Análisis funcional de mutaciones en el promotor del LDLR y su relación con la hipercolesterolemia familiar / Functional analysis of LDLR promoter mutations and their relationship with familial hypercholesterolemia

Introducción La hipercolesterolemia familiar (HF) es una enfermedad autosómica dominante, causada principalmente por mutaciones en la región codificante del gen del receptor de las LDL (LDLR). Sin embargo, varias mutaciones situadas en el promotor de LDLR se han asociado con la HF. La búsqueda de mutaciones en sujetos clínicamente diagnosticados como HF reveló la presencia en la zona promotora de LDLR de 4 mutaciones nuevas en heterocigosidad. Objetivo Estudiar la funcionalidad de las 4 mutaciones nuevas en el promotor del LDLR (c.-36T>G, c.-136C>G, c.-140C>G y c.-208A>T) encontradas en España mediante el uso de la plataforma LIPOchip® en pacientes con sospecha clínica de HF. Métodos Se realizó el análisis funcional de las mutaciones mediante ensayos de retardo de la movilidad electroforética (EMSA) y transfección de promotores mutados con el gen reportero de la luciferasa en cultivos celulares de HepG2. Resultados Las mutaciones c.-136G y c.-140G localizadas en el elemento regulador R3 mostraron un cambio significativo en el patrón de afinidad por las proteínas nucleares en los EMSA. Además, estas mutaciones produjeron una reducción de la actividad del promotor LDLR del 88-93%. Las mutaciones c.-36G y c.-208T no provocaron cambios significativos ni en los experimentos EMSA ni con genes reporteros. Conclusiones Las mutaciones localizadas en el elemento R3 se asocian con HF, mientras que las que se encuentran fuera de los elementos reguladores del promotor de LDLR no son causa directa de hipercolesterolemia. Nuestros resultados revelan la importancia de realizar análisis de funcionalidad de las variantes encontradas en la región promotora de LDLR con objeto de conocer su implicación con el fenotipo HF (AU)

Introduction: Familial hypercholesterolemia (FH) is an autosomal dominant disorder mainly caused by mutations in the coding region of the LDLR gene. However, a variety of mutations within the LDLR promoter have been associated with FH. Genetic screening in persons clinically diagnosed with HF revealed the presence of four new heterozygous mutations within the promoter region. Objective: To study the functionality of the four new LDLR promoter mutations (c.-36T>G, c.-136C>G, c.-140C>G and c.-208A>T) found in Spain, using the LIPOchip® platform in patients with clinically suspected FH. Methods: The functional analysis of mutations was carried out by using electrophoretic mobility shift assays (EMSA) and luciferase reporter gene expression in HepG2 transfected cells with the mutated promoters. Results: Two mutations, c.-136G and c.-140, located within the R3 regulatory element, showed a significant change in the pattern of nuclear binding protein affinity. Moreover, these mutations reduced the residual activity of the LDLR promoter by 88-93%. However, mutations c.-36Gand c.-208T, located outside the response elements, produced no significant changes in EMSA experiments or reporter genes. Conclusions: Mutations within the R3 element are associated with FH, while those located outside the regulatory elements of the LDLR promoter are not a direct cause of FH. Our results reveal the importance of functional analysis of the new variants in the LDLR promoter region to identify their role in the FH phenotype (AU)

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.

Promoter variant -204A > C of the cholesterol 7α-hydroxylase gene: association with response to plant sterols in humans and increased transcriptional activity in transfected HepG2 cells.

