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1.
Hum Mol Genet ; 28(9): 1463-1473, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576445

RESUMO

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of CAG repeats in the ATXN3 gene leading to an elongated polyglutamine tract in the ataxin-3 protein. Previously, we demonstrated that symptoms of SCA3 are reversible in the first conditional mouse model for SCA3 directing ataxin-3 predominantly to the hindbrain. Here, we report on the effects of transgenic ataxin-3 expression in forebrain regions. Employing the Tet-off CamKII-promoter mouse line and our previously published SCA3 responder line, we generated double transgenic mice (CamKII/MJD77), which develop a neurological phenotype characterized by impairment in rotarod performance, and deficits in learning new motor tasks as well as hyperactivity. Ataxin-3 and ubiquitin-positive inclusions are detected in brains of double transgenic CamKII/MJD77 mice. After turning off the expression of pathologically expanded ataxin-3, these inclusions disappear. However, the observed phenotype could not be reversed, very likely due to pronounced apoptotic cell death in the frontal brain. Our data demonstrate that cerebellar expression is not required to induce a neurological phenotype using expanded ATXN3 as well as the pronounced sensibility of forebrain neurons for toxic ataxin-3.


Assuntos
Ataxina-3/genética , Lobo Frontal/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Neurônios/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Ataxina-3/metabolismo , Comportamento Animal , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Modelos Animais de Doenças , Lobo Frontal/patologia , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Imuno-Histoquímica , Doença de Machado-Joseph/patologia , Camundongos , Camundongos Transgênicos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Especificidade de Órgãos/genética , Agregados Proteicos , Agregação Patológica de Proteínas , Desempenho Psicomotor
2.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360608

RESUMO

Achromatopsia (ACHM) is a rare autosomal recessively inherited retinal disease characterized by congenital photophobia, nystagmus, low visual acuity, and absence of color vision. ACHM is genetically heterogeneous and can be caused by biallelic mutations in the genes CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, or ATF6. We undertook molecular genetic analysis in a single female patient with a clinical diagnosis of ACHM and identified the homozygous variant c.778G>C;p.(D260H) in the CNGA3 gene. While segregation analysis in the father, as expected, identified the CNGA3 variant in a heterozygous state, it could not be displayed in the mother. Microsatellite marker analysis provided evidence that the homozygosity of the CNGA3 variant is due to partial or complete paternal uniparental isodisomy (UPD) of chromosome 2 in the patient. Apart from the ACHM phenotype, the patient was clinically unsuspicious and healthy. This is one of few examples proving UPD as the underlying mechanism for the clinical manifestation of a recessive mutation in a patient with inherited retinal disease. It also highlights the importance of segregation analysis in both parents of a given patient or especially in cases of homozygous recessive mutations, as UPD has significant implications for genetic counseling with a very low recurrence risk assessment in such families.


Assuntos
Cromossomos Humanos Par 2/genética , Defeitos da Visão Cromática/patologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Pai , Mutação , Dissomia Uniparental , Adolescente , Defeitos da Visão Cromática/genética , Feminino , Genes Recessivos , Humanos , Masculino , Linhagem , Fenótipo
3.
Brain ; 142(6): 1528-1534, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31009037

RESUMO

Herein we present a consanguineous family with three children affected by foveal hypoplasia with infantile nystagmus, following an autosomal recessive mode of inheritance. The patients showed normal electroretinography responses, no signs of albinism, and no anterior segment or brain abnormalities. Upon whole exome sequencing, we identified a homozygous mutation (c.1861C>T;p.Q621*) in the aryl hydrocarbon receptor (AHR) gene that perfectly co-segregated with the disease in the larger family. AHR is a ligand-activated transcription factor that has been intensively studied in xenobiotic-induced toxicity. Further, it has been shown to play a physiological role under normal cellular conditions, such as in immunity, inflammatory response and neurogenesis. Notably, knockout of the Ahr gene in mouse impairs optic nerve myelin sheath formation and results in oculomotor deficits sharing many features with our patients: the eye movement disorder in Ahr-/- mice appears early in development and presents as conjugate horizontal pendular nystagmus. We therefore propose AHR to be a novel disease gene for a new, recessively inherited disorder in humans, characterized by infantile nystagmus and foveal hypoplasia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Homozigoto , Nistagmo Congênito/genética , Hipoplasia do Nervo Óptico/genética , Receptores de Hidrocarboneto Arílico/genética , Animais , Criança , Eletrorretinografia/métodos , Feminino , Humanos , Masculino , Camundongos , Mutação/genética , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Nistagmo Congênito/diagnóstico , Hipoplasia do Nervo Óptico/patologia , Linhagem
4.
Genet Med ; 21(8): 1998, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30297699

RESUMO

The original version of this Article contained an error in the spelling of the author Anja K. Mayer, which was incorrectly given as Anja Kathrin Mayer. This has now been corrected in both the PDF and HTML versions of the Article.

5.
Hum Mutat ; 38(11): 1579-1591, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28795510

RESUMO

Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease-causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real-time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray-based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4-18 was identified, highlighting the importance of CNV analysis for this gene.


