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The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective.

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INTRODUCTION: The C1236T, G2677T/A, and C3435T variants of the ABCB1 gene alter the functioning of P-glycoprotein and the transport of endogenous and exogenous substances across the blood-brain barrier, and act as risk factors for some neurodegenerative diseases. This study aimed to determine the association between demyelinating disease and the C1236T, G2677T/A, and C3435T variants of ABCB1 and its haplotypes and combinations of genotypes. METHODS: Polymerase chain reaction with restriction fragment length polymorphism analysis (PCR-RFLP) and Sanger sequencing were used to genotype 199 patients with demyelinating disease and 200 controls, all Mexicans of mixed race; frequencies of alleles, genotypes, haplotypes, and genotype combinations were compared between patients and controls. We conducted a logistic regression analysis and calculated chi-square values and 95% confidence intervals (CI); odds ratios (OR) were calculated to evaluate the association with demyelinating disease. RESULTS: The TTT and CGC haplotypes were most frequent in both patients and controls. The G2677 allele was associated with demyelinating disease (OR: 1.79; 95% CI, 1.12-2.86; P =  .015), as were the genotypes GG2677 (OR: 2.72; 95% CI, 1.11-6.68; P =  .025) and CC3435 (OR: 1.82; 95% CI, 1.15-2.90; P =  .010), the combination GG2677/CC3435 (OR: 2.02; 95% CI, 1.17-3.48; P =  .010), and the CAT haplotype (OR: 0.21; 95% CI, 0.05-0.66; P =  .001). TTTTTT carriers presented the earliest age of onset (23.0 ±â€¯7.7 years, vs 31.6 ±â€¯10.7; P =  .0001). CONCLUSIONS: The GG2677/CC3435 genotype combination is associated with demyelinating disease in this sample, particularly among men, who may present toxic accumulation of P-glycoprotein substrates. In our study, the G2677 allele of ABCB1 may differentially modulate age of onset of demyelinating disease in men and women.

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INTRODUCTION: The C1236T, G2677T/A, and C3435T variants of the ABCB1 gene alter the functioning of P-glycoprotein and the transport of endogenous and exogenous substances across the blood-brain barrier, and act as risk factors for some neurodegenerative diseases. This study aimed to determine the association between demyelinating disease and the C1236T, G2677T/A, and C3435T variants of ABCB1 and its haplotypes and combinations of genotypes. METHODS: Polymerase chain reaction with restriction fragment length polymorphism analysis (PCR-RFLP) and Sanger sequencing were used to genotype 199 patients with demyelinating disease and 200 controls, all Mexicans of mixed race; frequencies of alleles, genotypes, haplotypes, and genotype combinations were compared between patients and controls. We conducted a logistic regression analysis and calculated chi-square values and 95% confidence intervals (CI); odds ratios (OR) were calculated to evaluate the association with demyelinating disease. RESULTS: The TTT and CGC haplotypes were most frequent in both patients and controls. The G2677 allele was associated with demyelinating disease (OR: 1.79; 95% CI: 1.12-2.86; P=.015), as were the genotypes GG2677 (OR: 2.72; 95% CI: 1.11-6.68; P=.025) and CC3435 (OR: 1.82; 95% CI: 1.15-2.90; P=.010), the combination GG2677/CC3435 (OR: 2.02; 95% CI, 1.17-3.48; P=.010), and the CAT haplotype (OR: 0.21; 95% CI: 0.05-0.66; P=.001). TTTTTT carriers presented the earliest age of onset (23.0±7.7 years, vs. 31.6±10.7; P=.0001). CONCLUSIONS: The GG2677/CC3435 genotype combination is associated with demyelinating disease in this sample, particularly among men, who may present toxic accumulation of P-glycoprotein substrates. In our study, the G2677 allele of ABCB1 may differentially modulate age of onset of demyelinating disease in men and women.

