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Background Genetic diagnosis of familial hypercholesterolemia (FH) has not been universally performed in the Canary Islands (Spain). Objectives This study aimed to genetically characterize a cohort of patients with FH in the island of Gran Canaria. Methods Study subjects were 70 unrelated index cases attending a tertiary hospital in Gran Canaria, with a clinical diagnosis of FH, according to the criteria of the Dutch Lipid Clinic Network. Given that 7 of the first 10 cases with positive genetic study were carriers of a single mutation in the LDLR gene [p.(Tyr400_Phe402del)], a specific polymerase chain reaction-based assay was developed for the detection of this variant as a first screening step on the remaining subjects. In those without this mutation, molecular diagnosis was completed using a next-generation sequencing panel including LDLR, APOB, PCSK9, LDLRAP1, APOE, STAP1, and LIPA genes and incorporating copy number variation detection in LDLR. Results On the whole, 44 subjects (62%) had a positive genetic study, of whom 30 (68%) were heterozygous carriers of the p.(Tyr400_Phe402del) variant. Eleven subjects carried other mutations in LDLR, including the novel mutation NM_000527.4: c.877dupG; NP_000518.1: p.(Asp293Glyfs*8). An unclassified PCSK9 gene variant was found in one subject [(NM_174936.3:c.1496G>A; NP_777596.2: p.(Arg499His)]. Other single patients had mutations in APOB (heterozygous) and in LIPA (homozygous). All identified variants co-segregated with the disease phenotype. Conclusions These findings suggest a founder effect for the p.(Tyr400_Phe402del) LDLR mutation in Gran Canaria. A cost-effective local screening strategy for genetic diagnosis of FH could be implemented in this region.

Background: The development of biomarkers for use in diagnosing, monitoring disease progression and analyzing therapeutic trials response in amyotrophic lateral sclerosis (ALS) is essential. Objective: The aim of this study was to identify inflammatory factors in plasma or cerebrospinal fluid (CSF) from patients with ALS with particular attention to specific markers of microglia activation as chitotriosidase (ChT) and chemokine (C-C motif) ligand 18 (CCL18) to determine its potential as ALS biomarkers. Methods: We studied CSF and plasma samples from 32 patients and 42 healthy controls. We assayed the ChT activity by a spectrofluorometric method and protein levels of other inflammatory ­biomarkers (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6 and CCL18) by enzyme-linked immunosorbent assay. CHIT1 gene polymorphism in exon 10 (c.1049_1072dup24) encoding inactive ChT enzyme was genotyped in all subjects. Results: ChT activity and TNF-alpha protein levels were significantly higher in CSF of ALS patients, but we found no correlation with the severity and progression of the disease. Nevertheless, we did not found any differences in CCL18 or IL-6 protein levels between both groups in CSF or plasma. In our sample, only 3% of subjects were homozygous carriers for the CHIT1 exon 10 duplication associated with defective enzyme. Conclusions: High ChT activity in CSF of patients with ALS may reflect microglia activation and could be a potential biomarker of the disease. We did not find any significant difference regarding CCL-18, another specific marker of microglia activation that is related with M2-like microglia phenotype. Deepening the understanding of the activation state of microglia (M1 and M2) may contribute to the knowledge about the specific role of neuroinflammation in ALS and future therapeutic strategies.

