RESUMEN
Mutations in the transcription factor PAX9 which plays a critical role in the switching of odontogenic potential from the epithelium to the mesenchyme during tooth development cause autosomal dominant non-syndromic hypodontia primarily affecting molars. Linkage analysis on a family segregating autosomal dominant molar hypodontia with markers flanking and within PAX9 yielded a maximum multipoint LOD score of 3.6. No sequence variants were detected in the coding or 5'- and 3'-untranslated regions (UTRs) of PAX9. However, we identified a novel g.-1258G>A sequence variant in all affected individuals of the family but not in the unaffected family members or in 3088 control chromosomes. This mutation is within a putative 5'-regulatory sequence upstream of PAX9 highly conserved in primates, somewhat conserved in ungulates and carnivores but not conserved in rodents. Bioinformatics analysis of the sequence determined that there was no abolition or creation of a putative binding site for known transcription factors. Based on our previous findings that haploinsufficiency for PAX9 leads to hypodontia, we postulate that the g.-1258G>A variant reduces the expression of PAX9 which underlies the hypodontia phenotype in this family.
Asunto(s)
Región de Flanqueo 5' , Anodoncia/genética , Trastornos de los Cromosomas , Cromosomas Humanos Par 14 , Secuencia Conservada , Diente Molar/patología , Odontogénesis/genética , Factor de Transcripción PAX9/genética , Animales , Anodoncia/patología , Secuencia de Bases , Carnívoros , Biología Computacional/métodos , Femenino , Genes Dominantes , Estudios de Asociación Genética , Ligamiento Genético , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Roedores , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
AIM: Congenital absence of teeth is a complex condition affecting several parameters of oral development. This is the first study to measure tooth crown dimensions using image analysis in a family with hypodontia in whom the mutation has been identified, and compare them with a control group. METHODS AND RESULTS: Study models were obtained from 10 family members from three generations affected by severe hypodontia with a missense mutation in PAX9 and 10 unaffected, unrelated controls. Using established image analysis techniques all teeth up to and including the first permanent molars were digitally imaged by two operators from the occlusal (O) and buccal (B) aspects three times and an average made for the mesio-distal (MDO and MDB) bucco-lingual (BL), area (A) and perimeter (P) measurements. Intra-class correlation coefficients (ICCC) were calculated to assess intra- and inter-operator reliability. Two-sample t-tests were then used to compare these dimensions with those of the controls. Reliability of the technique was high (mean r>0.95). The majority of tooth types throughout the dentition were significantly smaller in the family members with hypodontia than in the control group for all parameters measured. The levels of significance were very high for upper lateral incisors (p<0.0001) whilst the canines and first molars were less different. The greatest number of significant differences were found in BL and P, closely followed by MD and A measurements. CONCLUSIONS: The significantly smaller tooth crown dimensions recorded in the affected family members show that the effect of the PAX9 mutation is seen not only in the congenitally missing teeth but also in smaller crown size throughout the dentition.
Asunto(s)
Anodoncia/genética , Mutación , Factor de Transcripción PAX9/genética , Corona del Diente/anomalías , Estudios de Casos y Controles , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Modelos Dentales , Fenotipo , Fotograbar , Reproducibilidad de los ResultadosRESUMEN
When performing association studies in populations that have not been the focus of large-scale investigations of haplotype variation, it is often helpful to rely on genomic databases in other populations for study design and analysis - such as in the selection of tag SNPs and in the imputation of missing genotypes. One way of improving the use of these databases is to rely on a mixture of database samples that is similar to the population of interest, rather than using the single most similar database sample. We demonstrate the effectiveness of the mixture approach in the application of African, European, and East Asian HapMap samples for tag SNP selection in populations from India, a genetically intermediate region underrepresented in genomic studies of haplotype variation.