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A 4-bp deletion (c.230_233delATAG) of the ABCB1 gene, frequently found in various dog breeds, results in intolerance to certain drugs routinely used in veterinary medicine, including many chemotherapeutic agents and macrocyclic lactones. The use of rapid and reliable genetic testing is fundamental for early detection of the mutation and prevention of undesirable toxicoses. We developed and compared 2 genotyping tests: PCR-high-resolution melting (PCR-HRM) and PCR-restriction-fragment length polymorphism (PCR-RFLP) to identify the 4-bp deletion in the ABCB1 gene of canine breeds. Amplified PCR products were sequenced in order to confirm different genotypes. Both techniques were efficient in discriminating homozygous wild-type, homozygous mutated, and heterozygous ABCB1 genotypes, and proved to be reproducible and economical methods. The HRM analysis, a sensitive and specific method for the molecular detection of genetic disorders, does not require labeled probes, processing, or separations after PCR.

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Las variantes de los genes ABCB1 y ABCC2 han sido asociadas a mayor riesgo de epilepsia farmacorresistente pero tal proceso ha sido poco estudiado mediante comparaciones entre poblaciones. En Latinoamérica solo se han realizado 3 estudios. Objetivo: Evaluar la asociación entre las variantes C3435T del gen ABCB1 y -24C>T del gen ABCC2 con epilepsia farmacorresistente en pacientes peruanos atendidos en la Unidad de Epilepsia del Hospital Nacional Edgardo Rebagliati Martins (HNERM). Material y Métodos: Se analizaron muestras sanguíneas de 22 pacientes con epilepsia farmacorresistente y ocho pacientes con epilepsia de respuesta favorable a tratamiento farmacológico, entre Mayo 2016 y Junio 2017. La identificación de la variante C3435T del gen ABCB1 se realizó mediante reacción de cadena de la polimerasa y posterior digestión enzimática; la variante -24C>T del gen ABCC2 se obtuvo por secuenciación. Resultados: Se obtuvo una frecuencia alélica de 0,717 para C en la variante C3435T del gen ABCB1 y 0,967 para C en la variante -24C>T del gen ABCC2. La comparación de las frecuencias alélicas y genotípicas entre pacientes farmacorresistentes y farmacorrespondedores no mostró diferencia significativa, de lo cual se infiere ausencia de asociación entre la epilepsia farmacorresistente y las variantes C3435T del gen ABCB1 y -24C>T del gen ABCC2 (p>0.05). Conclusiones: En una muestra de pacientes peruanos con epilepsia, no se encontró asociación entre epilepsia farmacorresistente y los polimorfismos C3435T del gen ABCB1 y -24C>T del gen ABCC2.

Variants of the genes ABCB1 and ABCC2 have been associated with an increased risk of drug-resistant epilepsy; this phenomenon, however, has been scarcely tested by means of comparisons between populations: In Latin America there have only been 3 studies. Objective: To evaluate the association between the variants C3435T of the gene ABCB1, and --24C> T of the gene ABCC2 with drug-resistant epilepsy in Peruvian patients treated at the Epilepsy Unit of a Peruvian Hospital. Material and Methods: Blood samples from 22 patients with drug-resistant epilepsy and eight patients with pharmaco-responsive epilepsy were analyzed between May 2016 and June 2017. The identification of the C3435T variant of the ABCB1 gene was performed by polymerase chain reaction (PCR) and subsequent enzymatic digestion; the -24C>T variant of the ABCC2 gene was obtained by sequencing. Results: An allelic frequency of 0.717 was obtained for C in the C3435T variant of the gene ABCB1, and 0.967 for C in the-24C> T variant of the gene ABCC2. When genetic and allelic frequencies were compared between drug-resistant and drug-responsive patients no significant difference was observed, from which a lack of association between drug-resistant epilepsy and the C3435T variant of the gene ABCB1 and the -24C> T variant of the gene ABCC2 (p>0.05) was inferred. Conclusions: In a sample of Peruvian patients with epilepsy, no association was found between drug-resistant epilepsy and the C3435T and -24C>T polymorphisms of the genes ABCB1 and ABCC2, respectively.