Background Autosomal recessive hypercholesterolemia (ARH) is a rare lipid disorder characterized by premature atherosclerotic cardiovascular disease (ASCVD). There are sparse data for clinical management and cardiovascular outcomes in ARH. Objectives Evaluation of changes in lipid management, achievement of low-density lipoprotein cholesterol (LDL-C) goals and cardiovascular outcomes in ARH. Methods Published ARH cases were identified by electronic search. All corresponding authors and physicians known to treat these patients were asked to provide follow-up information, using a standardized protocol. Results We collected data for 52 patients (28 females, 24 males; 31.1 ± 17.1 years of age; baseline LDL-C: 571.9 ± 171.7 mg/dl). During a mean follow-up of 14.1 ± 7.3 years, there was a significant increase in the use of high-intensity statin and ezetimibe in combination with lipoprotein apheresis; in 6 patients, lomitapide was also added. Mean LDL-C achieved at nadir was 164.0 ± 85.1 mg/dl (−69.6% from baseline), with a better response in patients taking lomitapide (−88.3%). Overall, 23.1% of ARH patients reached LDL-C of Conclusions Despite intensive treatment, LDL-C in ARH patients remains far from targets, and this translates into a poor long-term cardiovascular prognosis. Our data highlight the importance of an early diagnosis and treatment and confirm the fact that an effective treatment protocol for ARH is still lacking.

Background Most primary severe hypertriglyceridemias (HTGs) are diagnosed in adults, but their molecular foundations have not been completely elucidated. Objective We aimed to identify rare dysfunctional mutations in genes encoding regulators of lipoprotein lipase (LPL) function in patients with familial and non-familial primary HTG. Methods We sequenced promoters, exons, and exon–intron boundaries of LPL , APOA5 , LMF1 , and GPIHBP1 in 118 patients with severe primary HTG (triglycerides >500 mg/dL) and 53 normolipidemic controls. Variant functionality was analyzed using predictive software and functional assays for mutations in regulatory regions. Results We identified 29 rare variants, 10 of which had not been previously described: c.(-16A>G), c.(1018+2G>A), and p.(His80Arg) in LPL ; p.(Arg143Alafs*57) in APOA5 ; p.(Val140Ile), p.(Leu235Ile), p.(Lys520*), and p.(Leu552Arg) in LMF1 ; and c.(-83G>A) and c.(-192A>G) in GPIHBP1 . The c.(1018+2G>A) variant led to deletion of exon 6 in LPL cDNA, whereas the c.(-16A>G) analysis showed differences in the affinity for nuclear proteins. Overall, 20 (17.0%) of the patients carried at least one allele with a rare pathogenic variant in LPL , APOA5 , LMF1 , or GPIHBP1 . The presence of a rare pathogenic variant was not associated with lipid values, family history of HTG, clinical diagnosis, or previous pancreatitis. Conclusions Less than one in five subjects with triglycerides >500 mg/dL and no major secondary cause for HTG may carry a rare pathogenic mutation in LPL , APOA5 , LMF1 , or GPIHBP1 . The presence of a rare pathogenic variant is not associated with a differential phenotype.

Background Early subclinical atherosclerosis has been mainly researched in carotid arteries. The potential value of femoral arteries for improving the predictive capacity of traditional risk factors is an understudied area. Objectives This study sought to evaluate the association of subclinical carotid and femoral plaques with risk factors and coronary artery calcium score (CACS) in middle-aged men. Methods Participants (n = 1,423) of the AWHS (Aragon Workers’ Health Study), a study designed to assess cardiovascular risk and subclinical atherosclerosis in a cohort of middle-aged men (40 to 59 years of age), underwent carotid and femoral ultrasound plus noncontrast coronary computed tomography. Subclinical atherosclerosis was defined as the presence of any plaque in carotid or femoral arteries and/or CACS ≥1. Logistic regression models were used to estimate the prevalence of atherosclerosis adjusted for risk factors and age, to evaluate the association of atherosclerosis with risk factors, and to calculate areas under the receiver-operating characteristic curves for the presence of positive CACS. Results Subclinical atherosclerosis was found in 72% of participants. Plaques were most common in femoral arteries (54%), followed by coronary calcification (38%) and carotid plaques (34%). Association of atherosclerosis with risk factors was stronger in femoral arteries than carotid or coronary arteries. The area under the receiver-operating characteristic curve for prediction of positive CACS increased from 0.665 when considering only risk factors (dyslipidemia, current smoking, hypertension, diabetes, and age) to 0.719 when adding femoral and carotid plaques (p Conclusions Subclinical atherosclerosis was highly prevalent in this middle-aged male cohort. Association with risk factors and positive CACS was stronger in femoral than carotid arteries. Screening for femoral plaques may be an appealing strategy for improving cardiovascular risk scales and predicting coronary disease.