Assuntos
Defeitos da Visão Cromática/diagnóstico , Defeitos da Visão Cromática/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Variações do Número de Cópias de DNA , Mutação , Alelos , Mapeamento Cromossômico , Segregação de Cromossomos , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Éxons , Efeito Fundador , Genótipo , Humanos , Taxa de Mutação
6.
Eur J Hum Genet ; 28(6): 742-753, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31896775

RESUMO

Visual impairment due to inherited ophthalmic disorders is amongst the most common disabilities observed in populations practicing consanguineous marriages. Here we investigated the molecular genetic basis of an unselected broad range of ophthalmic disorders in 20 consanguineous families from Arab villages of Israel and the Palestinian Authority. Most patients had little or very poor prior clinical workup and were recruited in a field study. Homozygosity mapping followed by candidate gene sequencing applying conventional Sanger sequencing or targeted next generation sequencing was performed in six families. In the remaining 14 families, one affected subject per family was chosen for whole exome sequencing. We discovered likely disease-causing variants, all homozygous, in 19 of 20 independent families (95%) including a previously reported novel disease gene for congenital nystagmus associated with foveal hypoplasia. Moreover, we found a family in which disease-causing variants for two collagenopathies - Stickler and Knobloch syndrome - segregate within a large sibship. Nine of the 19 distinct variants observed in this study were novel. Our study demonstrated a very high molecular diagnostic yield for a highly diverse spectrum of rare ophthalmic disorders in Arab patients from Israel and the Palestinian Authority, even with very limited prior clinical investigation. We conclude that 'genetic testing first' may be an economic way to direct clinical care and to support proper genetic counseling and risk assessment in these families.


Assuntos
Árabes/genética , Oftalmopatias Hereditárias/genética , Mutação , Oftalmopatias Hereditárias/epidemiologia , Oftalmopatias Hereditárias/etnologia , Feminino , Loci Gênicos , Humanos , Israel , Masculino , Linhagem
7.
J Clin Invest ; 128(12): 5663-5675, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30418171

RESUMO

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.


Assuntos
Defeitos da Visão Cromática , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Heterozigoto , Ativação do Canal Iônico , Mutação de Sentido Incorreto , Células Fotorreceptoras Retinianas Cones , Doenças Retinianas , Substituição de Aminoácidos , Animais , Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/metabolismo , Defeitos da Visão Cromática/patologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia
8.
Arch Immunol Ther Exp (Warsz) ; 55(6): 353-62, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18060372

RESUMO

Epithelial cells are the first line of defense against invading microbial pathogens. They are important contributors to innate mucosal immunity and generate various and sophisticated anti-microbial defense mechanisms, including the formation of a tight barrier and secretion of anti-microbial substances as well as inflammatory mediators. To provide these active defense mechanisms, epithelial cells functionally express various pattern-recognition receptors. Toll-like receptors have been shown to recognize conserved microbial patterns mediating inducible activation of innate immunity. Mucosal surfaces, however, are prone to contact with pathogenic as well as non-pathogenic microbes and, therefore, immune-recognition principles have to be strictly regulated to avoid uncontrolled permanent activation. This review will focus on mechanisms by which epithelial cells regulate mucosal immune responses, thus creating an organ-specific microenvironment. This includes local adaptations in microbial recognition, regulation of local immune homeostasis, and modulation of antigen-presenting cells and adaptive immune responses. These regulatory mechanisms serve the special needs of controlled microbial recognition in mucosal compartments.


Assuntos
Brônquios/imunologia , Células Epiteliais/imunologia , Retroalimentação Fisiológica/imunologia , Retroalimentação Fisiológica/fisiologia , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Animais , Infecções Bacterianas/imunologia , Humanos , Imunidade Inata , Especificidade de Órgãos , Receptores Toll-Like/imunologia
9.
Sci Rep ; 7(1): 6992, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765526

RESUMO

We report ophthalmic and genetic findings in patients with autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy (CRD) or cone dystrophy (CD) harboring potential pathogenic variants in the CDHR1 gene. Detailed ophthalmic examination was performed in seven sporadic and six familial subjects. Mutation screening was done using a customized next generation sequencing panel targeting 105 genes implicated in inherited retinal disorders. In one family, homozygosity mapping with subsequent candidate gene analysis was performed. Stringent filtering for rare and potentially disease causing variants following a model of autosomal recessive inheritance led to the identification of eleven different CDHR1 variants in nine index cases. All variants were novel at the time of their identification. In silico analyses confirmed their pathogenic potential. Minigene assays were performed for two non-canonical splice site variants and revealed missplicing for the mutant alleles. Mutations in CDHR1 are a rare cause of retinal dystrophy. Our study further expands the mutational spectrum of this gene and the associated clinical presentation.


