scholarly journals Wie Designer-Rekombinasen Erbkrankheiten heilen könnten

BIOspektrum ◽  
2021 ◽  
Vol 27 (2) ◽  
pp. 139-141
Author(s):  
Felix Lansing ◽  
Jenna Hoersten ◽  
Frank Buchholz
Keyword(s):  
Genome Editing ◽  
Hemophilia A ◽  
Chromosomal Rearrangements ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Severe Hemophilia ◽  
Site Specific ◽  
Large Gene ◽  
Recent Advances

AbstractRecent advances in nuclease-based genome editing allow for the correction of many point-mutations causing diseases. However, correcting genetic alterations caused by larger chromosomal rearrangements remain challenging with this approach. Designer-recombinases promise to fill this gap as demonstrated by the development of a heterodimeric Cre-based site-specific recombinase system. This system can functionally correct a large gene inversion frequently found in patients with severe Hemophilia A.

scholarly journals Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species

2020 ◽  
Vol 48 (10) ◽  
pp. e57-e57
Author(s):  
Xiaozhen He ◽  
Wenfeng Chen ◽  
Zhen Liu ◽  
Guirong Yu ◽  
Youbang Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Peripheral Blood ◽  
Point Mutations ◽  
Peripheral Blood Lymphocytes ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Low Efficiency ◽  
Multiple Species ◽  
Balancing Efficiency

Abstract Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

The Molecular Genetics of Hemophilia A Stylianos

1987 ◽  
Author(s):  
E Antonarakis
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
Hemophilia A ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Leader Peptide ◽  
Molecular Defect ◽  
Severe Hemophilia ◽  
Cpg Dinucleotides ◽  
Oligonucleotide Hybridization

Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.

Double Strand Conformation Polymorphism (DSCP) Detects Two Point Mutations at Codon 280 (AAC→ATC) and at Codon 431 (TAC→AAC) of the Blood Coagulation Factor VIII Gene

1993 ◽  
Vol 69 (05) ◽  
pp. 473-475 ◽  
Author(s):  
W C Pieneman ◽  
P H Reitsma ◽  
E Briët
Keyword(s):  
Factor Viii ◽  
Electrophoretic Mobility ◽  
Hemophilia A ◽  
Point Mutations ◽  
Coagulation Factor ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Exon 9 ◽  
Blood Coagulation Factor Viii ◽  
Conformation Polymorphism

SummaryHemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC→AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A→T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP).At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.

Description of a Large Duplication in F8 Gene Responsible for Severe Hemophilia A.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1149-1149
Author(s):  
Christine Vinciguerra ◽  
Jenny Goudemand ◽  
Christophe Zawadzki ◽  
Dorothee Pellecchia ◽  
Claude Negrier
Keyword(s):  
Factor Viii ◽  
Quantitative Method ◽  
Hemophilia A ◽  
High Titer ◽  
Genetic Disorders ◽  
Point Mutations ◽  
Genetic Mutation ◽  
Severe Hemophilia ◽  
Intron 1 ◽  
Large Rearrangements

Abstract Hemophilia A is a common inherited bleeding disorder, caused by factor VIII (FVIII) deficiency as a result of mutations in the factor VIII gene (F8 gene). Intron 1 and 22 inversions, small insertions/deletions and point mutations are the most common genetic defects responsible for severe hemophilia A. However, a F8 gene mutation is not found in 2–5% of patients with severe hemophilia A (FVIII:C<1IU/dL). Large rearrangements are frequent in other genetic disorders and only one case of exon 13 duplication was described in a patient with mild hemophilia (Casula et al, Blood 1990). We described here a large duplication in F8 gene in a CRM− patient with severe hemophilia A: (FVIII:C < 1 UI/dL, FVIII:Ag < 1%) who developed a high-titer inhibitor (peak 10 BU). A previous investigation did not find intron 1 and 22 inversion or exons /splice sites sequencing abnormalities. The promoter region was also sequenced, but no genetic mutation was then characterized. The duplication was detected by MP/LC (Multiplex PCR/Liquid chromatography) which is a quantitative method able to detect large rearrangements. Initially described by Dehainault et al (Nucl Acids Res 2004) in retinoblastoma patients, this method showed that a duplication affected a large part of F8 gene, e.g. the 3′ part of intron 10, exons 11 to 14 and a part of intron 14. To our knowledge, this is the first duplication responsible for severe hemophila A described so far. This finding suggests that the detection of large rearrangements with quantitative method as MP/LC, QMPSF or MLPA would be useful in hemophilic patients where the screening for inversions or genetic events in the coding regions are unsuccessful. This kind of large gene modifications may indeed account for at least some of the 2–5% cases where the coding sequence does not appeared to be altered. In this case, the truncation of the primary polypeptide sequence did not result in any secretion of the FVIII protein, that may have increased the risk of inhibitor development.

scholarly journals Genome-wide mapping of spontaneous genetic alterations in diploid yeast cells

