RESUMO
Chromosomal abnormalities are a common cause of human miscarriage but rarely reported in any other species. As a result, there are currently inadequate animal models available to study this condition. Horses present one potential model since mares receive intense gynecological care. This allowed us to investigate the prevalence of chromosomal copy number aberrations in 256 products of conception (POC) in a naturally occurring model of pregnancy loss (PL). Triploidy (three haploid sets of chromosomes) was the most common aberration, found in 42% of POCs following PL over the embryonic period. Over the same period, trisomies and monosomies were identified in 11.6% of POCs and subchromosomal aberrations in 4.2%. Whole and subchromosomal aberrations involved 17 autosomes, with chromosomes 3, 4, and 20 having the highest number of aberrations. Triploid fetuses had clear gross developmental anomalies of the brain. Collectively, data demonstrate that alterations in chromosome number contribute to PL similarly in women and mares, with triploidy the dominant ploidy type over the key period of organogenesis. These findings, along with highly conserved synteny between human and horse chromosomes, similar gestation lengths, and the shared single greatest risk for PL being advancing maternal age, provide strong evidence for the first animal model to truly recapitulate many key features of human miscarriage arising due to chromosomal aberrations, with shared benefits for humans and equids.
Assuntos
Aborto Espontâneo , Aberrações Cromossômicas , Animais , Cavalos , Feminino , Aborto Espontâneo/genética , Gravidez , Modelos Animais de Doenças , Humanos , TriploidiaRESUMO
Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental programme in mouse, cow and human embryos. However, the mechanisms transducing cell polarity into cell fate in cow and human embryos are unknown. Here, we have examined the evolutionary conservation of Hippo signalling, which is thought to function downstream of aPKC activity, in four different mammalian species: mouse, rat, cow and human. In all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2. However, the timing and localisation of molecular markers differ across species, with rat embryos more closely recapitulating human and cow developmental dynamics, compared with the mouse. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.
Assuntos
Via de Sinalização Hippo , Transdução de Sinais , Bovinos , Humanos , Feminino , Gravidez , Camundongos , Ratos , Animais , Transdução de Sinais/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Blastocisto/metabolismo , Placenta/metabolismo , Mamíferos/metabolismo , Linhagem da CélulaRESUMO
Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.
Assuntos
Evolução Biológica , Ectoderma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Trofoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Bovinos , Linhagem da Célula , Polaridade Celular , Ectoderma/citologia , Embrião de Mamíferos/enzimologia , Feminino , Fator de Transcrição GATA3/metabolismo , Via de Sinalização Hippo , Humanos , Camundongos , Mórula/citologia , Mórula/enzimologia , Mórula/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Trofoblastos/citologia , Proteínas de Sinalização YAP , Saco Vitelino/citologia , Saco Vitelino/metabolismoRESUMO
Among the rare bleeding disorders factor VII deficiency is the most common, but correlating deficiency with bleeding phenotype is challenging. In their study Lou and colleagues investigate a large cohort of unrelated factor VII deficient patients providing a further perspective on the link between genotype and phenotype in this disorder. Commentary on: Lou et al. Structural and functional characterization of novel F7 mutations identified in Chinese factor VII deficient patients. Br J Haematol. 2023;202:623-635.
Assuntos
Deficiência do Fator VII , Humanos , Fator VII , Genótipo , Fenótipo , Hemorragia , Fatores de Risco , MutaçãoRESUMO
Hereditary blood coagulation factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder resulting from variants in the gene encoding FVII (F7). Integration of genetic variation with functional consequences on protein function is essential for the interpretation of the pathogenicity of novel variants. Here, we describe the integration of previous locus-specific databases for F7 into a single curated database with enhanced features. The database provides access to in silico analyses that may be useful in the prediction of variant pathogenicity as well as cross-species sequence alignments, structural information, and functional and clinical severity described for each variant, where appropriate. The variant data is shared with the F7 Leiden Open Variation Database. The updated database now includes 221 unique variants, representing gene variants identified in 728 individuals. Single nucleotide variants are the most common type (88%) with missense representing 74% of these variants. A number of variants are found with relatively high minor allele frequencies that are not pathogenic but contribute significantly to the likely pathogenicity of coinherited variants due to their effect on FVII plasma levels. This comprehensive collection of curated information significantly aids the assessment of pathogenicity.
Assuntos
Bases de Dados Genéticas , Fator VII/genética , Frequência do Gene , Variação Genética , Humanos , Mutação , Estrutura Secundária de ProteínaRESUMO
INTRODUCTION: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes and access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity. AIM: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web-accessible resource for variants in genes involved in clinical bleeding disorders. RESULTS: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF). CONCLUSION: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity.
