants affecting the protein have been characterized. In the present study, we amplified and sequenced the proximal promoter and the first six exons containing the entire coding region for the β-lactoglobulin gene in eleven goat breeds from Spain, France, Italy, Switzerland, Senegal and Asia to identify genetic variants. Fifteen polymorphisms were detected, nine in the promoter region and six in the exons of the β-lactoglobulin gene. All polymorphisms were single nucleotide substitutions with the exception of one deletion/insertion in the promoter region. The polymorphisms in the coding region did not produce any amino acid change. In addition, pyrosequencing technology was used to genotype four polymorphisms in the promoter region in 200 goats belonging to eleven breeds. Differences in allelic frequencies for these polymorphisms between breeds are described and a specific polymorphism for the Italian populations was identified. Finally, the analysis of association between these four promoter point mutations was investigated resulting in five haplotypes, GCGC being the most frequent haplotype in all breeds analysed.

scholarly journals Tandem Substitutions in Somatic Hypermutation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 996-996
Author(s):  
Marvyn T. Koning ◽  
Julieta Haydee Sepulveda Yanez ◽  
Diego Alvarez-Saravia ◽  
Bas Pilzecker ◽  
Pauline Van Schouwenburg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Somatic Hypermutation ◽  
Adaptive Immune Response ◽  
Affinity Maturation ◽  
Reference Sequence ◽  
Nucleotide Substitutions ◽  
Sequence Context ◽  
Single Nucleotide ◽  
Adaptive Immune ◽  
Amino Acid Codon

Abstract Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. While tandem substitutions have been described in mice and other species, the incidence of this phenomenon and its underlying mechanism in humans is currently unknown. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors generated by unbiased ARTISAN PCR. Selection of unique, clonally unrelated, antigen-experienced sequences carrying up to 5% mutations yielded 13.532 VDJ, 7.952 VJ-kappa and 7.598 VJ-lambda. Comparison to the closest germline allele allowed for identification of a total of 122.878 single nucleotide substitutions (SNS), 10.735 tandem dinucleotide substitutions (TDNS) and 2.615 longer contiguous substitutions. After correcting for expected clusters of adjacent SNS, tandem substitutions comprised 5,7% of all AID-induced mutations. The mutation of more than one nucleotide in a single event, was shown to overcome amino acid codon redundancy and may therefore enhance the adaptive immune response. Clustering of such mutations around AID hotspots and their overall distribution indicates that tandem substitutions are an integral part of the SHM spectrum. In the majority of tandem substitutions, the mutated sequence may be identified in the directly adjacent reference sequence context. Tandem substitutions in humans therefore represent single nucleotide juxtalocations. Such juxtalocations appear to be favored in polydipyramidine stretches. These observations could be confirmed in patients with MSH2/6 deficiency, but were absent in a VDJ library from an UNG-deficient patient, indicating a strict dependence on abasic sites as an instigating mechanism. Together, these findings delineate a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Our study shows that a significant portion of mutations acquired during SHM are caused by tandem substitutions, and that this mechanism may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously. Figure legend. Corrected incidence of tandem dinucleotide substitutions in healthy donors. (A) Dinucleotide substitutions from unique IGHV, IGKV and IGLV sequences and corrected after in silico predictions of dinucleotide substitutions that did not occur in tandem. Burgundy cells represent sequence inversions, light and dark purple cells represent juxtalocations of the 5' and 3' base in the pair (as seen from the non-transcribed strand), respectively. For unshaded cells, juxtalocation could not be assessed due to one or more nucleotides in the reference sequence matching the mutated sequence. (B) Relative contribution of sequence inversions and juxtalocations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased oxidative stress tolerance of a spontaneously-occurring perR gene mutation in Streptococcus mutans UA159

2020 ◽  
Author(s):  
Jessica K. Kajfasz ◽  
Peter Zuber ◽  
Tridib Ganguly ◽  
Jacqueline Abranches ◽  
José A. Lemos
Keyword(s):  
Oxidative Stress ◽  
Streptococcus Mutans ◽  
Stress Responses ◽  
Transcriptional Repressor ◽  
Loss Of Function ◽  
Coding Region ◽  
Major Player ◽  
The Impact ◽  
Peroxide Stress

