RESUMEN
OBJECTIVES: The severe forms of hypertriglyceridaemia (HTG) are caused by mutations in genes that lead to the loss of function of lipoprotein lipase (LPL). In most patients with severe HTG (TG > 10 mmol L(-1) ), it is a challenge to define the underlying cause. We investigated the molecular basis of severe HTG in patients referred to the Lipid Clinic at the Academic Medical Center Amsterdam. METHODS: The coding regions of LPL, APOC2, APOA5 and two novel genes, lipase maturation factor 1 (LMF1) and GPI-anchored high-density lipoprotein (HDL)-binding protein 1 (GPIHBP1), were sequenced in 86 patients with type 1 and type 5 HTG and 327 controls. RESULTS: In 46 patients (54%), rare DNA sequence variants were identified, comprising variants in LPL (n = 19), APOC2 (n = 1), APOA5 (n = 2), GPIHBP1 (n = 3) and LMF1 (n = 8). In 22 patients (26%), only common variants in LPL (p.Asp36Asn, p.Asn318Ser and p.Ser474Ter) and APOA5 (p.Ser19Trp) could be identified, whereas no mutations were found in 18 patients (21%). In vitro validation revealed that the mutations in LMF1 were not associated with compromised LPL function. Consistent with this, five of the eight LMF1 variants were also found in controls and therefore cannot account for the observed phenotype. CONCLUSIONS: The prevalence of mutations in LPL was 34% and mostly restricted to patients with type 1 HTG. Mutations in GPIHBP1 (n = 3), APOC2 (n = 1) and APOA5 (n = 2) were rare but the associated clinical phenotype was severe. Routine sequencing of candidate genes in severe HTG has improved our understanding of the molecular basis of this phenotype associated with acute pancreatitis and may help to guide future individualized therapeutic strategies.
Asunto(s)
Hipertrigliceridemia , Adulto , Apolipoproteína A-V , Apolipoproteína C-II/genética , Apolipoproteínas A/genética , Proteínas Portadoras/genética , Femenino , Pruebas Genéticas , Humanos , Hipertrigliceridemia/epidemiología , Hipertrigliceridemia/genética , Hipertrigliceridemia/fisiopatología , Lipoproteína Lipasa/genética , Masculino , Proteínas de la Membrana/genética , Epidemiología Molecular , Mutación Missense , Países Bajos/epidemiología , Prevalencia , Receptores de Lipoproteína , Índice de Severidad de la EnfermedadRESUMEN
Mice carrying mutations in the fatty liver dystrophy (fld) gene have features of human lipodystrophy, a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertriglyceridemia and insulin resistance. Through positional cloning, we have isolated the gene responsible and characterized two independent mutant alleles, fld and fld(2J). The gene (Lpin1) encodes a novel nuclear protein which we have named lipin. Consistent with the observed reduction of adipose tissue mass in fld and fld(2J)mice, wild-type Lpin1 mRNA is expressed at high levels in adipose tissue and is induced during differentiation of 3T3-L1 pre-adipocytes. Our results indicate that lipin is required for normal adipose tissue development, and provide a candidate gene for human lipodystrophy. Lipin defines a novel family of nuclear proteins containing at least three members in mammalian species, and homologs in distantly related organisms from human to yeast.
Asunto(s)
Lipodistrofia/genética , Mutación/genética , Proteínas Nucleares/genética , Células 3T3 , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Alelos , Animales , Diferenciación Celular , Línea Celular , Núcleo Celular/química , Clonación Molecular , Secuencia Conservada , Evolución Molecular , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Perfilación de la Expresión Génica , Humanos , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/patología , Resistencia a la Insulina/genética , Leptina/análisis , Lipodistrofia/metabolismo , Lipodistrofia/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Fosfatidato Fosfatasa , ARN Mensajero/análisis , ARN Mensajero/genética , Mapeo de Híbrido por Radiación , Células Madre/citología , Células Madre/metabolismoRESUMEN
Lipodystrophies are a group of heterogeneous diseases characterized by the loss of adipose tissue and by abnormalities of carbohydrate and lipid metabolism, including insulin resistance, diabetes, and hyperlipidemia. In this review, we describe several mouse models that recapitulate various aspects of the lipodystrophy syndrome, offering insights into the etiology of this condition and potential therapeutic approaches. Studies on these mice suggest that adipose is the primary tissue affected in lipodystrophy, and that secondary leptin deficiency may be responsible for the associated insulin resistance.