Asunto(s)
Bases de Datos Genéticas , Desequilibrio de Ligamiento , Grupos de Población/genética , Bases de Datos Genéticas/normas , Genética de Población , Genoma Humano , Haplotipos , Humanos , India , Polimorfismo de Nucleótido Simple , Proyectos de InvestigaciónRESUMEN
Smith-Magenis syndrome (SMS) is a multisystem disorder characterized by developmental delay and mental retardation, a distinctive behavioral phenotype, and sleep disturbance. We undertook a comprehensive meta-analysis to identify genotype-phenotype relationships to further understand the clinical variability and genetic factors involved in SMS. Clinical and molecular information on 105 patients with SMS was obtained through research protocols and a review of the literature and analyzed using Fisher's exact test with two-tailed p values. Several differences in these groups of patients were identified based on genotype and gender. Patients with RAI1 mutation were more likely to exhibit overeating, obesity, polyembolokoilamania, self-hugging, muscle cramping, and dry skin and less likely to have short stature, hearing loss, frequent ear infections, and heart defects when compared with patients with deletion, while a subset of small deletion cases with deletions spanning from TNFRSF13B to MFAP4 was less likely to exhibit brachycephaly, dental anomalies, iris abnormalities, head-banging, and hyperactivity. Significant differences between genders were also identified, with females more likely to have myopia, eating/appetite problems, cold hands and feet, and frustration with communication when compared with males. These results confirm previous findings and identify new genotype-phenotype associations including differences in the frequency of short stature, hearing loss, ear infections, obesity, overeating, heart defects, self-injury, self-hugging, dry skin, seizures, and hyperactivity among others based on genotype. Additional studies are required to further explore the relationships between genotype and phenotype and any potential discrepancies in health care and parental attitudes toward males and females with SMS.
Asunto(s)
Anomalías Múltiples/genética , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Discapacidad Intelectual/patología , Anomalías Múltiples/patología , Femenino , Genotipo , Humanos , Masculino , Fenotipo , Mutación Puntual , Factores Sexuales , Síndrome , Transactivadores , Factores de Transcripción/genéticaRESUMEN
Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.
Asunto(s)
Proteínas de la Mielina/genética , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/farmacología , Regiones Promotoras Genéticas/genética , Animales , Emparejamiento Base , Unión Competitiva , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/terapia , Cricetinae , ADN/química , Huella de ADN , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Fibroblastos/metabolismo , Terapia Genética , Humanos , Manganeso/farmacología , Vaina de Mielina/fisiología , Conformación de Ácido Nucleico/efectos de los fármacos , Oligodesoxirribonucleótidos/genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Over- and under expression of the 22 kDa peripheral myelin protein (PMP22) results in dysmyelinating peripheral neuropathies, such as Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy, with the liability to pressure palsies (HNPP). Expression of the PMP22 gene is driven by two alternative promoters, P1 and P2, with transcripts originating from P1 associated with peripheral nerve myelination by Schwann cells. Transient transfections of constructs containing P1 (3.5 kb) or P2 (2.5 kb) resulted in high levels of reporter gene expression in the RT4-D6P2T schwannoma cell line. Serial deletions of P1 revealed that region P1-A (-105 to -43), situated upstream of the minimal promoter, contained a positive regulatory element. The 62 bp P1-A region conferred in cis a sevenfold increase in expression of luciferase driven by a heterologous promoter in an orientation-dependent manner. Interspecies comparison of the P1-A region revealed a 98% degree of identity between the human, mouse, and rat sequences. A prominent sequence-dependent DNA-protein complex (C-I) was detected in electrophoretic mobility shift assays with P1-A using RT4-D6P2T nuclear extract and was localized to a minimal 21 bp region within P1-A. Site-directed mutagenesis of this region revealed nucleotides at positions -46 to -43 as being necessary for formation of C-I. Functional analysis of the mutated P1-A element indicated that positions -46 and -45 were essential for transactivation mediated by this element. Characterization of the transacting factor(s) interacting with this key regulatory element will shed light on its role in regulating peripheral nerve myelination.