BACKGROUND & AIMS: The bile acid pool influences intestinal cholesterol absorption because this process is strictly dependent on micellar solubilization, which is disrupted by plant sterols (PS). Plasma lipid variation relates to promoter variant -204A > C (rs3808607) of the CYP7A1 gene encoding for 7α-hydroxylase, an enzyme for bile acid synthesis. We hypothesized that this polymorphism would be associated with variability in lipid responses to PS. METHODS: We investigated 67 subjects (31 AA and 36 AC + CC) with lipid responses to PS documented in two studies. To assess the functionality of the -204A > C variant, electrophoretic mobility gel shift assays were performed and luciferase reporter plasmids containing the promoter were transfected into HepG2 cells. RESULTS: Compared to AA-subjects, C-carriers showed significantly higher adjusted mean reductions in total cholesterol (0.14 versus 0.43 mmol/L, P = 0.042) and increases in lathosterol-to-cholesterol ratios (0.10 versus 0.75, P = 0.013). The C-construct caused a 78% promoter activity increase and gel-shift assays showed lower affinity for nuclear transcription factors, while in silico experiments predicted a binding site for inhibitory nuclear factors RXR-CAR. CONCLUSIONS: Results suggest that promoter -204A > C variant is associated with enhanced CYP7A1 activity. Increased intestinal bile acids and ensuing more efficient cholesterol absorption might explain why C-allele carriers show enhanced cholesterol lowering and increased feedback cholesterol synthesis to PS intervention.

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.

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 / -1131 T>C polymorphism of the apolipoprotein A5 gene promoter alters the binding of NRF2 (nuclear respiratory factor 2) and decreases promoter activity

Introducción. El papel que desempeñan los triglicéridos (TG) ingeridos en la dieta en la aparición 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 aparición de esta enfermedad. Recientemente, se ha identificado la apolipoproteína (Apo) AV como una proteína clave en la regulación del metabolismo de los TG. El polimorfismo ¿1131 T>C del gen de la ApoA5 se ha asociado con cambios en los valores plasmáticos de TG y de ApoAV. El objetivo de este trabajo fue determinar el papel del polimorfismo ¿1131 T>C en la expresión del gen ApoA5. Material y métodos. Con el fin de estudiar la influencia de este single nucleotide polymorphisms (SNP) en la expresión de este promotor, se han utilizado construcciones de genes reporteros de ambos alelos. Mediante experimentos de retardo en gel se demostró que este polimorfismo también altera la secuencia de unión 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. Además, este cambio de nucleótido se traduce en un cambio de afinidad proteínas-ácido desoxirribonucleico. Estos resultados indican que un cambio en la actividad del promotor del gen ApoA5 podría ser la causa del incremento de los valores plasmáticos de TG asociados con el alelo C (AU)

Introduction. Postprandial triglycerides (TG) have been established as an independent risk factor for atherosclerosis, even though the role of fasting TG in the pathogenesis of this disease remains controversial. Recently, apolipoprotein AV (ApoAV) has been identified as a key player in TG metabolism. The ¿1131 T>C polymorphism in the ApoA5 gene promoter has been associated with changes in plasma triglyceride and ApoAV levels. Our objective was the functional analysis of the SNP ¿1131 T>C located on ApoA5 promoter to clarify his effect on ApoA5 expression. Material and methods. In order to test if this SNP influences promoter function, we have performed reporter gene assays. Our results indicated that the promoter carrying the T allele is stronger than its C- allele variant. Additionally electrophoretic ¿ mobility shift assays showed that the variant also produces a change in a NRF-2 binding site. Conclusions. Together, these results suggest that a change in the activity of ApoA5 promoter could be responsible for the increases in plasma TG levels associated with the C allele (AU)

Estudio funcional del promotor del gen NPC1L1 / Functional analysis of the NPC1L1 gene promoter