AbstractThe ability of bacteria such as the dental pathogen Streptococcus mutans to coordinate a response against damage-inducing oxidants is a critical aspect of their pathogenicity. The oxidative stress regulator SpxA1 has been proven to be a major player in the ability of S. mutans to withstand both disulfide and peroxide stresses. While studying spontaneously-occurring variants of an S. mutans ΔspxA1 strain, we serendipitously discovered that our S. mutans UA159 host strain bore a single nucleotide deletion within the coding region ofperR, resulting in a premature truncation of the encoded protein. PerR is a metal-dependent transcriptional repressor that senses and responds to peroxide stress such that loss of PerR activity results in activation of oxidative stress responses. To determine the impact of loss of PerR regulation, we obtained a UA159 isolated bearing an intact perR copy and created a clean perR deletion mutant. Our findings indicate that loss of PerR activity results in a strain that is primed to tolerate oxidative stresses in the laboratory setting. Interestingly, RNA-Seq and targeted transcriptional expression analyses reveal that PerR has a minor contribution to the ability of S. mutans to orchestrate a transcriptional response to peroxide stress. Furthermore, we detected loss-of-function perR mutations in two other commonly used laboratory strains of S. mutans suggesting that this may be not be an uncommon occurrence. This report serves as a cautionary warning regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices.ImportanceA resident of the human oral biofilm, Streptococcus mutans is one of the major bacterial pathogens associated with dental caries. This report highlights a spontaneously-occurring mutation within the laboratory strain S. mutans UA159, found in the coding region of perR, a gene encoding a transcriptional repressor associated with peroxide tolerance. Though perR mutant strains of S. mutans showed a distinct growth advantage and enhanced tolerance toward H2O2, a ΔperR deletion strain showed a small number of differentially expressed genes as compared to the parent strain, suggesting few direct regulatory targets. In addition to characterizing the role of PerR in S. mutans, our findings serve as a warning to laboratory researchers regarding bacterial adaptation to in vitro growth conditions.

The Impact of Factor VIII/von Willebrand Factor Concentrate in Inhibitor Development and Management of Patients with Hemophilia A with Inhibitors

2009 ◽  
Vol 02 ◽  
pp. 13
Author(s):  
Caroline Cromwell ◽  
Louis M Aledort ◽  
Margaret Heisel Kurth ◽  
◽  
◽  
...  
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Von Willebrand Factor ◽  
Active Sites ◽  
Hemophilia A ◽  
Safety Issue ◽  
Inhibitor Development ◽  
Von Willebrand ◽  
The Impact ◽  
Willebrand Factor

The development of inhibitor antibodies that bind to active sites on the factor VIII (FVIII) molecule and neutralize its function and/or accelerate its clearance is the most serious adverse event and safety issue associated with the treatment of hemophilia A. Inhibitor development complicates hemostasis management and increases morbidity and the cost of treatment because bleeding episodes do not respond to standard replacement therapy. Risk factors for inhibitor development include genetic and non-genetic factors. Immunogenicity of the type of product used for replacement therapy may also play a role. Within the category of human-derived products, the presence of von Willebrand factor (vWF) bound to FVIII (vWF/FVIII products) may reduce its immunogenicity. The challenge for inhibitors is to reduce their incidence and, when present, to facilitate their eradication. Factor-bypassing agents have been used to treat acute bleeds in patients who have inhibitors. Immune tolerance induction (ITI) therapy is an alternative approach whose goal is to create tolerance to inhibitors and return patients to their native state. The use of ITI therapy has raised many questions, including the optimal regimen and cost. The basic science data on reduced immunogenicity of vWF/FVIII-containing products and their success in achieving ITI have given us an incentive to continue to explore this approach to both primary and secondary ITI.

Sequence characterization and polymorphism detectionin AQP7 gene of Murrah buffalo

2016 ◽  
Author(s):  
Ragini Kumari ◽  
K.P. Ramesha ◽  
Rakesh Kumar ◽  
Divya Divya ◽  
Anjali Kumari ◽  
...  
Keyword(s):  
Semen Quality ◽  
Single Strand Conformation Polymorphism ◽  
Sscp Analysis ◽  
Reference Sequence ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Single Nucleotide ◽  
Sequence Characterization ◽  
Murrah Buffalo ◽  
Aquaporin 7

Aquaporin 7 (AQP7) gene is a member of aqua-glyceroporins which transports glycerol and water to spermatids. The present study aimed to investigate the polymorphisms within exons 2, 3, 4, 5 and their flanking intronic regions in AQP7 gene of Murrah bulls. Genomic DNA was extracted from 69 Murrah bulls blood samples and was subjected to polymerase chain reaction- single strand conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP analysis revealed a total of eight different variants for amplicons of exons 4 and 5. Amplicon of exon 4 revealed three different patterns viz. E4P1, E4P2 and E4P3 with the frequency of 0.30, 0.22 and 0.48, respectively. Analysis of exon 5 revealed five unique SSCP patterns viz. E5P1, E5P2, E5P3, E5P4 and E5P5, with the frequency of 0.10, 0.37, 0.20, 0.20 and 0.13, respectively. Sequence analysis showed 16 single nucleotide polymorphisms, 7 of which were observed in coding region. Amplicons of exons 2 and 3 showed monomorphic patterns. However, compared to the reference sequence of taurine cattle one transition (C6878T) in exon 3 and 4 transitions (G2099A, C2116T, A2117G, G6848C) in intron 2 were observed for all the bulls under study. The genetic variability identified in the AQP7 gene may serve as potential genetic marker(s) for semen quality traits in buffalo.

