RESUMEN
PURPOSE: Recessive mutations of the myosin VIIA (MYO7A) gene are reported to be responsible for both a deaf-blindness syndrome (Usher type 1B [USH1B] and atypical Usher syndrome) and nonsyndromic hearing loss (HL; Deafness, Neurosensory, Autosomal Recessive 2 [DFNB2]). The existence of DFNB2 is controversial, and often there is no relationship between the type and location of the MYO7A mutations corresponding to the USH1B and DFNB2 phenotype. We investigated the molecular determinant of a mild form of retinopathy in association with a subtle splicing modulation of MYO7A mRNA. METHODS: Affected members underwent detailed audiologic and ocular characterization. DNA samples from family members were genotyped with polymorphic microsatellite markers. Sequencing of MYO7A was performed. Endogenous lymphoid RNA analysis and a splicing minigene assay were used to study the effect of the c.1935G>A mutation. RESULTS: Funduscopy showed mild retinitis pigmentosa in adults with HL. Microsatellite analysis showed linkage to markers in the region on chromosome 11q13.5. Sequencing of MYO7A revealed a mutation in the last nucleotide of exon 16 (c.1935G>A), which corresponds to a substitution of a methionine to an isoleucine residue at amino acid 645 of the myosin VIIA. However, structural prediction of the molecular model of myosin VIIA shows that this amino acid replacement induces only minor structural changes in the immediate environment of the mutation and thus does not alter the overall native structure. We found that, although predominantly included in mature mRNA, exon 16 is in fact alternatively spliced in control cells and that the mutation at the very last position is associated with a switch toward a predominant exclusion of that exon. This observation was further supported using a splicing minigene transfection assay; the c.1935G>A mutation was found to trigger a partial impairment of the adjacent donor splice site, suggesting that the unique change at the last position of the exon is responsible for the enhanced exon exclusion in this family. CONCLUSIONS: This study shows how an exonic mutation that weakens the 5' splice site enhances a minor alternative splicing without abolishing a complete exclusion of the exon and therefore causes a less severe retinitis pigmentosa than the USH1B-associated alleles. It would be interesting to examine a possible correlation between intrafamilial phenotypic variability and the subtle variation in exon 16 inclusion, probably related to genetic background specificities.
Asunto(s)
Empalme Alternativo/genética , Sordera/complicaciones , Sordera/genética , Mutación Missense/genética , Miosinas/genética , Enfermedades de la Retina/complicaciones , Enfermedades de la Retina/genética , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos/genética , Niño , Segregación Cromosómica/genética , Análisis Mutacional de ADN , Exones/genética , Familia , Femenino , Genes Recesivos/genética , Heterogeneidad Genética , Genotipo , Células HeLa , Humanos , Masculino , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Miosina VIIa , Linaje , Sitios de Empalme de ARN/genética , Homología Estructural de ProteínaRESUMEN
Among phenolic compounds, Agave americana L. extract contained puerarin (38.4%) and p-coumaric acid (12.29%) (pCa). From the Lineweaver-Burk plots, pCa and puerarin demonstrated a competitive and a non competitive inhibitions towards human α-amylase activity, respectively. PCa exhibited a higher human inhibitory activity with an IC50 of 98.8 µM which was about 2.3 times than acarbose. Puerarin (IC50 = 3.87 µM) and pCa (IC50 = 10.16 µM) also showed an excellent inhibition for Aspergillus oryzae S2 α-amylase activity. The inhibitions of the described biocatalysts compounds towards both amylases were significantly decreased when they were pre-incubated with starch. The binding modes of these compounds were evaluated in silico. The binding efficiency order of these molecules in terms of polar contact numbers for both enzymes was in agreement with the in vitro studies. These findings provided a rational reason to establish the isolated compounds capability as therapeutic target for hyperglycaemia modulation and antifungal therapy.
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Agave/química , Aspergillus oryzae/enzimología , Isoflavonas/farmacología , Fenoles/farmacología , Propionatos/farmacología , alfa-Amilasas/antagonistas & inhibidores , Acarbosa/farmacología , Ácidos Cumáricos , Humanos , Fitoquímicos/farmacología , Extractos Vegetales/químicaRESUMEN
PURPOSE: Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin. METHODS: Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes. RESULTS: Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q. CONCLUSIONS: Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q.