Asunto(s)
Genes Reguladores/genética , Proteínas de la Mielina/genética , Vaina de Mielina/metabolismo , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Sitios de Unión/genética , Regulación de la Expresión Génica/fisiología , Vectores Genéticos/fisiología , Humanos , Datos de Secuencia Molecular , Mutación/genética , Proteínas de la Mielina/biosíntesis , Nucleótidos/genética , Unión Proteica/genética , Homología de Secuencia de Ácido Nucleico , Virus 40 de los Simios/genética , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas/metabolismo , Regulación hacia Arriba/genéticaAsunto(s)
Ataxia de Friedreich/genética , Proteínas de Unión a Hierro , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Expansión de Repetición de Trinucleótido/genética , Animales , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patología , Ataxia de Friedreich/terapia , Mutación de Línea Germinal/genética , Homeostasis , Humanos , Hierro/metabolismo , Ratones , Mitocondrias/metabolismo , Modelos Genéticos , Conformación de Ácido Nucleico , Estrés Oxidativo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transcripción Genética/genética , FrataxinaAsunto(s)
Anodoncia/genética , Investigación Dental , Salud de la Familia , Humanos , Selección de PacienteRESUMEN
Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome associated with an interstitial deletion of chromosome 17 involving band p11.2. SMS is hypothesised to be a contiguous gene syndrome in which the phenotype arises from the haploinsufficiency of multiple, functionally-unrelated genes in close physical proximity, although the true molecular basis of SMS is not yet known. In this study, we have generated the first overlapping and contiguous transcription map of the SMS critical interval, linking the proximal 17p11.2 region near the SMS-REPM and the distal region near D17S740 in a minimum tiling path of 16 BACs and two PACs. Additional clones provide greater coverage throughout the critical region. Not including the repetitive sequences that flank the critical interval, the map is comprised of 13 known genes, 14 ESTs, and six genomic markers, and is a synthesis of Southern hybridisation and polymerase chain reaction data from gene and marker localisation to BACs and PACs and database sequence analysis from the human genome project high-throughput draft sequence. In order to identify possible candidate genes, we performed sequence analysis and determined the tissue expression pattern analysis of 10 novel ESTs that are deleted in all SMS patients. We also present a detailed review of six promising candidate genes that map to the SMS critical region.
Asunto(s)
Anomalías Múltiples/genética , Mapeo Cromosómico , Cromosomas Humanos Par 17/genética , Eliminación de Gen , Genoma Humano , Discapacidad Intelectual/genética , Aberraciones Cromosómicas , Clonación Molecular , Etiquetas de Secuencia Expresada , Facies , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , SíndromeRESUMEN
Frataxin is a nuclear-encoded mitochondrial protein widely conserved among eukaryotes. Human frataxin (fxn) is severely reduced in Friedreich ataxia (FRDA), a frequent autosomal recessive neuro- and cardio-degenerative disease. Whereas the function of fxn is unknown, the yeast frataxin homolog (Yfh1p) has been shown to be involved in mitochondrial iron homeostasis and protection from free radical toxicity. Evidence of iron accumulation and oxidative damage in cardiac tissue from FRDA patients suggests that fxn may have a similar function, but whether yeast and human frataxin actually have interchangeable roles in mitochondrial iron homeostasis is unknown. We show that a wild-type FRDA cDNA can complement Yfh1p-deficient yeast (yfh1 delta) by preventing the mitochondrial iron accumulation and oxidative damage associated with loss of Yfh1p. We analyze the functional effects of two FRDA point mutations, G130V and W173G, associated with a mild and a severe clinical presentation, respectively. The G130V mutation affects protein stability and results in low levels of mature (m) fxn, which are nevertheless sufficient to rescue yfh1 delta yeast. The W173G mutation affects protein processing and stability and results in severe m-fxn deficiency. Expression of the FRDA (W173G) cDNA in yfh1 delta yeast leads to increased levels of mitochondrial iron which are not as elevated as in Yfh1p-deficient cells but are above the threshold for oxidative damage of mitochondrial DNA and iron-sulfur centers, causing a typical yfh1 delta phenotype. These results demonstrate that fxn functions like Yfh1p, providing experimental support to the hypothesis that FRDA is a disorder of mitochondrial iron homeostasis.
Asunto(s)
Ataxia de Friedreich/genética , Proteínas de Unión a Hierro , Hierro/metabolismo , Mitocondrias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/metabolismo , Línea Celular , Clonación Molecular , Expresión Génica , Prueba de Complementación Genética , Heterocigoto , Homeostasis , Humanos , Estrés Oxidativo , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Mutación Puntual , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , FrataxinaRESUMEN
A frameshift mutation recently identified within the paired domain of the transcription factor, PAX9, has been linked to a unique form of oligodontia in a single, multigenerational family (Stockton et al., 2000). We now describe the phenotypic and segregation analyses of this remarkable kindred, the initial approach taken to identify a candidate gene involved in this form of oligodontia, and the power of this single-family pedigree to generate significant linkage in a genome search. Of the 43 family members enrolled in this study, 21 individuals were affected with several congenitally missing permanent teeth. The pattern of inheritance of the oligodontia trait suggested the involvement of a single gene bearing a dominant mutation. To various degrees, affected members lacked permanent first, second, and third molars in all four quadrants. Several individuals with missing molars also lacked second premolars- most commonly, maxillary second premolars and mandibular central incisors. To the best of our knowledge, this pattern of non-syndromic, familial tooth agenesis has not been previously described in the literature. Since a missense mutation in the homeobox gene, MSX1, was previously linked to tooth agenesis in a single family lacking second premolars and third molars, we performed a mutational analysis of MSX1 by PCR. The absence of a mutation in exons 1 and 2 of MSX1 suggested that allelic mutations in the coding region of MSX1 are not associated with this phenotypically distinct form of oligodontia. Computer simulation of linkage analysis further proved that this pedigree alone was sufficient to generate a significant result for a total genome scan.