Assuntos
Hemofilia A/epidemiologia , Hemostasia/fisiologia , Pesquisa Biomédica , Bases de Dados Factuais , Europa (Continente) , HumanosRESUMO
With the advent of large-scale next-generation sequencing initiatives, there is an increasing importance to interpret and understand the potential phenotypic influence of identified genetic variation and its significance in the human genome. Bioinformatics analyses can provide useful information to assist with variant interpretation. This review provides an overview of tools/resources currently available, and how they can help predict the impact of genetic variation at the deoxyribonucleic acid, ribonucleic acid, and protein level.
Assuntos
Biologia Computacional/métodos , Educação a Distância/métodos , Variação Genética/genética , HumanosRESUMO
Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect â¼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.
Assuntos
Transtornos Plaquetários/genética , Predisposição Genética para Doença , Hemorragia/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Trombose/genética , Estudos de Casos e Controles , Variações do Número de Cópias de DNA , Feminino , Estudos de Associação Genética/métodos , Humanos , Masculino , Mutação , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodosRESUMO
Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.
Assuntos
Encéfalo/anatomia & histologia , Centrossomo/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Animais , Proteínas de Ciclo Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Recessivos , Células HeLa , Homozigoto , Humanos , Masculino , Camundongos , Mitose/fisiologia , Dados de Sequência Molecular , Neurônios/fisiologia , Linhagem , Fuso Acromático/fisiologiaRESUMO
Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.
Assuntos
Mutação , Proteínas rab de Ligação ao GTP/metabolismo , Domínio Catalítico , Sistema Nervoso Central/anormalidades , Anormalidades do Olho/patologia , Genitália/anormalidades , Humanos , Dados de Sequência Molecular , Síndrome , Proteínas rab de Ligação ao GTP/genéticaRESUMO
One of the most notable trends in mammalian evolution is the massive increase in size of the cerebral cortex, especially in primates. Humans with autosomal recessive primary microcephaly (MCPH) show a small but otherwise grossly normal cerebral cortex associated with mild to moderate mental retardation. Genes linked to this condition offer potential insights into the development and evolution of the cerebral cortex. Here we show that the most common cause of MCPH is homozygous mutation of ASPM, the human ortholog of the Drosophila melanogaster abnormal spindle gene (asp), which is essential for normal mitotic spindle function in embryonic neuroblasts. The mouse gene Aspm is expressed specifically in the primary sites of prenatal cerebral cortical neurogenesis. Notably, the predicted ASPM proteins encode systematically larger numbers of repeated 'IQ' domains between flies, mice and humans, with the predominant difference between Aspm and ASPM being a single large insertion coding for IQ domains. Our results and evolutionary considerations suggest that brain size is controlled in part through modulation of mitotic spindle activity in neuronal progenitor cells.
Assuntos
Córtex Cerebral/anatomia & histologia , Proteínas de Drosophila , Proteínas do Tecido Nervoso/genética , Antropometria , Northern Blotting , Códon sem Sentido , Feminino , Humanos , Masculino , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Linhagem , Análise de Sequência de DNARESUMO
We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5' of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.
Assuntos
Regiões Promotoras Genéticas , Deleção de Sequência , Ativação Transcricional , Doença de von Willebrand Tipo 1/genética , Fator de von Willebrand/genética , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Fatores de Transcrição GATA/metabolismo , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Ligação Proteica , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Transgenes , Doença de von Willebrand Tipo 1/metabolismo , Fator de von Willebrand/metabolismoRESUMO
The online locus-specific database for von Willebrand disease (VWFdb) acts as a repository for sequence variant data and associated resources for those with an interest in the disorder. It currently holds details of 561 mutations and 217 polymorphisms in the von Willebrand factor (VWF) gene. Lists can be queried and displayed by VWF region or disease type. A total of 42% of the mutations are located in the large exon 28, the most heavily studied VWF region, and mutations have been reported in all but 4 of the 51 protein-coding exons. Polymorphisms are reported in the 5' and 3' untranslated regions and in 33 exons and 35 introns. Additional resources include references linked to sequence variation entries, descriptors of each VWD type, genomic and cDNA sequences, nomenclature for VWF and its attributes, Human Genome Variation Society sequence variant nomenclature recommendations, multimer images, and related densitometry traces for type 2 VWD. Analysis of recessively inherited VWD indicates that whereas the majority (69%) of type 3 VWD patients are homozygous for their mutations, the majority (62%) of 2N patients are compound heterozygous. Comparison of missense substitutions reported as mutations with those reported as polymorphisms suggests that loss or gain of cysteine, tryptophan, methionine, or glutamate residues are more likely to result in a pathogenic effect than loss/gain of other VWF residues.
Assuntos
Bases de Dados Genéticas/estatística & dados numéricos , Mutação , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Bases de Dados Genéticas/tendências , Frequência do Gene , Humanos , Cooperação Internacional , Polimorfismo Genético , Sociedades Médicas , Doenças de von Willebrand/classificaçãoRESUMO
Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.