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Síndromes de Usher/genética , Adolescente , Adulto , Anciano , Segregación Cromosómica , Electrorretinografía , Familia , Femenino , Pruebas Genéticas , Genoma Humano/genética , Haplotipos , Pérdida Auditiva Sensorineural/genética , Homocigoto , Humanos , Escala de Lod , Masculino , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Linaje , Túnez , Síndromes de Usher/fisiopatología , Campos VisualesRESUMEN
We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (ß/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.
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Aspergillus oryzae/enzimología , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Simulación del Acoplamiento Molecular , Proteolisis , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , alfa-Amilasas/químicaRESUMEN
Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by deficiency of mitochondrial isovaleryl-CoA dehydrogenase (IVD). Accumulation of isovaleryl-CoA derivatives to toxic levels results in clinical symptoms of the disease. Here, we investigate the clinical and molecular features of Arab patients with IVA. Patients from five unrelated families were evaluated clinically and for defects in the IVD gene. Four novel mutations (p.F382fs, p.R392H, p.R395Q and p.E408K) have been identified with p.R395Q occurring in two families. In addition, molecular modeling of the identified missense mutations predicted their damaging effects on the protein and computational analysis of the p.F382fs mutation predicted the disruption of a 3' splicing site resulting in inactive or unstable gene product. Furthermore, we found an unusual case of a 17 years old female homozygous for the p.R392H mutation with no clinical symptoms. Our results illustrate a heterogeneous mutation spectrum and clinical presentation in the relatively small UAE population.
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Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/genética , Isovaleril-CoA Deshidrogenasa/genética , Mutación , Fenotipo , Adolescente , Niño , Preescolar , Consanguinidad , Exones , Femenino , Humanos , Enlace de Hidrógeno , Isovaleril-CoA Deshidrogenasa/química , Isovaleril-CoA Deshidrogenasa/deficiencia , Masculino , Modelos Moleculares , Linaje , Conformación Proteica , Sitios de Empalme de ARN , Emiratos Árabes UnidosRESUMEN
Branchio-oto-renal (BOR) and Branchio-otic (BO) syndromes are dominant disorders characterized by variable hearing impairment (HI) and branchial defects. BOR includes additional kidney malformations. BO/BOR syndromes are genetically heterogeneous and caused by mutations in EYA1 and SIX1 genes. Mutation in SIX1 is responsible also for DFNA23, a locus for non-syndromic HI. Strikingly, the severity of the phenotype did not seem to correlate with the type of SIX1 mutation. Herein, we identified a novel mutation in SIX1 (p.E125K) in a Tunisian family with variable HI and preauricular pits. This mutation is located at the same position as the mutation identified in the Catwhesel (Cwe) mouse. No renal and branchial defects were observed in our family nor in Cwe/+ mice. A homology model revealed that the replacement of the Glutamate by a Lysine alters the electrostatic potential surface propriety which may affect the DNA-binding activity.
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Oído/anomalías , Pérdida Auditiva/genética , Proteínas de Homeodominio/genética , Mutación/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Síndrome Branquio Oto Renal/genética , Secuencia Conservada , Femenino , Humanos , Riñón/anomalías , Masculino , Repeticiones de Microsatélite/genética , Modelos Moleculares , Datos de Secuencia Molecular , Linaje , Fenotipo , Conformación Proteica , Alineación de SecuenciaRESUMEN
R-spondin 4 is a secreted protein mainly associated with embryonic nail development. R-spondins have been recently identified as heparin-binding proteins with high affinity. Proteoglycan binding has been associated with both the TSR and the C terminal basic amino acid rich domains. In this paper, molecular modelling techniques were used to construct the model of R-spondin 4 TSR domain based on the structure of the F-spondin TSR domain 4 (30-40 cent sequence identity). Beside a positively charged surface in the TSR domain, presence of the basic amino acid rich domain which could forms a continuous heparin binding surface may explain the high affinity of R-spondins for heparin. Our results provide a framework for understanding the possible regulatory role of heparin in R-spondins signalling.
RESUMEN
Many proteins necessary for sound transduction have been identified through positional cloning of genes that cause deafness. We report here that mutations of LRTOMT are associated with profound nonsyndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, detected by protein blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, new transcripts can arise through partial or complete coalescence of genes. We provide evidence that in the primate lineage LRTOMT evolved from the fusion of two neighboring ancestral genes, which exist as separate genes (Lrrc51 and Tomt) in rodents.