Asunto(s)
Anodoncia/genética , Adolescente , Anodoncia/diagnóstico por imagen , Anodoncia/patología , Niño , Simulación por Computador , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Femenino , Mutación del Sistema de Lectura , Genes Dominantes , Genes Homeobox , Ligamiento Genético , Proteínas de Homeodominio/genética , Humanos , Factor de Transcripción MSX1 , Masculino , Factor de Transcripción PAX9 , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Radiografía , Factores de Transcripción/genéticaAsunto(s)
Anomalías Múltiples/genética , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Proteínas de Unión al GTP/genética , Ligamiento Genético/genética , Discapacidad Intelectual/genética , Southern Blotting , Mapeo Cromosómico , Etiquetas de Secuencia Expresada , Feto/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Híbridas , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , Lugares Marcados de Secuencia , SíndromeRESUMEN
To better understand the mechanism by which glucocorticosteroids (GLUC) could enhance myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P(0)) genes. GLUC stimulated the activity of the P(0) promoter and the PMP22 promoters 1 and 2. The effect of GLUC was specific as estradiol and testosterone did not activate the promoters. The antagonist RU486 did not abolish the effect of GLUC, but instead stimulated promoter activities by itself. In the mammary carcinoma cell line 34i, which expresses GLUC receptors, GLUC did not stimulate the P(0) and PMP22 promoters while the promoter of the mouse mammary tumor virus was strongly activated. Thus, the activation by GLUC of the promoter activities of two peripheral myelin protein genes is Schwann cell-specific.
Asunto(s)
Glucocorticoides/farmacología , Proteína P0 de la Mielina/genética , Proteínas de la Mielina/genética , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Factores de Edad , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glucocorticoides/metabolismo , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Sistema Nervioso Periférico/citología , Regiones Promotoras Genéticas/fisiología , Ratas , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Most patients with Friedreich ataxia (FRDA) have abnormal GAA triplet repeat expansions in both X25 genes. The size of the GAA expansion in the shorter of the 2 expanded alleles correlates significantly with parameters of clinical severity and is inversely related to the age at onset. OBJECTIVES: To describe the clinical and molecular genetic findings in a patient with very late-onset FRDA and to review the literature. PATIENT AND METHODS: A 58-year-old white woman with mild progressive gait disturbance of 15 years' duration whose examination revealed mild incoordination was analyzed for mutations in the X25 gene. A combination of long-range polymerase chain reaction and genomic Southern blot analyses were used to identify GAA expansions in intron 1 of the X25 gene. To uncover evidence of somatic variability in triplet repeat length, DNA isolated from several tissue samples was similarly analyzed. Single-strand conformational polymorphism analysis was used to screen for mutations spanning the entire coding sequence of frataxin and all intron-exon junctions of the X25 gene. RESULTS: DNA isolated from blood leukocytes revealed GAA triplet repeat expansions in both X25 genes, which were estimated to contain 835 and 1200 repeats. Similar expansions were detected in DNA isolated from lymphoblasts, fibroblasts, buccal cells, and sural nerve, with estimated mean (+/- SD) lengths of the shorter and longer expansions being 854 (+/-69) and 1283 (+/-72) triplets, respectively. A review of reported cases of late-onset Friedreich ataxia (25-39 years) and very late-onset Friedreich ataxia (> or =40 years) demonstrated that this is the first instance of a patient presenting with very late-onset FRDA despite carrying more than 800 GAA repeats in both expanded X25 alleles. CONCLUSIONS: This unique case of very late-onset FRDA highlights a limitation in our ability to accurately predict the phenotype in FRDA based solely on the size of the GAA expansion. Other genetic or environmental factors may significantly modify disease severity in FRDA.
