Sandra Pampín

Functional analysis of LDLR promoter and 5' UTR mutations in subjects with clinical diagnosis of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5′ UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.−155_‐150delACCCCinsTTCTGCAAACTCCTCCC, c.−136C>G, c.−140C>G, and c.−140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.−208T, c.−88A, and c.−36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.Hum Mutat 32:1–5, 2011.

An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia

BACKGROUND AND AIMS A substantial number of subjects with autosomal dominant hypercholesterolemia (ADH) do not have LDL receptor (LDLR) or apolipoprotein B (APOB) mutations. Some ADH subjects appear to hyperabsorb sterols from the intestine, thus we hypothesized that they could have variants of the Niemann-Pick C1-Like 1 gene (NPC1L1). NPC1L1 encodes a crucial protein involved in intestinal sterol absorption. METHODS AND RESULTS Four NPC1L1 variants (-133A>G, -18C>A, 1679C>G, 28650A>G) were analyzed in 271 (155 women and 116 men) ADH bearers without mutations in LDLR or APOB aged 30-70years and 274 (180 women and 94 men) control subjects aged 25-65years. The AC haplotype determined by the -133A>G and -18C>A variants was underrepresented in ADH subjects compared to controls (p=0.01). In the ADH group, cholesterol absorption/synthesis markers were significantly lower in AC homozygotes that in all others haplotypes. Electrophoretic mobility shift assay (EMSA) results revealed that the -133A-specific oligonucleotide produced a retarded band stronger than the -133G allele. Luciferase activity with NPC1L1 -133G variant was 2.5-fold higher than with the -133A variant. CONCLUSION The -133A>G polymorphism exerts a significant effect on NPC1L1 promoter activity. NPC1L1 promoter variants might explain in part the hypercholesterolemic phenotype of some subjects with nonLDLR/nonAPOB ADH.

Functional analysis of regulatory single-nucleotide polymorphisms.

Purpose of review The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. Recent findings New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. Summary The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed ‘haploChIP’ allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.

Characterization of the c.(-203)A>G variant in the glucocerebrosidase gene and its association with phenotype in Gaucher disease

BACKGROUND Gaucher disease (GD) is a rare autosomal recessive disorder caused mainly by mutations in the glucocerebrosidase (GBA) gene. Great phenotypic variability has been observed among patients with the same genotype, suggesting other factors, such as polymorphic variants, might influence GD phenotypes. We previously reported the c.(-203)A>G (g.1256A>G) variant in exon 1 of the GBA gene in Spanish GD patients. METHODS We analyzed the frequency and transcriptional activity of the promoter carrying the G-allele using restriction isotyping, electrophoretic mobility shift assay, cell culture, transfection, and luciferase assays. RESULTS We found the variant is present at a similar frequency to the control group. In our patients, the G-allele was always found in combination with another mutation in the same allele, and patients carrying the c.(-203)A>G variant showed a more severe GD phenotype. The promoter containing the G-allele showed a 35% reduction in promoter activity when transfected into HepG2 cells. CONCLUSION The c.(-203)A>G variant seems to be a polymorphism resulting in a decrease in activity of the GBA promoter. The change, per se, is not enough to elicit a GD phenotype, but it may produce a more severe phenotype in GD patients when combined with an already defective GBA protein.

Estudio funcional del promotor del gen NPC1L1

Introduccion En la absorcion intestinal de colesterol intervienen distintos transportadores, uno de gran importancia y diana del farmaco ezetimiba, la proteina NPC1L1. Se ha demostrado una asociacion entre distintas variantes geneticas de NPC1L1, la eficiencia en la absorcion de esteroles y la concentracion plasmatica de colesterol unido a lipoproteinas de baja densidad. El objetivo de este estudio fue identificar y analizar el efecto funcional de los polimorfismos geneticos potencialmente mas relevantes del gen NPC1L1 sobre su actividad transcripcional. Material y metodos Como zona de estudio se selecciono 2 kb de la region promotora del gen NPC1L1. Se realizo una busqueda en bases de datos para localizar variantes descritas en las secuencias seleccionadas. Se disenaron 3 fragmentos, que se amplificaron mediante PCR y posteriormente se secuenciaron. La funcionalidad del polimorfismo −133A>G se determino mediante ensayos de retardo en gel y medida de la actividad luciferasa. Resultados El analisis de la zona promotora de 102 sujetos normolipidemicos mostro 5 nuevas variantes polimorficas (−1485C>T, −1425C>G, −982G>C, −292T>C y −18C>A) no identificadas previamente. Los resultados de los ensayos de retardo en gel con el polimorfismo −133A>G revelaron mayor afinidad de union de las proteinas nucleares con la sonda portadora de la variante −133A. Por otra parte, la actividad del promotor de NPC1L1 con la variante −133G mostro un aumento de 2,5 veces respecto a la variante −133A. Conclusion Nuestros resultados demuestran que hay diferencias en la afinidad de union y actividad transcripcional de NPC1L1 en funcion del polimorfismo −133A>G. Esta variante genica podria contribuir a la variabilidad interindividual de la absorcion intestinal de esteroles.

El polimorfismo ¿1131 T>C del promotor del gen de la apolipoproteína A5 altera la unión de NRF2 ( nuclear respiratory factor-2) y disminuye la actividad del promotor

Introduccion: El papel que desempenan los trigliceridos (TG) ingeridos en la dieta en la aparicion de aterosclerosis es controvertido. Sin embargo, recientemente se ha descrito que los valores de TG en estado posprandial son un factor de riesgo independiente para la aparicion de esta enfermedad. Recientemente, se ha identificado la apolipoproteina (Apo) AV como una proteina clave en la regulacion del metabolismo de los TG. El polimorfismo -1131 T>C del gen de la ApoA5 se ha asociado con cambios en los valores plasmaticos de TG y de ApoAV. El objetivo de este trabajo fue determinar el papel del polimorfismo -1131 T>C en la expresion del gen ApoA5. Material y metodos: Con el fin de estudiar la influencia de este single nucleotide polymorphisms (SNP) en la expresion de este promotor, se han utilizado construcciones de genes reporteros de ambos alelos. Mediante experimentos de retardo en gel se demostro que este polimorfismo tambien altera la secuencia de union a NRF-2. Conclusiones: Nuestros resultados indicaron que la actividad del promotor de ApoA5 que porta la variante T del SNP �1131 T>C es mayor que la del promotor que lleva el alelo C. Ademas, este cambio de nucleotido se traduce en un cambio de afinidad proteinas-acido desoxirribonucleico. Estos resultados indican que un cambio en la actividad del promotor del gen ApoA5 podria ser la causa del incremento de los valores plasmaticos de TG asociados con el alelo C.