Asunto(s)
Modelos Animales de Enfermedad , Lipodistrofia/fisiopatología , Ratones Transgénicos , Animales , Lipodistrofia/genética , RatonesRESUMEN
We describe a genomic DNA-based signal sequence trap method, signal-exon trap (SET), for the identification of genes encoding secreted and membrane-bound proteins. SET is based on the coupling of an exon trap to the translation of captured exons, which allows screening of the exon-encoded polypeptides for signal peptide function. Since most signal sequences are expected to be located in the 5'-terminal exons of genes, we first demonstrate that trapping of these exons is feasible. To test the applicability of SET for the screening of complex genomic DNA, we evaluated two critical features of the method. Specificity was assessed by the analysis of random genomic DNA and efficiency was demonstrated by screening a 425 kb YAC known to contain the genes of four secretory or membrane-bound proteins. All trapped clones contained a translation initiation signal followed by a hydrophobic stretch of amino acids representing either a known signal peptide, transmembrane domain or novel sequence. Our results suggest that SET is a potentially useful method for the isolation of signal sequence-containing genes and may find application in the discovery of novel members of known secretory gene clusters, as well as in other positional cloning approaches.
Asunto(s)
Clonación Molecular/métodos , Técnicas Genéticas , Señales de Clasificación de Proteína/genética , Animales , Células COS , Complemento C3/genética , ADN , Dipeptidil Peptidasa 4/genética , Exones , Genes Reporteros , Vectores Genéticos , Genoma , Humanos , Interleucinas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Señales de Clasificación de Proteína/metabolismo , Proteínas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Transcripción GenéticaRESUMEN
Dipeptidyl peptidase IV (CD26) is a membrane-associated enzyme that is expressed on the surface of T cells and on the hepatocyte brush border. In a soluble form it is present in serum. CD26 has been implicated in the regulation of T cell activation and in the metabolism of hormones and cytokines. Dipeptidyl peptidase (DPP) activity is elevated in the urine and serum of patients with biliary atresia (BA). To clarify the role of cholestasis in the development of increased serum and urinary DPP/CD26 activity, we studied the mechanism of activity increase in experimentally induced cholestasis of CD26-deficient and wild-type rats. The clinical utility of serum and urinary DPP/CD26 activity measurements was tested in adult and pediatric patients with hepatobiliary diseases and in liver transplant recipients. The results establish CD26-associated serum DPP activity as a novel, clinically useful marker of cholestasis and demonstrate that in contrast with alkaline phosphatase levels, DPP levels do not change in metastatic bone disease. Additionally, DPP activity is useful as a urinary test of cholestasis in infants who are not receiving nephrotoxic medication.
Asunto(s)
Biomarcadores/sangre , Biomarcadores/orina , Colestasis/enzimología , Dipeptidil Peptidasa 4/metabolismo , Fosfatasa Alcalina/sangre , Animales , Bilirrubina/sangre , Neoplasias Óseas/sangre , Neoplasias Óseas/enzimología , Neoplasias Óseas/secundario , Estudios de Casos y Controles , Colestasis/sangre , Colestasis/orina , Dipeptidil Peptidasa 4/sangre , Dipeptidil Peptidasa 4/orina , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Hepatopatías/sangre , Hepatopatías/enzimología , Hepatopatías/orina , Ratas , Ratas Endogámicas BUF , Ratas Endogámicas F344RESUMEN
The fatty liver dystrophy (fld) mutation is manifested in abnormalities of lipid and glucose metabolism and peripheral neuropathy. To identify the gene affected by this mutation, we generated a genetic map of the fld region on chromosome 12 by the analysis of F2 offspring from an intersubspecific cross between strains BALB/cByJ-fld and CAST/EiJ. The results localize fld to the 0.42-cM interval between the microsatellite markers D12Mit170 and D12Mit184. A contig of YACs and BACs covering the nonrecombinant genomic region has been constructed and used for the identification of genes. Expressed sequence tag mapping and exon trapping identified three transcripts within the critical interval: Ctla2b, which encodes a cysteine protease inhibitor, and mouse homologs of KIAA0188 and KIAA0575, two long human transcripts of unknown function. Expression analysis revealed that Kiaa0188 is expressed in wildtype but not in fld liver, implicating this gene as a candidate for harboring the fld mutation.