Assuntos
Doença de von Willebrand Tipo 1 , Doenças de von Willebrand , Variações do Número de Cópias de DNA , Humanos , Corpos de Weibel-Palade , Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/genética , Fator de von Willebrand/genéticaAssuntos
Mutação Puntual , Polimorfismo de Nucleotídeo Único , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Negro ou Afro-Americano , Substituição de Aminoácidos , Povo Asiático , Humanos , Índia , Estados Unidos , População Branca , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Plasma levels of von Willebrand factor (VWF) vary considerably in the general population and this variation has been linked to several genetic and environmental factors. Genetic factors include 2 common single nucleotide variants (SNVs) located in VWF, rs1063856 (c.2365A>G) and rs1063857 (c.2385T>C), although to date the mechanistic basis for their association with VWF level is unknown. Using genotypic/phenotypic information from a European healthy control population, in vitro analyses of recombinant VWF expressing both SNVs, and in vivo murine models, this study determined the precise nature of their association with VWF level and investigated the mechanism(s) involved. Possession of either SNV corresponded with a significant increase in plasma VWF in healthy controls (P < .0001). In vitro expression confirmed this observation and highlighted an independent effect for each SNV (P < .0001 and P < .01, respectively), despite close proximity and strong linkage disequilibrium between them both. The influence of c.2365A>G on VWF levels was also confirmed in vivo. This increase in VWF protein corresponded to an increase in VWF messenger RNA (mRNA) resulting, in part, from prolonged mRNA half-life. In addition, coinheritance of both SNVs was associated with a lower VWF propeptide-to-VWF antigen ratio in healthy controls (P < .05) and a longer VWF half-life in VWF knockout mice (P < .0001). Both SNVs therefore directly increase VWF plasma levels through a combined influence on VWF biosynthesis and clearance, and may have an impact on disease phenotype in both hemostatic and thrombotic disorders.
Assuntos
Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , RNA Mensageiro , Fator de von Willebrand , Animais , Feminino , Humanos , Masculino , Camundongos , Estabilidade de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genéticaRESUMO
Intrauterine infusion of peanut oil at Day 10 post-ovulation has been reported to prolong dioestrus in mares. However, the effects of peanut oil treatment on the endometrium and whether the technique is painful have not been assessed. The objectives of this study were, (i) to determine the effect of intrauterine infusion of peanut oil on endometrial health, (ii) to determine whether use of intrauterine peanut oil is painful and (iii) to confirm that peanut oil causes prolonged dioestrus. Six mares aged 3-12 years old were used in a cross-over design with each mare administered both 1 ml of intrauterine peanut oil and a sham treatment on different oestrous cycles. The effect of intrauterine infusion of 1 ml peanut oil or sham treatment were measured using interovulatory period, uterine fluid accumulation as determined by transrectal ultrasonography, serum progesterone levels, endometrial Kenney biopsy scores and histological features, endometrial eosinophil numbers and salivary cortisol measurements. The individual mare response to intrauterine infusion of peanut oil was variable. Peanut oil infusion did not statistically prolong the luteal phase, nor elevate salivary cortisol levels but did cause superficial erosion of the endometrial surface epithelium in all mares and significantly increased eosinophil numbers in the endometrium (P = 0.0068). The Kenney grade for biopsies from 2/6 mares worsened transiently following infusion. In conclusion, intra-uterine peanut oil does not statistically increase the duration of the luteal phase but results in an inflammatory response and increase in endometrial eosinophil numbers suggesting treatment may be associated with a hypersensitivity-type reaction. Those contemplating using peanut oil to suppress oestrus should also be aware of the legislative and regulatory implications.
Assuntos
Endométrio/efeitos dos fármacos , Ciclo Estral/efeitos dos fármacos , Cavalos/fisiologia , Hidrocortisona/química , Óleo de Amendoim/farmacologia , Animais , Estudos Cross-Over , Feminino , Cavalos/sangue , Óleo de Amendoim/administração & dosagem , Progesterona/sangueRESUMO
A consanguineous pedigree is described where 14 individuals are affected with a novel autosomal recessive disorder, which causes static moderate mental retardation, truncal obesity, a congenital nonprogressive retinal dystrophy and micropenis in males. We have tentatively named this condition MORM syndrome. It shows similarities to Bardet-Biedl syndrome and Cohen syndrome, but can be distinguished by clinical features; the age of onset and nonprogressive nature of the visual impairment, the lack of characteristic facies, skin or gingival infection, microcephaly, 'mottled retina', polydactyly and small penis without testicular anomalies. Furthermore, linkage to the known Bardet-Biedl (BBS1-8) and Cohen syndrome loci was excluded. Autozygosity mapping identified a single homozygous subtelomeric region shared by all affecteds on chromosome 9q34.3, with a maximum LOD score of 5.64. We believe this to be the first example of the identification of a subtelomeric recessive locus by autozygosity mapping.