RESUMO
Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ('rescues') using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01).
Assuntos
Fator V/genética , Predisposição Genética para Doença/genética , Mutagênese/genética , Fosfolipase C beta/genética , Tromboembolia Venosa/genética , Animais , Proteínas de Transporte , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Feminino , Proteínas de Choque Térmico , Humanos , Estimativa de Kaplan-Meier , Lipoproteínas/genética , Masculino , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese/efeitos dos fármacos , Linhagem , Penetrância , Tromboembolia Venosa/mortalidade , Sequenciamento do ExomaRESUMO
Factor V Leiden (F5L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/- ) suppressed F5L/LTfpi+/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.
Assuntos
Fator V/genética , Trombose/genética , Proteína 2 Relacionada a Actina/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Modelos Animais de Doenças , Etilnitrosoureia , Fator VIII/genética , Feminino , Testes Genéticos , Haploinsuficiência , Homozigoto , Humanos , Lipoproteínas/deficiência , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese , Gravidez , Fatores de Risco , Trombose/prevenção & controle , Sequenciamento do ExomaRESUMO
Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1' substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.
Assuntos
Proteínas ADAM/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteína ADAMTS13 , Sequência de Aminoácidos , Sítios de Ligação/genética , Coagulação Sanguínea , Clonagem Molecular , Epistasia Genética , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Mutação , Biblioteca de Peptídeos , Ligação Proteica/genética , Proteólise , Especificidade por Substrato , Fator de von Willebrand/químicaRESUMO
Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.
Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Animais , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Mutação INDEL , CamundongosRESUMO
Hypertension is a complex disease that affects a large proportion of adult population. Although approximately half of the inter-individual variance in blood pressure (BP) level is heritable, identification of genes responsible for its regulation has remained challenging. Genome-wide association study (GWAS) is a novel approach to search for genetic variants contributing to complex diseases. We conducted GWAS for three BP traits [systolic and diastolic blood pressure (SBP and DBP); hypertension (HYP)] in the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) S3 cohort (n = 1644) recruited from general population in Southern Germany. GWAS with 395,912 single nucleotide polymorphisms (SNPs) identified an association between BP traits and a common variant rs11646213 (T/A) upstream of the CDH13 gene at 16q23.3. The initial associations with HYP and DBP were confirmed in two other European population-based cohorts: KORA S4 (Germans) and HYPEST (Estonians). The associations between rs11646213 and three BP traits were replicated in combined analyses (dominant model: DBP, P = 5.55 x 10(-5), effect -1.40 mmHg; SBP, P = 0.007, effect -1.56 mmHg; HYP, P = 5.30 x 10(-8), OR = 0.67). Carriers of the minor allele A had a decreased risk of hypertension. A non-significant trend for association was also detected with severe family based hypertension in the BRIGHT sample (British). The novel susceptibility locus, CDH13, encodes for an adhesion glycoprotein T-cadherin, a regulator of vascular wall remodeling and angiogenesis. Its function is compatible with the BP biology and may improve the understanding of the pathogenesis of hypertension.