2020 ◽  
Vol 117 (45) ◽  
pp. 28191-28200
Author(s):  
Yang Sui ◽  
Lei Qi ◽  
Jian-Kun Wu ◽  
Xue-Ping Wen ◽  
Xing-Xing Tang ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Point Mutations ◽  
Mitotic Cell ◽  
Genetic Alterations ◽  
Mitotic Recombination ◽  
Tumor Formation ◽  
Yeast Cells ◽  
Nucleotide Polymorphisms ◽  
Individual Snps ◽  
Diploid Cells

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain ofSaccharomyces cerevisiaeby whole-genome sequencing of many independent isolates (n= 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n= 1,337) and mitotic recombination events (n= 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.

scholarly journals A genome editing primer for the hematologist

Blood ◽  
2016 ◽  
Vol 127 (21) ◽  
pp. 2525-2535 ◽  
Author(s):  
Megan D. Hoban ◽  
Daniel E. Bauer
Keyword(s):  
Genome Editing ◽  
Genetic Alterations ◽  
Double Strand Breaks ◽  
Blood Disorders ◽  
Site Specific ◽  
Strand Breaks ◽  
Epigenome Editing ◽  
Genome Modification ◽  
A Genome ◽  
Experimental Hematology

Abstract Gene editing enables the site-specific modification of the genome. These technologies have rapidly advanced such that they have entered common use in experimental hematology to investigate genetic function. In addition, genome editing is becoming increasingly plausible as a treatment modality to rectify genetic blood disorders and improve cellular therapies. Genome modification typically ensues from site-specific double-strand breaks and may result in a myriad of outcomes. Even single-strand nicks and targeted biochemical modifications that do not permanently alter the DNA sequence (epigenome editing) may be powerful instruments. In this review, we examine the various technologies, describe their advantages and shortcomings for engendering useful genetic alterations, and consider future prospects for genome editing to impact hematology.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

Dose Requirement for Replacement Therapy in Hemophilia A

1979 ◽  
Vol 42 (03) ◽  
pp. 825-831 ◽  
Author(s):  
Jean-Pierre Allain
Keyword(s):  
Clinical Effect ◽  
Hemophilia A ◽  
Clinical Results ◽  
Severe Hemophilia ◽  
Dose Requirement ◽  
Viii Level ◽  
The Relationship ◽  
In Vivo Recovery ◽  
Different Doses

SummaryIn order to determine the correlation between different doses of F. VIII and their clinical effect,. 70 children with severe hemophilia A were studied after treatment with single doses of cryoprecipitate. The relationship between plasma F. VIII levels or doses calculated in u/ kg of body weight and clinical results followed an exponential curve. Plasma F. VIII levels of 0.35 and 0.53 u/ml corresponded to 95 and 99% satisfactory treatment, respectively. Similar clinical results were obtained with 20 and 31 u/kg. When the in vivo recovery of F. VIII after lyophilized cryoprecipitate was 0.015 u/ml for each u/kg injected, plasma F. VIII levels of 0.30 and 0.47 u/ml respectively were achieved. Since home treatment is largely based on single infusions of F. VIII, it is suggested that moderate and severe hemorrhages be treated with a dose which will provide a plasma F. VIII level of 0.5 u/ml.

The Impact of Bioconjugation on the Interfacial Activity of a Protein Biosurfactant

2018 ◽  
Author(s):  
Hossam H Tayeb ◽  
Marina Stienecker ◽  
Anton Middelberg ◽  
Frank Sainsbury
Keyword(s):  
Polyethylene Glycol ◽  
Colloidal Stability ◽  
Point Mutations ◽  
Pharmaceutical Formulations ◽  
Industrial Processes ◽  
Parent Molecule ◽  
Site Specific ◽  
Interfacial Activity ◽  
Surface Active ◽  
The Impact

Biosurfactants, are surface active molecules that can be produced by renewable, industrially scalable biologic processes. DAMP4, a designer biosurfactant, enables the modification of interfaces via genetic or chemical fusion to functional moieties. However, bioconjugation of addressable amines introduces heterogeneity that limits the precision of functionalization as well as the resolution of interfacial characterization. Here we designed DAMP4 variants with cysteine point mutations to allow for site-specific bioconjugation. The DAMP4 variants were shown to retain the structural stability and interfacial activity characteristic of the parent molecule, while permitting efficient and specific conjugation of polyethylene glycol (PEG). PEGylation results in a considerable reduction on the interfacial activity of both single and double mutants. Comparison of conjugates with one or two conjugation sites shows that both the number of conjugates as well as the mass of conjugated material impacts the interfacial activity of DAMP4. As a result, the ability of DAMP4 variants with multiple PEG conjugates to impart colloidal stability on peptide-stabilized emulsions is reduced. We suggest that this is due to constraints on the structure of amphiphilic helices at the interface. Specific and efficient bioconjugation permits the exploration and investigation of the interfacial properties of designer protein biosurfactants with molecular precision. Our findings should therefore inform the design and modification of biosurfactants for their increasing use in industrial processes, and nutritional and pharmaceutical formulations.

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