Background and aims Few studies have evaluated the association of sleep duration with subclinical atherosclerosis, and with heterogeneous findings. We evaluated the association of sleep duration with the presence of coronary, carotid, and femoral subclinical atherosclerosis in healthy middle-age men with low prevalence of clinical comorbidities. Methods We performed a cross-sectional analysis of 1968 men, 40–60 years of age, participating in the Aragon Workers' Health Study (AWHS). Duration of sleep during a typical work week was assessed by questionnaire. Coronary artery calcium scores (CACS) was assessed by computed tomography and the presence of carotid plaque and femoral plaque by ultrasound. Results In fully adjusted models, the odds ratios (95% CI) for CACS >0 comparing sleep durations of ≤5, 6, and ≥8 h with 7 h were 1.34 (0.98–1.85), 1.35 (1.08–1.69) and 1.21 (0.90–1.62), respectively (p = 0.04). A similar U-shaped association was observed for CACS ≥100 and for CACS. The corresponding odds ratios for the presence of at least one carotid plaque were ≤5, 6, and ≥8 h with 7 h were 1.23 (0.88–1.72), 1.09 (0.86–1.38), and 0.86 (0.63–1.17), respectively (p = 0.31), and for the presence of at least one femoral plaque were 1.25 (0.87–1.80), 1.19 (0.93–1.51) and 1.17 (0.86–1.61), respectively (p = 0.39). Conclusions Middle-aged men reporting 7 h of sleep duration had the lowest prevalence of subclinical coronary atherosclerosis as assessed by CACs. Our results support that men with very short or very long sleep durations are at increased risk of atherosclerosis.

We report data from a prospective, observational study (ZAGAL) evaluating miglustat 100mg three times daily orally. in treatment-naïve patients and patients with type 1 Gaucher Disease (GD1) switched from previous enzyme replacement therapy (ERT). Clinical evolution, changes in organ size, blood counts, disease biomarkers, bone marrow infiltration (S-MRI), bone mineral density by broadband ultrasound densitometry (BMD), safety and tolerability annual reports were analysed. Between May 2004 and April 2016, 63 patients received miglustat therapy; 20 (32%) untreated and 43 (68%) switched. At the time of this report 39 patients (14 [36%] treatment-naïve; 25 [64%] switch) remain on miglustat. With over 12-year follow-up, hematologic counts, liver and spleen volumes remained stable. In total, 80% of patients achieved current GD1 therapeutic goals. Plasma chitotriosidase activity and CCL-18/PARC concentration showed a trend towards a slight increase. Reductions on S-MRI (p=0.042) with an increase in BMD (p

The majority of severe primary hypertriglyceridemia (HTG) are diagnosed in adults, and their molecular bases have not yet been fully defined. The promoter, coding regions and intron-exon boundaries of LMF1 were sequenced in 112 patients with severe primary hipertrigliceridemia (defined as TG above 500 mg/dl). Five patients (4.46%) were carriers of four rare variants in the LMF1 gene associated with HTG, which participate in lipoprotein lipase (LpL) function. Also, we have identified two common variants, c.194-28 T>G and c.729+18C>G that were associated with HTG, with a different allelic frequency to that observed in the general population. A bioinformatic analysis of all found variants was conducted, defining the following as potentially harmful: p.Arg364Gln, p.Arg451Trp, p.Pro562Arg and p.Leu85Leu. Our results suggest that LMF1 mutations are involved in a substantial proportion of cases with severe HTG, putting together the moderate-aggressive effect of rare mutations with polymorphisms classically associated with this disease.