Assuntos
Pressão Sanguínea , Caderinas/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hipertensão/genética , População Branca/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Humanos , Hipertensão/fisiopatologia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
BACKGROUND: More than one third of adult population in Estonia has problems with elevated blood pressure (BP). The Hypertension in Estonia (HYPEST) study represents the country's first hypertension-targeted sample collection aiming to examine the epidemiological and genetic determinants for hypertension (HTN) and related cardiovascular diseases (CVD) in Estonian population. The HYPEST subjects (n = 1,966) were recruited across Estonia between 2004-2007 including clinically diagnosed HTN cases and population-based controls. The present report is focused on the clinical and epidemiological profile of HYPEST cases, and gender-specific effects on the pathophysiology of hypertension. METHODS: Current analysis was performed on 1,007 clinically diagnosed HTN patients (617 women and 390 men) aged 18-85 years. The hypertensives were recruited to the study by BP specialists at the North Estonia Medical Center, Centre of Cardiology, Tallinn or at the Cardiology Clinic, Tartu University Hospital, Estonia. Longitudinal BP data was extracted retrospectively from clinical records. Current and retrospective data of patient's medical history, medication intake and lifestyle habits were derived from self-administrated questionnaire and each variable was examined separately for men and women. Eleven biochemical parameters were measured from fasting serum samples of 756 patients. RESULTS: The distribution of recruited men and women was 39% and 61% respectively. Majority of Estonian HTN patients (85%) were overweight (BMI ≥ 25 kg/m2) and a total of 79% of patients had additional complications with cardiovascular system. In men, the hypertension started almost 5 years earlier than in women (40.5 ± 14.5 vs 46.1 ± 12.7 years), which led to earlier age of first myocardial infarction (MI) and overall higher incidence rate of MI among male patients (men 21.2%, women 8.9%, P < 0.0001). Heart arrhythmia, thyroid diseases, renal tubulo-intestinal diseases and hyperlipidemia were more prevalent in hypertensive women compared to men (P < 0.0001). An earlier age of HTN onset was significantly associated with smoking (P = 0.00007), obesity (BMI ≥ 30 kg/m2; P = 0.0003), increased stress (P = 0.0003) and alcohol consumption (P = 0.004). CONCLUSION: Understanding the clinical profile of HTN patients contributes to CVD management. Estonian hypertension patients exhibited different disease and risk profiles of male and female patients. This well-characterized sample set provides a good resource for studying hypertension and other cardiovascular phenotypes.
Assuntos
Hipertensão/diagnóstico , Hipertensão/etnologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estônia/etnologia , Feminino , Humanos , Hipertensão/epidemiologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores Sexuais , Adulto JovemRESUMO
The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.
Assuntos
Desenvolvimento Embrionário , Teste de Complementação Genética , Proteínas de Transporte Vesicular , Animais , Camundongos , Camundongos Transgênicos , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/genéticaRESUMO
CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Deleção de Sequência , Animais , Genótipo , Humanos , Camundongos , Mutagênese , Reação em Cadeia da Polimerase/métodosRESUMO
We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >107 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.
Assuntos
Proteína ADAMTS13/metabolismo , Trombina/metabolismo , Proteína ADAMTS13/genética , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Ensaios de Triagem em Larga Escala/métodos , Humanos , Cinética , Modelos Moleculares , Biblioteca de Peptídeos , Proteômica/métodos , Especificidade por Substrato , Trombina/genéticaRESUMO
During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.
Assuntos
Pareamento de Bases/genética , Proteínas Sanguíneas/genética , Mutação da Fase de Leitura , Síndrome da Plaqueta Cinza/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Sanguíneas/química , Medula Óssea/imunologia , Emperipolese/genética , Exoma/genética , Éxons/genética , Feminino , Fertilidade/genética , Síndrome da Plaqueta Cinza/complicações , Síndrome da Plaqueta Cinza/imunologia , Síndrome da Plaqueta Cinza/fisiopatologia , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Neutropenia/complicações , Neutrófilos/citologia , Baço/imunologia , Trombocitopenia/complicaçõesRESUMO
In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.
Assuntos
Células Germinativas/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Morte Perinatal , Proteínas de Transporte Vesicular/deficiência , Células Acinares/metabolismo , Envelhecimento/patologia , Alelos , Animais , Linfócitos B/metabolismo , Medula Óssea/patologia , Cromossomos Artificiais Bacterianos/genética , Cromossomos de Mamíferos/genética , Cruzamentos Genéticos , Eritrócitos/metabolismo , Eritrócitos/patologia , Células Eritroides/metabolismo , Células Eritroides/patologia , Feminino , Hematopoese , Humanos , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites/genética , Mutação/genética , Fenótipo , Transgenes , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
This paper was withdrawn at the request of the editors due to uncertainties inherent in the statistical analysis.