RESUMO
Molecular diagnosis is important to provide accurate genetic counseling of skeletal dysplasias (SD). Although next-generation sequencing (NGS) techniques are currently the preferred methods for analyzing these conditions, some of the published results have not shown a detection rate as high as it would be expected. The present study aimed to assess the diagnostic yield of targeted NGS combined with Sanger sequencing (SS) for low-coverage exons of genes of interest and exome sequencing (ES) in a series of patients with rare SD and use two patients as an example of our strategy. This study used two different in-house panels. Of 93 variants found in 88/114 (77%) patients, 57 are novel. The pathogenic variants found in the following genes: B3GALT6, PCYT1A, INPPL1, LIFR, of four patients were only detected by SS. In conclusion, the high diagnostic yield reached in the present study can be attributed to both a good selection of patients and the utilization of the SS for the insufficiently covered regions. Additionally, the two case reports-a patient with acrodysostosis related to PRKAR1A and another with ciliopathy associated with KIAA0753, add new and relevant clinical information to the current knowledge.
Assuntos
Disostoses , Osteocondrodisplasias , Colina-Fosfato Citidililtransferase , Galactosiltransferases , Aconselhamento Genético , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento do ExomaRESUMO
Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location.
Assuntos
Núcleo Celular/enzimologia , Colina-Fosfato Citidililtransferase/metabolismo , Células Epiteliais/efeitos dos fármacos , Membrana Nuclear/enzimologia , Prostaglandina D2/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Western Blotting , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/metabolismo , Indometacina/farmacologia , Rim/citologia , Masculino , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Ratos WistarRESUMO
In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.
Assuntos
Bufo arenarum/embriologia , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Embrião não Mamífero/enzimologia , Fosfatidilcolinas/metabolismo , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Bufo arenarum/metabolismo , Colina Quinase/genética , Colina-Fosfato Citidililtransferase/genética , Citosol/enzimologia , Feminino , Masculino , Óvulo/enzimologia , Fosfatidilcolinas/biossíntese , Fosfatidiletanolaminas/metabolismoRESUMO
Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-ß (C/EBPß) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPß expression. Elevated levels of C/EBPß bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Fosfolipídeos/biossíntese , Animais , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proliferação de Células , Colina Quinase/biossíntese , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Humanos , Camundongos , Neuritos/metabolismo , Neurônios/citologia , Fosfatidilcolinas/metabolismo , Fosfolipídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
Spondylometaphyseal dysplasia with cone-rod dystrophy is a rare autosomal-recessive disorder characterized by severe short stature, progressive lower-limb bowing, flattened vertebral bodies, metaphyseal involvement, and visual impairment caused by cone-rod dystrophy. Whole-exome sequencing of four individuals affected by this disorder from two Brazilian families identified two previously unreported homozygous mutations in PCYT1A. This gene encodes the alpha isoform of the phosphate cytidylyltransferase 1 choline enzyme, which is responsible for converting phosphocholine into cytidine diphosphate-choline, a key intermediate step in the phosphatidylcholine biosynthesis pathway. A different enzymatic defect in this pathway has been previously associated with a muscular dystrophy with mitochondrial structural abnormalities that does not have cartilage and/or bone or retinal involvement. Thus, the deregulation of the phosphatidylcholine pathway may play a role in multiple genetic diseases in humans, and further studies are necessary to uncover its precise pathogenic mechanisms and the entirety of its phenotypic spectrum.
Assuntos
Colina-Fosfato Citidililtransferase/genética , Osteocondrodisplasias/genética , Retinose Pigmentar/genética , Adolescente , Brasil , Criança , Pré-Escolar , Colina-Fosfato Citidililtransferase/metabolismo , Feminino , Genes Recessivos , Homozigoto , Humanos , Lactente , Masculino , Oftalmologia/métodos , LinhagemRESUMO
Lipid synthesis is a complex process regulated at multiple levels. Here, we will discuss nongenomic regulatory mechanisms, particularly the activation and/or recruitment of key enzymes to membranes. The phospholipid synthesis enzymes Lipin and CTP:phosphocholine cytidylyltransferase are taken as examples of these mechanisms that are mediated by posttranslational modifications or by an intrinsic property of the enzyme that senses lipid composition. In addition, special emphasis will be put on another relevant non genomic lipid synthesis regulation mechanism that is dependent on c-Fos, a protein that has deserved less attention so far. This latter regulatory mechanism is emerging as an important determinant for processes that require high rates of lipid synthesis such as those of growth and proliferation.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Fosfolipídeos/biossíntese , Processamento de Proteína Pós-Traducional , Membrana Celular/enzimologia , Proliferação de Células , Humanos , Lipídeos/biossíntese , Compostos Orgânicos/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.
Assuntos
Crithidia/efeitos dos fármacos , Crithidia/microbiologia , Fosforilcolina/análogos & derivados , Simbiose , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Crithidia/metabolismo , Crithidia/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Fosfatidilcolinas/biossíntese , Isótopos de Fósforo/metabolismo , Fosforilcolina/farmacologiaRESUMO
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.
Assuntos
Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Animais , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Diacilglicerol Colinofosfotransferase/metabolismo , Humanos , Mamíferos , Modelos Biológicos , Neurônios/citologiaRESUMO
Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.
Assuntos
Núcleo Celular/metabolismo , Colina-Fosfato Citidililtransferase/fisiologia , Enzimas/química , Fosfatidilcolinas/biossíntese , Animais , Linhagem Celular , Colina-Fosfato Citidililtransferase/química , Citoplasma/metabolismo , Cães , Retículo Endoplasmático/metabolismo , Inativação Gênica , Lamina Tipo A/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Fatores de TempoRESUMO
Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes that are dependent on membrane biosynthesis. Thus, the production of phosphatidylcholine (PtdCho), the major membrane phospholipid, should be stimulated during neuronal differentiation. We demonstrate that during retinoic acid (RA)-induced differentiation of Neuro-2a cells, PtdCho synthesis was promoted by an ordered and sequential activation of choline kinase alpha (CK(alpha)) and choline cytidylyltransferase alpha (CCT(alpha)). Early after RA stimulation, the increase in PtdCho synthesis is mainly governed by the biochemical activation of CCT(alpha). Later, the transcription of CK(alpha)- and CCT(alpha)-encoding genes was induced. Both PtdCho biosynthesis and neuronal differentiation are dependent on ERK activation. A novel mechanism is proposed by which PtdCho biosynthesis is coordinated during neuronal differentiation. Enforced expression of either CK(alpha) or CCTalpha increased the rate of synthesis and the amount of PtdCho, and these cells initiated differentiation without RA stimulation, as evidenced by cell morphology and the expression of genes associated with neuritogenesis. The differentiation resulting from enforced expression of CCT(alpha) or CK(alpha) was dependent on persistent ERK activation. These results indicate that elevated PtdCho synthesis could mimic the RA signals and thus determine neuronal cell fate. Moreover, they could explain the key role that PtdCho plays during neuronal regeneration.