RESUMEN
We report the molecular characterization of two splice mutations in two different French families affected with a late onset form of Charcot-Marie-Tooth disease type 1B (CMT1B), an autosomal dominant inherited disorder caused by mutations in the myelin protein zero gene. The first substitution, c.306G>A, located in exon 3, does not change the codon p.Val102Val but is co-transmitted with the disease in the first family. The second substitution, c.675+3dup, is an insertion of a T at position +3 of intron 5. To identify the functional impact of these nucleotide changes on splicing and because no RNA sample was available, we used in silico prediction and in vitro splicing assay. Mutation c.306G>A increases the strength of a preexisting cryptic donor site at position c.304 which becomes stronger than the normal donor site of intron 3. This variation creates a sequence that better matches the U1 small nuclear RNA (snRNA) binding consensus, and HeLa cells, transfected with the mutant minigene, produce a truncated exon 3 messenger RNA (mRNA). Mutation c.675+3dup was predicted to abolish the donor site of intron 5, and, indeed, HeLa cells transfected with the mutant minigene completely skip exon 5 from the transcript. The mutated sequence abolishes U1 snRNA binding and co-transfection of a mutated complementary U1 snRNA restored exon 5 inclusion in the mRNA. This work provides valuable information regarding the molecular basis of two forms of late onset of CMT1B, U1 snRNA mis-binding, and provides more evidence that a "silent" polymorphism may be a disease causing mutation.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , ARN Nuclear Pequeño/metabolismo , Adulto , Exones , Femenino , Células HeLa , Humanos , Intrones , Masculino , Persona de Mediana Edad , Mutación , Linaje , Polimorfismo Genético , Empalme del ARNRESUMEN
The cytochromes 2 C19 2* loss of function polymorphism has recently been shown to be associated with decreased platelet reactivity (PR) inhibition after clopidogrel loading dose (LD) and an excess in thrombotic events following percutaneous coronary intervention (PCI). We aimed to investigate if an optimal PR inhibition could be obtained in patients homozygotes for this alleles using adjusted LD of clopidogrel according to platelet reactivity monitoring. Post-treatment PR was measured using the VASP index. Dose adjustment was performed in order to obtain a PR <50% using additional clopidogrel LD. Direct sequencing was used to select homozygotes for CYP 2C19 2* alleles. Six patients with the loss of function polymorphism were recruited. The mean VASP index was 53.7 ± 16.2% after the first LD of clopidogrel. Four patients had a VASP index >50% and were therefore considered to have high on treatment PR. Using up to 3 additional 600 mg LD of clopidogrel we were able to obtain a VASP <50% in all these patients. The present study is the first to suggest that in homozygotes for CYP 2C19 2* loss of function polymorphism, increased loading dose of 55 clopidogrel is efficient to obtain an optimal level PR inhibition in patients undergoing PCI.
Asunto(s)
Síndrome Coronario Agudo/enzimología , Síndrome Coronario Agudo/genética , Hidrocarburo de Aril Hidroxilasas/genética , Homocigoto , Agregación Plaquetaria/genética , Polimorfismo Genético/genética , Síndrome Coronario Agudo/sangre , Hidrocarburo de Aril Hidroxilasas/sangre , Clopidogrel , Citocromo P-450 CYP2C19 , Humanos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Estudios Prospectivos , Ticlopidina/administración & dosificación , Ticlopidina/análogos & derivadosRESUMEN
OBJECTIVES: We aimed to investigate the biological impact of a tailored clopidogrel loading dose (LD) according to platelet reactivity monitoring in carriers of the cytochrome (CYP) 2C19*2 loss-of-function polymorphism undergoing percutaneous coronary intervention for an acute coronary syndromes. BACKGROUND: CYP2C19*2 polymorphism is associated with reduced clopidogrel metabolism and a worse prognosis after percutaneous coronary intervention. METHOD: A prospective multicenter study enrolling 411 patients with non-ST-segment elevation acute coronary syndrome undergoing percutaneous coronary intervention was performed. Platelet reactivity was measured using the vasodilator-stimulated phosphoprotein (VASP) index, and a cutoff value of ≥ 50% was used to define high on-treatment platelet reactivity (HTPR). The genetic polymorphism of CYP2C19 was determined by allele-specific polymerase chain reaction. In patients carrying CYP2C19*2 and exhibiting HTPR after a first 600-mg LD of clopidogrel, dose adjustment was performed by using up to 3 additional 600 mg LDs to obtain a VASP index <50%. RESULTS: One hundred thirty-four patients (35.3%) carried at least one 2C19*2 allele (11 homozygotes [2.7%] and 123 heterozygotes [32.6%]). The VASP index in these patients was significantly higher than in homozygotic patients for the wild-type alleles (61.7 ± 18.4% vs. 49.2 ± 24.2%; p < 0.001). Of the 134 carriers of the loss-of-function polymorphism, 103 were considered to have HTPR. After a second clopidogrel LD, the VASP index was significantly decreased in these patients (69.7 ± 10.1% vs. 50.6 ± 17.6%; p < 0.0001). Finally, dose adjustment according to platelet reactivity monitoring, enabled 88% of 2C19*2 carriers exhibiting HTPR to reach a VASP index <50%. CONCLUSIONS: Increased and tailored clopidogrel loading dose according to platelet reactivity monitoring overcome HTPR in carriers of the loss-of-function CYP2C19*2 polymorphism.