Assuntos
Caderinas/genética , Distrofias de Cones e Bastonetes/genética , Mutação , Proteínas do Tecido Nervoso/genética , Retinose Pigmentar/genética , Proteínas Relacionadas a Caderinas , Distrofias de Cones e Bastonetes/patologia , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Retinose Pigmentar/patologia
10.
Eur J Hum Genet ; 24(3): 459-62, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26153215

RESUMO

Several genes have been implicated in the autosomal recessive form of cone-rod dystrophy (CRD), but the majority of cases remain unsolved. We identified a homozygous interval comprising two known genes associated with the autosomal recessive form of CRD, namely RAB28 and PROM1, in a consanguineous family with clinical evidence of CRD. Both genes proved to be mutation negative upon sequencing of exons and canonical splice sites but whole-genome sequencing revealed a private variant located deep in intron 18 of PROM1. In silico and functional analyses of this variant using minigenes as splicing reporters revealed the integration of a pseudoexon in the mutant transcript, thereby leading to a premature termination codon and presumably resulting in a functional null allele. This is the first report of a deep intronic variant that acts as a splicing mutation in PROM1. The detection of such variants escapes the exon-focused techniques typically used in genetic analyses. Sequencing the entire genomic regions of known disease genes might identify more causal mutations in the autosomal recessive form of CRD.


Assuntos
Antígenos CD/genética , Éxons/genética , Predisposição Genética para Doença , Genoma Humano , Glicoproteínas/genética , Íntrons/genética , Mutação/genética , Peptídeos/genética , Retinose Pigmentar/genética , Antígeno AC133 , Sequência de Aminoácidos , Antígenos CD/química , Sequência de Bases , Análise Mutacional de DNA , Feminino , Glicoproteínas/química , Células HEK293 , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Peptídeos/química , Splicing de RNA/genética
11.
PLoS One ; 11(1): e0145951, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26766544

RESUMO

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.


Assuntos
Mutação , Distrofias Retinianas/genética , Variações do Número de Cópias de DNA , Exoma , Proteínas do Olho/genética , Feminino , Estudos de Associação Genética , Heterogeneidade Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Taxa de Mutação , Linhagem , Fenótipo , Distrofias Retinianas/diagnóstico
12.
Eur J Immunol ; 38(6): 1689-99, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18421791

RESUMO

The upper airways are prone to contact with pathogenic as well as non-pathogenic microbes, therefore immune recognition principles have to be tightly controlled. Here we show that human BEAS-2B bronchial epithelial cells inhibited secretion of the pro-inflammatory cytokines TNF-alpha and IL-12 by monocytes, macrophages and dendritic cells. This inhibitory effect could be transferred by supernatant of resting BEAS-2B cells and was also observed when primary murine tracheal epithelial cells were prepared. In contrast to inhibition of pro-inflammatory cytokine secretion epithelial cell-conditioned dendritic cells showed increased expression of IL-10 and arginase-1, thus displaying properties of alternative activation. Accordingly, Toll-like receptor-mediated up-regulation of CD40, CD86 and PD-L2 (CD273) on murine dendritic cells was reduced in the presence of bronchial epithelial cell supernatant. However, expression of negative regulatory PD-L1 (CD274) was increased and dendritic cell induced proliferation of T lymphocytes was diminished. Epithelial cells also showed a direct inhibitory effect on T lymphocyte proliferation and this was due to the constitutive secretion of TGF-beta by bronchial epithelial cells. Moreover, epithelial cell-conditioned T lymphocytes showed increased differentiation towards IL-10-producing Tr1 cells. The results indicate that bronchial epithelial cells induce a non-inflammatory microenvironment that regulates local immune homeostasis.


Assuntos
Células Epiteliais/imunologia , Imunidade nas Mucosas/imunologia , Monócitos/metabolismo , Mucosa Respiratória/imunologia , Animais , Antígenos CD/metabolismo , Arginase/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , Mucosa Respiratória/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Receptores Toll-Like/agonistas , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
13.
J Immunol ; 178(5): 3134-42, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17312161

RESUMO

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1-6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.


Assuntos
Brônquios/imunologia , Antígenos CD36/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Infecções Respiratórias/imunologia , Receptores Toll-Like/imunologia , Animais , Antígenos CD36/genética , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Humanos , Imunidade Inata/genética , Ligantes , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/genética , Receptores Toll-Like/deficiência
14.
Genesis ; 33(3): 125-30, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12124945

RESUMO

We describe the generation of transgenic mouse lines expressing Cre recombinase in epithelial cells of the lactating mammary gland. As an expression vector, we used a P1-derived bacterial artificial chromosome (PAC) which harbors the gene for the secretory milk protein, whey acidic protein (Wap). Using homologous recombination in E. coli, the PAC was modified to carry the improved coding sequence of Cre recombinase (iCre). Transgenic lines carrying the WAPiCre PAC express Cre recombinase efficiently in the majority of mammary epithelial cells upon lactation. Of only four transgenic lines produced, three express Cre recombinase to a high efficiency. LoxP-flanked DNA sequences are recombined in virtually all epithelial cells of WAPiCre transgenic mice at lactation day 3.


Assuntos
Mama/citologia , Mama/metabolismo , Cromossomos Artificiais Bacterianos/genética , Células Epiteliais/metabolismo , Marcação de Genes/métodos , Animais , Linhagem Celular , Expressão Gênica , Vetores Genéticos/genética , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Especificidade de Órgãos , Proteínas Virais/genética , Proteínas Virais/metabolismo
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