Asunto(s)
Ataxia de Friedreich/genética , Expansión de Repetición de Trinucleótido/genética , Edad de Inicio , Alelos , Southern Blotting , ADN/análisis , ADN/genética , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with an interstitial deletion of chromosome band 17p11.2. The critical region is extremely gene-rich and spans approximately 1.5-2.0 Mb of DNA. Here we report the localization and partial characterization of the gene for subunit 3 of the COP9 signalosome, SGN3. SGN3 maps to the distal portion of the SMS critical interval, between SREBF1 and cCI17-638. We assessed the potential effect of haploinsufficiency of SGN3 in SMS patient lymphoblastoid cell lines through transfection studies and western analysis. Our results indicate that the COP9 signalosome assembles properly in these cells and appears to have normal expression and a kinase function intact. However, because the role of the COP9 signalosome in embryogenesis or differentiation is still uncertain, we cannot rule out the involvement of this gene in the Smith-Magenis syndrome.
Asunto(s)
Anomalías Múltiples/genética , Cromosomas Humanos Par 17/genética , Discapacidad Intelectual/genética , Proteínas Quinasas/genética , Animales , Western Blotting , Células CHO , Complejo del Señalosoma COP9 , Células Cultivadas , Deleción Cromosómica , Mapeo Cromosómico , Cricetinae , ADN/análisis , ADN/genética , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Células Híbridas , Luciferasas/genética , Luciferasas/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SíndromeRESUMEN
The vast majority of Friedreich ataxia patients are homozygous for large GAA triplet repeat expansions in intron 1 of the X25 gene. Instability of the expanded GAA repeat was examined in 23 chromosomes bearing 97-1250 triplets in lymphoblastoid cell lines passaged 20-39 times. Southern analyses revealed 18 events of significant changes in length ranging from 69 to 633 triplets, wherein the de novo allele gradually replaced the original over 1-6 passages. Contractions and expansions occurred with equal frequency and magnitude. This behavior is unique in comparison with other large, non-coding triplet repeat expansions [(CGG)(n)and (CTG)(n)] which remain relatively stable under similar conditions. We also report a rare patient who, having inherited two expanded alleles, showed evidence of contracted GAA repeats ranging from nine to 29 triplets in DNA from two independent peripheral blood samples. The GAA triplet repeat is known to adopt a triplex structure, and triplexes in transcribed templates cause enhanced mutagenesis. The poly(A) tract and a 135 bp sequence, both situated immediately upstream of the GAA triplet repeat, were therefore examined for somatic mutations. The poly(A) tract showed enhanced instability when in cis with the GAA expansion. The 135 bp upstream sequence was found to harbor a 3-fold excess of point mutations in DNA derived from individuals homozygous for the GAA triplet repeat expansion compared with normal controls. These data are likely to have important mechanistic and clinical implications.
Asunto(s)
Ataxia de Friedreich/genética , Proteínas de Unión a Hierro , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Expansión de Repetición de Trinucleótido , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , Humanos , Activación de Linfocitos , Linfocitos/citología , Linfocitos/metabolismo , Datos de Secuencia Molecular , Poli A/genética , FrataxinaRESUMEN
Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an approximately 80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situ hybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.
Asunto(s)
Araquidonato 12-Lipooxigenasa/genética , Mapeo Cromosómico , Cromosomas Humanos Par 17 , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biosíntesis , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
In this Journal, we previously reported genetic linkage between loci on chromosome (chr)2p(ETM) and dominantly inherited essential tremor (ET) in a large American kindred of Czech ancestry. Other investigators reported another ET susceptibility locus on chr 3q (FET1) which accounted for over half of the Icelandic families that were studied. We now report evidence for linkage to the ETM locus in three additional, unrelated American families with ET and exclude the FET1 locus in these families. Fine mapping results, using an "affecteds-only" model in all four American families, demonstrate positive combined pairwise lod scores (Z) at the ETM locus with aZ(max) = 5.94 at a recombination fraction (theta) = 0.00 for locus D2S220. Haplotype reconstruction places the ETM gene in a 9.10 cM interval between the D2S224 and D2S405 loci. Multipoint linkage analysis suggests that the ETM gene is in the 2.18 cM interval between loci D2S2150 and D2S220 with a Z(max) = 8.12. These findings may facilitate the search for a gene that causes ET and may further our understanding of other disorders that are associated with tremor [corrected].