Background— The majority of hypertriglyceridemias are diagnosed as familial combined hyperlipidemia (FCHL) and primary isolated hypertriglyceridemias. The contribution of common genetic variants in primary hypertriglyceridemias and the genetic difference between FCHL and isolated hypertriglyceridemias have not been thoroughly examined. Methods and Results— This study involved 580 patients with hypertriglyceridemias and 403 controls. Of the 37 single nucleotide polymorphisms examined, 12 located in 10 genes showed allelic and genotype frequency differences between hypertriglyceridemias and controls. The minor alleles of APOE , APOA5 , GALNTN2 , and GCKR variants were positively correlated with plasma triglycerides, whereas minor alleles of ADIPOR2 , ANGPTL3 , LPL , and TRIB1 polymorphisms were inversely associated. Body mass index, glucose, sex, rs328 and rs7007797 in LPL , rs662799 and rs3135506 in APOA5 , and rs1260326 in GCKR explained 36% of the variability in plasma triglycerides, 7.3% of which was attributable to the genetic variables. LPL , GCKR , and APOA5 polymorphisms fit dominant, recessive, and additive inheritance models, respectively. Variants more frequently identified in isolated hypertriglyceridemias were rs7412 in APOE and rs1800795 in IL6 ; rs2808607 in CYP7A1 and rs3812316 and rs17145738 in MLXIPL were more frequent in FCHL. The other 32 single nucleotide polymorphisms presented similar frequencies between isolated hypertriglyceridemias and FCHL. Conclusions— Common genetic variants found in LPL , APOA5 , and GCKR are associated with triglycerides levels in patients with primary hypertriglyceridemias. FCHL and isolated hypertriglyceridemias are probably trace to an accumulation of genetic variants predisposing to familial and sporadic hypertriglyceridemias or to hypertriglyceridemias and hypercholesterolemia in case of FCHL.

s p l P e c De todos los resultados que el Proyecto Genoma Humano y los estudios adicionales han generado, el descubrimiento de la proprotein convertase subtilisin/kesin tipo 9 (PCSK9) es con toda seguridad uno de los que ha tenido el impacto mas rapido en la salud humana. Podemos afirmar que PCSK9 es un «ejemplo emblematico» de la medicina traslacional de la era genomica. En 1999 se identifico mediante analisis de ligamiento genetico el tercer locus mayor asociado con hipercolesterolemia autosomica dominante en el cromosoma 1p34.1-p321, y 4 anos mas tarde se encontro en este locus al gen causal de la misma, el PCSK92. Al poco tiempo de descubrirse, este gen nos dio la primera sorpresa que fue el descubrimiento de su otra cara, como las del dios Jano, es decir, la asociacion con una hipocolesterolemia de las variantes del gen que cursaban con perdida de funcion de la proteina y el hecho de que solo las mutaciones de ganancia de funcion se asociaban con hipercolesterolemia3. La proteina PCSK9 es secretada al plasma y se une al receptor de las lipoproteinas de baja densidad (rLDL) en el primer dominio (EGF-A) del homologo al precursor del factor de crecimiento epidermico (EGF)4. Aunque el dominio C-terminal de PCSK9 no es necesario para la union al rLDL, es fundamental para la degradacion del receptor5. El mecanismo completo mediante el cual PCSK9 se une al rLDL y lo dirige a la degradacion no se conoce con precision. Otra de las sorpresas de esta proteina fue el hecho de que al ser una proteasa de serina se creia que su accion proteolitica

