Oscar Puig

Seven DNA polymorphisms in the LDL receptor gene: application to the study of familial hypercholesterolemia in Spain

We have performed restriction fragment length polymorphism (RFLP) analysis at the low density lipoprotein receptor (LDLR) locus in order to investigate the molecular genetics of familial hypercholesterolemia (FH) in Spain. Firstly, a sample of 50 unrelated patients with a clinical diagnosis of FH was screened for the presence of major rearrangements at this locus by Southern blot analysis of BgtII digested genomic DNA. Four different mutations were detected, accounting for 8% of the mutant alleles in the Spanish FH sample. Then, we determined the relative allele frequency and estimated linkage disequilibrium between seven RFLPs of the LDLR gene in the remaining 46 FH patients and in 61 normolipidemic controls. Hindi, Avail, PvuII, MspI, and NcoI are the most polymorphic sites with individual PIC values higher than 0.28, whereas the TaqI and StuI sites display low levels of polymorphism. The usefulness of the seven RFLPs to confirm a clinical diagnosis of FH was investigated in 15 FH‐families, consisting of 118 individuals, in whom the presence of Familial Defective Apolipoprotein B‐100 (FDB) due to the apoB3500 mutation was excluded. Independent haplotypes were constructed for 71 chromosomes: 15 FH and 56 control haplotypes. A total of 14 different haplotypes was found. In 12 families, clinical diagnosis of FH was confirmed by cosegregation analysis, which makes these RFLPs useful for studying the inheritance of the LDLR gene in 80% of Spanish families with FH. Comparison of haplotypes found in the Spanish sample with those found in Swiss and Norwegians suggests heterogeneity of haplotypes among European populations.

Reduced penetrance of autosomal dominant hypercholesterolemia in a high percentage of families: importance of genetic testing in the entire family.

BACKGROUND Autosomal dominant hypercholesterolemias (ADHs) are characterised by increased plasma levels of total and LDL cholesterol, predisposing to premature atherosclerosis. ADHs comprise several diseases with undistinguishable phenotype, caused by mutations in different genes: LDLR, APOB and PCSK9. Genetic studies are usually performed in patients with altered cholesterol levels. However, some persons carrying pathogenic mutations are normocholesterolemic and there are no further studies about this subject. We have studied the frequency of families and individuals carrying ADH mutations who do not present the disease in Spanish population. METHODS We have analysed genes known to cause ADH by direct sequencing in 24 ADH families (215 members). Functional effect of some LDLR gene mutations was assessed by transfecting cultured cells with plasmids. RESULTS Six families with mutations presented 7 mutation carriers who did not show ADH phenotype: 30% of ADH families presented normocholesterolemic individuals, and 7% of carriers of pathogenic mutations did not show ADH phenotype. We have analysed the effect of some of these mutations and they are responsible for impaired LDL receptor function. We have excluded mutations in APOB and PCSK9 genes that could reduce LDLc levels. CONCLUSIONS An important percentage of ADH families presented individuals who do not show an ADH phenotype, but who are able to transmit the pathogenic mutation to their offspring. Genetic study of all subjects in ADH families should be performed in order to identify normocholesterolemic carriers that allow the detection of mutations in their descendants and the prevention of the disease consequences.

A three‐allelic polymorphic system in exon 12 of the LDL receptor gene is highly informative for segregation analysis of familial hypercholesterolemia in the Spanish population

We have screened exon 12 of the low density lipoprotein (LDL) receptor gene from 46 familial hypercholesterolemia (FH) heterozygotes and 64 normolipidemic controls for two polymorphisms, Hindi, which is caused by a T to C substitution at base 1773, and a C to T transition at base 1725, by using single strand conformation polymorphism (SSCP) analysis. Our results indicate that polymorphism at base 1725, previously reported as very rare from a Japanese sample, is quite frequent in the Spanish population and that it is closely linked to the presence of the Hindi site (HincII+). Thus, both polymorphisms constitute a system of three alleles, typed HincII‐C1725, HincII+C1725, and HincII+T1725, whose frequencies in the FH sample were 0.489,0.347, and 0.164, respectively. No significant differences were found in the allele frequencies between the FH and control samples. This three‐allelic polymorphic system provides a higher information content (PIC value) than the Hindi RFLP alone (0.537 versus 0.373, respectively); therefore, it is an extremely useful marker for linkage analysis of FH in Caucasian populations.

Asociación de factores lipídicos, genotipo de APOE y tipos de mutación del gen del receptor de LDL con el infarto agudo de miocardio en sujetos con hipercolesterolemia familiar heterocigota

Fundamento Evaluar la relacion de los lipidos, del genotipo de APOE y del tipo de mutacion delgen del receptor de LDL, clasificandolas en nulas y no nulas, sobre la prevalencia de infartoagudo de miocardio (IAM) en individuos heterocigotos con hipercolesterolemia familiar (HF) delsur de Europa, donde existen pocos datos al respecto. Pacientes y metodo Se trata de un estudio transversal que compara individuos con HF e IAM(n = 32) y a individuos con HF sin IAM (n = 76) mayores de 35 anos (41 varones y 67 mujeres).En 88 sujetos se establecio el diagnostico genetico, siendo divididos en portadores de mutacionesnulas o no nulas del gen del receptor de LDL. Se han comparado los factores clasicosde riesgo cardiovascular, concentraciones plasmaticas de lipidos y lipoproteinas, tipo de mutaciondel receptor de LDL y genotipo de APOE entre individuos con HF e IAM y sin IAM. Resultados Las variables relacionadas con el IAM fueron, en el analisis univariante, la edad, lapresencia de xantomas tendinosos o arco corneal, las concentraciones plasmaticas de colesteroltotal (CT) y colesterol unido a lipoproteinas de baja densidad (cLDL), el cociente CT/cHDL > de 5,3 y genotipo e4 de la APOE. Las odds ratio (OR) bipareadas para IAM fueron: la presenciade xantomas o arco corneal, 1,36 (intervalo de confianza [IC] del 95%, 1,08–1,71; p = 0,01);la edad > 54 anos (percentil 50 del total de individuos con HF) 1,56 (IC del 95%, 1,19–2,04;p = 0,001), y las concentraciones plasmaticas de CT > 332 mg/dl (percentil 50 del total de individuoscon HF), 1,34 (IC del 95%, 1,05–1,71; p = 0,019). En el analisis multivariante solola edad (p = 0,002) y el CT (p = 0,032) permanecieron en el modelo. Conclusiones El IAM en individuos con HF mayores de 35 anos de una poblacion del sur deEuropa se relaciona de forma univariante con la edad y las concentraciones plasmaticas de CTy cLDL, el cociente CT/cHDL y genotipo e4. El IAM se relaciona de forma independiente con laedad y las concentraciones plasmaticas de CT.