scholarly journals Polymorphisms in the cytotoxic T lymphocyte-associated protein-4 immune regulatory gene and their impact on inhibitor development in patients with hemophilia A

2019 ◽  
Vol 47 (10) ◽  
pp. 4981-4992
Author(s):  
Aveen M. Raouf Abdulqader ◽  
Ali Ibrahim Mohammed ◽  
Shwan Rachid
Keyword(s):  
T Lymphocyte ◽  
Hemophilia A ◽  
Fragment Length Polymorphism ◽  
Cytotoxic T Lymphocyte ◽  
Direct Sequencing ◽  
Regulatory Gene ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Inhibitor Development ◽  
The Impact

Objective The development of inhibitors against infused factor VIII represents the most severe complication of substitution therapy in hemophilia A (HA) patients. Data on risk factors for inhibitor formation in Iraqi Kurdish patients with HA are unavailable. This study aimed to evaluate the impact of two single nucleotide polymorphisms (SNPs) in an immune regulatory gene in the emergence of inhibitors. Methods We focused on 126 patients with either severe or mild/moderate HA presenting with and without inhibitors. We analyzed the frequency of two polymorphisms in the cytotoxic T lymphocyte-associated protein-4 gene ( CTLA-4; CTLA-4-318C > T and CTLA-4 + 49A > G). Genotyping was performed using restriction fragment length polymorphism–PCR and direct sequencing. Results We found no significant correlation between the CTLA-4-318 C > T T allele and inhibitor development among patients with severe or mild/moderate HA. However, a significantly high inhibitor risk was detected for the CTLA-4 + 49 A > G G allele (odds ratio [OR] = 3.1, 95% confidence interval [CI] = 1.383–7.024) and (OR = 4, 95% CI = 1.719–9.437) among patients with severe and mild/moderate HA, respectively. Conclusion We conclude that the CTLA-4 +49 A > G SNP plays a substantial role as a potential risk determinant for inhibitor formation in Iraqi Kurdish patients with HA.

scholarly journals Localized structural frustration for evaluating the impact of sequence variants

2013 ◽  
Vol 44 (21) ◽  
Author(s):  
Sushant Kumar ◽  
Declan Clarke ◽  
Mark Gerstein
Keyword(s):  
Protein Structures ◽  
Loss Of Function ◽  
Single Nucleotide Variants ◽  
Local Interactions ◽  
Opposite Pattern ◽  
Single Nucleotide ◽  
Coding Regions ◽  
Local Perturbations ◽  
Large Numbers ◽  
The Impact

Abstract Population-scale sequencing is increasingly uncovering large numbers of rare single-nucleotide variants (SNVs) in coding regions of the genome. The rarity of these variants makes it challenging to evaluate their deleteriousness with conventional phenotype–genotype associations. Protein structures provide a way of addressing this challenge. Previous efforts have focused on globally quantifying the impact of SNVs on protein stability. However, local perturbations may severely impact protein functionality without strongly disrupting global stability (e.g. in relation to catalysis or allostery). Here, we describe a workflow in which localized frustration, quantifying unfavorable local interactions, is employed as a metric to investigate such effects. Using this workflow on the Protein Databank, we find that frustration produces many immediately intuitive results: for instance, disease-related SNVs create stronger changes in localized frustration than non-disease related variants, and rare SNVs tend to disrupt local interactions to a larger extent than common variants. Less obviously, we observe that somatic SNVs associated with oncogenes and tumor suppressor genes (TSGs) induce very different changes in frustration. In particular, those associated with TSGs change the frustration more in the core than the surface (by introducing loss-of-function events), whereas those associated with oncogenes manifest the opposite pattern, creating gain-of-function events.