Background and aims Autosomal recessive hypercholesterolemia (ARH) is a very rare disease, caused by mutations in LDL protein receptor adaptor 1 (LDLRAP1). It is characterized by high levels of low-density lipoprotein cholesterol (LDL-C) and increased risk of premature cardiovascular disease. We aimed to characterize ARH in Spain. Methods Data were collected from the Dyslipidemia Registry of the Spanish Atherosclerosis Society. A literature search was performed up to June 2017, and all diagnostic genetic studies for familial hypercholesterolemia of Spain were reviewed. Results Seven patients with ARH were identified, 6 true homozygous and one compound heterozygous with a novel mutation: c.[863C>T];p.[Ser288Leu]. High genetic heterogeneity was found in this cohort. True homozygous subjects for LDLRAP1 have more severe phenotypes than the compound heterozygous patient, but similar to patients with homozygous familial hypercholesterolemia (HoFH). Cardiovascular disease was present in 14% of the ARH patients. LDL-C under treatment was above 185 mg/dl and the response to PCSK9 inhibitors was heterogeneous. Finally, the estimated prevalence in Spain is very low, with just 1 case per 6.5 million people. Conclusions ARH is a very rare disease in Spain, showing high genetic heterogeneity, similarly high LDL-C concentrations, but lower incidence of ASCVD than HoFH.

Purpose of review:Niemann-Pick disease type C (NP-C) is a neurovisceral disorder that may be more prevalent than earlier estimates. Diagnosis of NP-C is often delayed; a key aim for clinical practice is to reduce this delay. Recently, substantial progress has been made in the field of NP-C screening and diagnosis, justifying an update to the existing recommendations for clinical practice.Recent findings:New biomarker profiling and genetic analysis technologies are included as first-line diagnostic tests for NP-C. Most diagnoses can now be confirmed by combination of biomarker and genetic analyses. Filipin staining may facilitate diagnosis in uncertain cases. Recommendations are provided for psychiatrists, neuro-ophthalmologists, and radiologists, and on screening within specific at-risk patient cohorts. The NP-C diagnostic algorithm has been updated and simplified.Summary:This publication provides expert recommendations for clinicians who may see patients presenting with the signs and symptoms of NP-C, including general practitioners, pediatricians, neurologists, and psychiatrists.

Background: The enzymatic replacement therapy (ERT) availability for Gaucher disease (GD) has changed the landscape of the disease, several countries have screening programs. These actions have promoted the early diagnosis and avoided many complications in pediatric patients. In Spain ERT has been available since 1993 and 386 patients have been included in the Spanish Registry of Gaucher Disease (SpRGD). The aim of this study is to analyze the impact of ERT on the characteristics at time of diagnosis and initial complications in pediatric Gaucher disease patients. Aim: To analyze the impact of ERT on the characteristics at time of diagnosis and initial complications in pediatric Gaucher disease patients. Methods: A review of data in SpRGD from patients' diagnosed before 18 years old was performed. The cohort was split according the year of diagnosis (= 1995, cohort B). Results: A total of 98 pediatric patients were included, GD1: 80, GD3: 18; mean age: 7.2 (0.17-16.5) years, 58 (59.2%) males and 40 (40.8%) females. Forty-five were diagnosed = 1995. Genotype: N370S/N370S: 2 (2.0%), N370S/L444P: 27 (27.5%), N370S/other: 47 (48%), L444P/L444P: 7 (7.1%), L444P/D409H: 2 (2.0%), L444P/other: 3 (6.2%), other/other: 10 (10.2%). The mean age at diagnosis was earlier in patients diagnosed after 1995 (p < 0.001) and different between the subtypes, GD1: 8.2 (0.2-16.5) years and GD3: 2.8 (0.17-10.2) years (p < 0.001). There were more severe patients in the group diagnosed before 1994 (p = 0.045) carrying L444P (2), D409H (2), G377S (1), G195W (1) or the recombinant mutation. The patients' diagnosed = 1994 showed worse cytopenias, higher chance of bone vascular complications at diagnosis and previous spleen removal. The patients started ERT at a median time after diagnosis of 5.2 years [cohort A] and 1.6 years [cohort B] (p < 0.001). Conclusions: The early diagnosis of Gaucher disease in the era of ERT availability has permitted to reduce the incidence of severe and irreversible initial complication in pediatric patients, and this has permitted better development of these patients. This is the largest pediatric cohort from a national registry.