Introducción. En la absorción intestinal de colesterol intervienen distintos transportadores, uno de gran importancia y diana del fármaco ezetimiba, la proteína NPC1L1. Se ha demostrado una asociación entre distintas variantes genéticas de NPC1L1, la eficiencia en la absorción de esteroles y la concentración plasmática de colesterol unido a lipoproteínas de baja densidad. El objetivo de este estudio fue identificar y analizar el efecto funcional de los polimorfismos genéticos potencialmente más relevantes del gen NPC1L1sobre su actividad transcripcional. Material y métodos. Como zona de estudio se seleccionó 2 kb de la región promotora del genNPC1L1. Se realizó una búsqueda en bases de datos para localizar variantes descritas en las secuencias seleccionadas. Se diseñaron 3fragmentos, que se amplificaron mediante PCR y posteriormente se secuenciaron. La funcionalidad del polimorfismo –133A>G se determinó mediante ensayos de retardo en gel y medida de la actividadluciferasa. Resultados. El análisis de la zona promotora de102 sujetos normolipidémicos mostró 5 nuevas variantes polimórficas (–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>Grevelaron mayor afinidad de unión de las proteínas nucleares con la sonda portadora de la variante–133A. Por otra parte, la actividad del promotor deNPC1L1 con la variante –133G mostró un aumento de 2,5 veces respecto a la variante –133A.Conclusión. Nuestros resultados demuestran que hay diferencias en la afinidad de unión y actividad transcripcional de NPC1L1 en función del polimorfismo –133A>G. Esta variante génica podría contribuir a la variabilidad interindividual de la absorción intestinal de esteroles (AU)

Introduction. Cholesterol absorption is a process which involves the participation of numerous transporters. One of these transporters is NPC1L1,identified as a critical protein for intestinal cholesterol absorption and the molecular target of ezetimibe. It has been shown that NPC1L1 variants are associated with variations in sterol absorption and plasma levels of LDL-C. The aim of this work was to identify and analyse the potential functional effect on the transcriptional activity of NPC1L1gene polymorphisms. Material and methods. The previously describedNPC1L1 promoter variants were located by means of SNP database search. The NPC1L1 promoter was analysed by three PCR reactions followed by automatic capillary sequencing. Functional study of the –133A>G polymorphism was performed by EMSA and Luciferase assays. Results. NPC1L1 promoter analysis in 102normolipemic subjects showed five (..) (AU)

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland.

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5' flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

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.

Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations.

Gaucher disease (GD) is a disorder of glycosphinglipid metabolism caused by deficiency of lysosomal acid beta-glucosidase (GC), resulting in progressive deposition of glucosylceramide in macrophages. The glucose analogue, N-butyl-deoxynojirimycin (NB-DNJ, Miglustat), is an inhibitor of the ceramide-specific glucosyltransferase (CSG) which catalyzes the first step of glycosphingolipids biosynthesis and is currently approved for the oral treatment of type 1 GD. Using site-directed mutagenesis, we constructed plasmids containing wild-type and several mutations in glucocerebrosidase (GBA) gene. The plasmids were transfected into COS-7 cells and stable transfected cell lines were obtained by geneticin (G418) selection. Cells were cultured during 6 days with medium with or without 10 microM NB-DNJ. The addition of NB-DNJ to COS-7 cell medium leads to 1.3-, 2.1-, 2.3-, 3.6-, and 9.9-fold increase in the activity of S364R, wild-type, N370S, V15M, and M123T GC, respectively. However, no significant changes were observed in the activity of the L444P, L336P, and S465del mutated proteins, but a small decrease in the rare P266L variant was observed. These results suggest that NB-DNJ, in addition to the inhibitory effect on CSG, also works as a "chemical chaperone", increasing the activity of acid beta-glucosidase of wild-type and several GC mutated proteins, including the most frequent N370S mutation. The specific location of the Miglustat binding site in GC is unknown. Potential binding sites in the enzyme have been searched for using computational molecular docking. The searching strategy identified three potential GC binding sites for Miglustat, one being the substrate-binding site of the enzyme, which was the best-ranked site by AutoDock program. Therefore, it is possible that Miglustat exerts its chaperoning activity on acid beta-glucosidase by acting as an inhibitor bound at the active site. This increase on the activity of the acid beta-glucosidase would imply that Miglustat is not only a substrate reducer but also an inhibitor of the GC degradation, with very promising clinical implications for the treatment of GD patients.

TGF-beta-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-kappaB activation.

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-beta is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-beta induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-beta. By contrast, in cells in which p27 was overexpressed, TGF-beta had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-kappaB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program.