A single nucleotide polymorphism in the sheep κ-casein coding region

2005 ◽  
Vol 72 (3) ◽  
pp. 317-321 ◽  
Author(s):  
Maria Feligini ◽  
Slavica Vlaco ◽  
Vlatka Cubric Curik ◽  
Pietro Parma ◽  
GianFranco Greppi ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Genomic Region ◽  
Reference Sequence ◽  
Mature Protein ◽  
Coding Region ◽  
Nucleotide Polymorphism ◽  
Conventional Pcr ◽  
Homozygous State ◽  
Single Nucleotide ◽  
Forward Primer

Genetic polymorphisms in CSN3 gene in Pag (Croatia), Sarda (Italy) and Pramenka (Serbia) sheep breeds were investigated. A single nucleotide polymorphism (SNP) was localized by sequence analysis (sequence submitted to GenBank under accession AY237637) relying on an original primer pair. Primers for sequencing (κ-casF and κ-casR) were designed on the available CSN3 sequences to amplify the genomic region encoding the major part of the mature protein (exon 4). An SNP was detected at position 237 of the sheep κ-casein mRNA (reference sequence: GenBank X51822), where a thymine was substituted for a cytosine. The SNP was typed by conventional PCR and SYBR Green I-based real-time PCR. C and T alleles were discriminated using a dedicated set of primers that consisted of one common forward primer (SNP-TC) and two reverse primers (SNP-T and SNP-C), the latter two differing in the 3′ end base and in the presence of a 12 bp poly-G tail in SNP-C. The SNP was found in both the heterozygous and the homozygous state in Sarda and Pramenka breeds, and in the heterozygous state only in the Pag breed. The observed allelic frequencies of the SNP were 0·12 in Pag, 0·27 in Sarda and 0·45 in Pramenka.

Identification of Single Nucleotide Polymorphism in GnRHR gene in Murrah bulls

2018 ◽  
Author(s):  
J. K. Reen ◽  
K. P. Ramesha ◽  
Preeti . ◽  
D. Revanasiddu ◽  
M. Ahirwar
Keyword(s):  
Bos Taurus ◽  
Reference Sequence ◽  
Coding Region ◽  
Single Nucleotide ◽  
Training Centre ◽  
Blood Samples ◽  
Livestock Breeding ◽  
Single Stranded Conformational Polymorphism ◽  
Similar Band ◽  
And Training

The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphism in the GnRHR gene in Murrah bulls. Blood samples were collected from 109 Murrah bulls maintained at three different organized semen stations viz., Centralized Semen Collection Centre of Livestock Breeding and Training Centre, Dharwad; Nandini Sperm Station and State Livestock Breeding and Training Centre, Hessarghatta, Bengaluru, Karnataka, India. Genomic DNA was isolated from the blood samples within 24 hours of collection . Mutations were screened using Polymerase Chain Reaction – Single Stranded Conformational Polymorphism (PCR-SSCP) technique followed by Sanger Sequencing. PCR-SSCP method revealed similar band pattern within Murrah bulls. To confirm monomorphism in the studied population with respect to GnRHR gene, duplicate samples from each primer fragment were custom sequenced. PCR-SSCP and sequence analysis revealed monomorphism within the studied population that is coding region as well as exon-intron boundaries of GnRHR gene is highly conserved among Murrah bulls. However, a total of 28 Single Nucleotide variations (SNV’s) have been found when compared with Bos Taurus reference sequence for GnRHR gene.

scholarly journals Suppression of Nonsense Mutations by New Emerging Technologies

2020 ◽  
Vol 21 (12) ◽  
pp. 4394 ◽  
Author(s):  
Pedro Morais ◽  
Hironori Adachi ◽  
Yi-Tao Yu
Keyword(s):  
Clinical Trials ◽  
New Technologies ◽  
Genetic Disorders ◽  
Protein Translation ◽  
Premature Termination Codon ◽  
Nonsense Suppression ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Nonsense Mutations ◽  
The Impact

Nonsense mutations often result from single nucleotide substitutions that change a sense codon (coding for an amino acid) to a nonsense or premature termination codon (PTC) within the coding region of a gene. The impact of nonsense mutations is two-fold: (1) the PTC-containing mRNA is degraded by a surveillance pathway called nonsense-mediated mRNA decay (NMD) and (2) protein translation stops prematurely at the PTC codon, and thus no functional full-length protein is produced. As such, nonsense mutations result in a large number of human diseases. Nonsense suppression is a strategy that aims to correct the defects of hundreds of genetic disorders and reverse disease phenotypes and conditions. While most clinical trials have been performed with small molecules, there is an increasing need for sequence-specific repair approaches that are safer and adaptable to personalized medicine. Here, we discuss recent advances in both conventional strategies as well as new technologies. Several of these will soon be tested in clinical trials as nonsense therapies, even if they still have some limitations and challenges to overcome.

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