Asunto(s)
Mapeo Cromosómico , Cromosomas/genética , Hígado Graso/genética , Proteínas Nucleares , Proteínas/genética , Animales , Clonación Molecular , Mapeo Contig , Cruzamientos Genéticos , Exones , Femenino , Expresión Génica , Marcadores Genéticos , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Fosfatidato Fosfatasa , Lugares Marcados de SecuenciaRESUMEN
Isolated lissencephaly sequence and Miller-Dieker syndrome are related neurodevelopmental disorders caused by defects of the LIS1 gene encoding the alpha subunit of intracellular platelet-activating factor acetylhydrolase. In addition to the ortholog of the human LIS1 gene (Pafaha/Lis1), the mouse genome contains two more homologs. In order to characterize the new members of this gene family, we isolated both Pafaha/Lis1-related genes (Pafaha-ps1 and Pafaha-ps2) from a mouse genomic library. Pafaha-ps1 and Pafaha-ps2 are processed pseudogenes formed by the retroinsertion of 5'-truncated Pafaha/Lis1 cDNAs. Sequence analysis revealed a striking accumulation of retroelements at both loci, identifying two retroinsertion hotspots in the mouse genome. The recognition of tRNA genes flanking Pafaha-ps1 provides an example for the potential association of RNA polymerase III transcription and retroinsertion in mammals. Linkage mapping placed Pafaha-ps1 and Pafaha-ps2 to distal chromosome (Chr) 3 and proximal Chr 7, respectively. Our results indicate that only one of the three LIS1-related mouse loci (Pafaha/Lis1) is functional, in contrast with two closely related functional genes (LIS1 and LIS2) reported in humans. 1998 Elsevier Science B.V.
Asunto(s)
Mapeo Cromosómico , Genes de Partícula A Intracisternal/genética , Proteínas Asociadas a Microtúbulos , Proteínas/genética , Seudogenes/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Clonación Molecular , Cruzamientos Genéticos , Elementos Transponibles de ADN/genética , ADN Complementario/genética , Genoma , Ratones , Mutación/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/genéticaRESUMEN
Platelet-activating factor acetylhydrolases (PAF-AHs) play an important role in the metabolism of PAF, a potent phospholipid mediator affecting various physiological processes. The heterotrimeric form of intracellular PAF-AH consists of two catalytic subunits (PAF-AH Ib beta and PAF-AH Ib gamma) and a potential regulatory subunit (PAF-AH Ib alpha). Hemizygous deletion of the gene encoding the alpha subunit has been implicated in two related neurological disorders: isolated lissencephaly sequence and Miller-Dieker syndrome. Here we report the isolation and characterization of mouse Pafaha/Lis1 cDNAs and the corresponding Pafaha/Lis1 gene. We have cloned five cDNAs representing alternatively polyadenylated messages. Northern blot analysis revealed that the various Pafaha/Lis1 mRNAs are differentially expressed in mouse tissues. The Pafaha/Lis1 gene spans a genomic region of more than 50 kb and consists of 12 exons, the first 2 of which are embedded in CpG islands. We have identified two sites of alternative splicing of Pafaha/Lis1: one affecting the length of the 5' untranslated region, the other potentially resulting in a truncated form of the encoded protein.
Asunto(s)
Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Proteínas Asociadas a Microtúbulos , Fosfolipasas A/genética , Factor de Activación Plaquetaria/metabolismo , Proteínas/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Empalme Alternativo , Animales , Encéfalo/metabolismo , Exones , Intrones , Ratones , Datos de Secuencia Molecular , Fosfolipasas A/química , Reacción en Cadena de la Polimerasa , Proteínas/química , Análisis de Secuencia de ADNRESUMEN
Lissencephaly is a human brain malformation manifested by a smooth cerebral surface and severe mental retardation. Some of the patients have been shown to have deletions in chromosome 17p13.3, and recently, LIS-1 has been proposed to be the disease-associated gene. We have now mapped the mouse homolog of LIS-1 to mouse chromosome 11B3 by using fluorescence in situ hybridization to metaphase chromosomes. The analysis of yeast artificial chromosome clones placed Lis-1 in close proximity to the microsatellite marker D11Mit65.
