RESUMEN
Rare copy number variations by the nonrecurrent rearrangements involving PMP22 have been recently suggested to be associated with CMT1A peripheral neuropathy. As a mechanism of the nonrecurrent rearrangement, replication-based fork stalling template switching (FoSTeS) by microhomology-mediated break-induced replication (MMBIR) has been proposed. We found three Korean CMT1A families with putative nonrecurrent duplication. The duplications were identified by microsatellite typing and applying a CGH microarray. The breakpoint sequences in two families suggested an Alu-Alu-mediated rearrangement with the FoSTeS by the MMBIR, and a two-step rearrangement of the replication-based FoSTeS/MMBIR and meiosis-based recombination. The two-step mechanism has still not been reported. Segregation analysis of 17p12 microsatellite markers and breakpoint junction analysis suggested that the nonrecurrent rearrangements are stably inherited without alteration of junction sequence; however, they may allow some alteration of the genomic contents in duplication across generations by recombination event. It might be the first study on the pedigree analysis of the large CMT1A families with nonrecurrent rearrangements. It seems that the exact mechanism of the nonrecurrent rearrangements in the CMT1A may have a far more complex process than has been expected.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Reordenamiento Génico , Elementos Alu , Secuencia de Bases , Cromosomas Humanos Par 17/genética , Hibridación Genómica Comparativa , ADN/genética , Variaciones en el Número de Copia de ADN , Femenino , Humanos , Masculino , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , República de CoreaRESUMEN
Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are frequent forms of genetically heterogeneous peripheral neuropathies. Reciprocal unequal crossover between flanking CMT1A-REPs on chromosome 17p11.2-p12 is a major cause of CMT type 1A (CMT1A) and HNPP. The importance of a sensitive and rapid method for identifying the CMT1A duplication and HNPP deletion is being emphasized. In the present study, we established a molecular diagnostic method for the CMT1A duplication and HNPP deletion based on hexaplex PCR of 6 microsatellite markers (D17S921, D17S9B, D17S9A, D17S918, D17S4A and D17S2230). The method is highly time-, cost- and sample-saving because the six markers are amplified by a single PCR reaction and resolved with a single capillary in 3 h. Several statistical and forensic estimates indicated that most of these markers are likely to be useful for diagnosing the peripheral neuropathies. Reproducibility, as determined by concordance between independent tests, was estimated to be 100%. The likelihood that genotypes of all six markers are homozygous in randomly selected individuals was calculated to be 1.6 x 10(-4) which indicates that the statistical error rate for this diagnosis of HNPP deletion is only 0.016%.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Eliminación de Gen , Duplicación de Gen , Repeticiones de Microsatélite/genética , Enfermedades del Sistema Nervioso Periférico/genética , Reacción en Cadena de la Polimerasa/métodos , Alelos , Cromosomas Humanos Par 17/genética , Dosificación de Gen , Frecuencia de los Genes , Genoma Humano/genética , Humanos , Linaje , Fenotipo , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
We examined CMT1A duplication of 17p11.2-p12, mutations of PMP22, MPZ (P0), GJB1 (Cx32), EGR2 and NEFL genes in 57 Korean families with patients diagnosed as having Charcot-Marie-Tooth (CMT) disease. The CMT1A duplication was present in 53.6% of 28 CMT type 1 patients. In the 42 CMT families without CMT1A duplication, 10 pathogenic mutations were found in 9 families. The 10 mutations were not detected in 105 healthy controls. Seven mutations (c.318delT (p.Ala106fs) in PMP22, c.352G>A (p.Asp118Asn), c.449-1G>T (3'-splice site), c.706A>G (p.Lys236Glu) in MPZ, c.407T>C (p.Val136Ala)[corrected], c.502T>C (p.Cys168Arg) in GJB1, and c.1001T>C (p.Leu334Pro) in NEFL) were determined to be novel. The mutation frequencies of PMP22 and MPZ were similar to those found in several European populations, however, it appeared that mutations in GJB1 are less frequent in East Asian CMT patients than in Eur opean patients. We described the identified mutations and phenotype-genotype correlations based on nerve conduction studies.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Conexinas/genética , Análisis Mutacional de ADN/métodos , Proteínas de Unión al ADN/genética , Proteína P0 de la Mielina/genética , Proteínas de la Mielina/genética , Proteínas de Neurofilamentos/genética , Factores de Transcripción/genética , Proteína 2 de la Respuesta de Crecimiento Precoz , Duplicación de Gen , Humanos , Corea (Geográfico) , Proteína beta1 de Unión ComunicanteRESUMEN
Recombinant human tenascin peptide (hTNCIII3) that includes the Arg-Gly-Asp (RGD) cell recognition site was expressed in Escherichia coli using a prokaryotic expression system. Addition of recombinant hTNCIII3 peptide enhanced cell adhesion and survival of human chondrocytes by about 3-fold in each case.
Asunto(s)
Condrocitos/efectos de los fármacos , Condrocitos/fisiología , Oligopéptidos/biosíntesis , Oligopéptidos/farmacología , Ingeniería de Proteínas/métodos , Tenascina/biosíntesis , Tenascina/farmacología , Ingeniería de Tejidos/métodos , Adsorción , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Condrocitos/citología , Materiales Biocompatibles Revestidos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Ensayo de Materiales , Oligopéptidos/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacología , Tenascina/genéticaRESUMEN
The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.