Asunto(s)
Encéfalo/anomalías , Mapeo Cromosómico , Anomalías Congénitas/genética , Ratones/genética , Proteínas Asociadas a Microtúbulos , Proteínas/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Southern Blotting , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Discapacidad Intelectual/genéticaRESUMEN
The nucleotide (nt) sequence of the LIS-1 cDNA encoding the murine lissencephaly-1 (LIS-1) protein has been determined. The deduced protein shows a very high degree (99.8%) of homology with human LIS-1, having a single conservative amino acid (aa) change out of 410 aa and is identical to a subunit of bovine platelet-activating factor (PAF) acetylhydrolase.
Asunto(s)
Hominidae/genética , Ratones/genética , Proteínas Asociadas a Microtúbulos , Proteínas/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , ADN Complementario/química , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
The induction of interferon (IFN) by human immunodeficiency virus type 1 (HIV-1) in primary, nonstimulated monocyte-macrophage cultures was studied. HIV-1 infection, as confirmed by p24 antigen levels in the cell supernatant, led to the production of alpha interferon (IFN-alpha) over 7 to 21 days following infection. In two of seven experiments, the IFN detected was acid labile. Coupled reverse transcription-polymerase chain reaction analysis confirmed the induction of IFN-alpha mRNA in cells of HIV-1-infected cultures.
Asunto(s)
VIH-1/inmunología , Interferón-alfa/biosíntesis , Macrófagos/inmunología , Monocitos/inmunología , Adhesión Celular , Línea Celular , Transformación Celular Viral , Células Cultivadas , VIH-1/genética , Humanos , Interferón-alfa/análisis , Interferón-alfa/genética , Cinética , Macrófagos/microbiología , Monocitos/microbiología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.
Asunto(s)
Centrómero/ultraestructura , ADN/fisiología , Animales , Clonación Molecular , Humanos , Técnicas In Vitro , Kanamicina Quinasa , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosfotransferasas/genética , Mapeo RestrictivoRESUMEN
Central nervous system involvement of systemic lupus erythematosus was observed in 34 (36%) of the 94 patients studied between 1970-1990. A review of the diagnostic methods and therapy for central nervous system lupus is presented. The diagnosis of primary cerebral lupus was based on the history, physical examination and on the results of the cerebrospinal fluid analysis, CT-scan and EEG. Intractable headache (22/34), behavioural abnormalities (18/34), cranial neuropathy (16/34) occurred most frequently among neuropsychiatric symptoms. Immunoglobulin analysis of the cerebrospinal fluid proved to be the most sensitive method for detecting clinical activity (in 20/23). Central nervous system involvement was suggested by conventional serological test to a lesser degree. Alterations on CT scan and EEG were found in 17/27 and 14/26 of cases, respectively. IgM, IgA, and IgG indexes (indicators of intratechal immunglobulin synthesis) decreased when the central nervous system events subsided after successful treatment but the CT abnormalities (e. g. atrophy) were not altered.
Asunto(s)
Lupus Eritematoso Sistémico/fisiopatología , Enfermedades del Sistema Nervioso/fisiopatología , Electroencefalografía , Humanos , Lupus Eritematoso Sistémico/líquido cefalorraquídeo , Enfermedades del Sistema Nervioso/líquido cefalorraquídeo , Tomografía Computarizada por Rayos XRESUMEN
In a case of severe IgG kappa myeloma with cryoglobulinaemia usual concentrations of epinephrine, collagen, ADP, arachidonic acid, thrombin and ristocetin caused no aggregation of platelets in platelet rich plasma. However, in contrast to other agents ristocetin induced platelet aggregation in higher concentrations. The investigations showed, that the aggregating activity was inhibited by binding of ristocetin to the abnormal protein. Following saturation of the monoclonal protein, the surplus ristocetin caused normal aggregation. This indicates that platelets actually preserved their responsiveness to ristocetin. Possible causes of the phenomenon are discussed.