Background— Homozygous familial hypercholesterolemia (HoFH) is a rare disease characterized by elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) and extremely high risk of premature atherosclerotic cardiovascular disease. HoFH is caused by mutations in several genes, including LDL receptor ( LDLR ), apolipoprotein B ( APOB ), proprotein convertase subtilisin/kexin type 9 ( PCSK9 ), and LDL protein receptor adaptor 1 ( LDLRAP1 ). No epidemiological studies have assessed HoFH prevalence or the clinical and molecular characteristics of this condition. Here, we aimed to characterize HoFH in Spain. Methods and Results— Data were collected from the Spanish Dyslipidemia Registry of the Spanish Atherosclerosis Society and from all molecular diagnoses performed for familial hypercholesterolemia in Spain between 1996 and 2015 (n=16 751). Clinical data included baseline lipid levels and atherosclerotic cardiovascular disease events. A total of 97 subjects were identified as having HoFH—of whom, 47 were true homozygous (1 for APOB , 5 for LDLRAP1 , and 41 for LDLR ), 45 compound heterozygous for LDLR , 3 double heterozygous for LDLR and PSCK9 , and 2 double heterozygous for LDLR and APOB . No PSCK9 homozygous cases were identified. Two variants in LDLR were identified in 4.8% of the molecular studies. Over 50% of patients did not meet the classical HoFH diagnosis criteria. The estimated HoFH prevalence was 1:450 000. Compared with compound heterozygous cases, true homozygous cases showed more aggressive phenotypes with higher LDL-C and more atherosclerotic cardiovascular disease events. Conclusions— HoFH frequency in Spain was higher than expected. Clinical criteria would underestimate the actual prevalence of individuals with genetic HoFH, highlighting the importance of genetic analysis to improve familial hypercholesterolemia diagnosis accuracy.

Familial hypercholesterolemia (FH) is one of the most common and severe genetic diseases, causing disabilities and premature death to those who suffer it. Lipid-lowering therapy substantially improves the prognosis of FH patients and, therefore, appropriate pharmacological treatment is of the utmost importance. The Spanish Society of Arteriosclerosis (SEA) has always been a pioneer in the diagnosis and treatment of FH. Since its inception, FH has been one of the main areas of clinical and scientific interest, mainly for Lipids Units of the SEA, where most patients with this pathology are referred in Spain. This document arises from the willingness of our society to update the scientific knowledge on this subject and to provide physicians with clear clinical guidelines regarding diagnosis and treatment of FH. These guidelines can be summarized in two main aspects: early diagnosis of the disease and a rapid normalization of LDLcholesterol. In the coming years, health providers should accomplish that the majority of patients with FH are aware of their diagnosis and that adequate treatment is provided.

Evaluation of: Talmud PJ, Shah S, Whittall R et al. Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic hypercholesterolaemia: a case–control study. Lancet 381(9874), 1293–1301 (2013). Familial hypercholesterolemia (FH) is a common disorder caused by mutations in LDLR, APOB and PCSK9. However, many subjects with primary hypercholesterolemia did not demonstrate functional mutations in any of these genes. Genome-wide association studies have identified single nucleotide polymorphisms that influence the variability of LDL-C concentration. Talmud et al. have genotyped 12 common LDL cholesterol (LDL-C)-raising alleles in 640 patients with the clinical diagnosis of FH and in 3020 healthy subjects from the UK Whitehall II study. Approximately 50% of the FH patients did not possess a causative mutation and the mean weighted LDL-C gene score was significantly higher than in the controls, indicating a polygenic cause for their disorder. These results indicate t...

Background Familial hypercholesterolemia (FH) is the best-described autosomal dominant genetic hypercholesterolemia (GH). Mutations in candidate genes can explain a high proportion of FH cases, but for many, no causative mutations are detected (designed non-FG-GH), suggesting the existence of additional genetic variants associated with the disease. Objective We aimed to identify new single-nucleotide variants (SNVs) located at the 3′ untranslated regions (3′UTRs) of the low-density lipoprotein receptor, low-density lipoprotein receptor–related protein-associated protein 1, ATP-binding cassette sub-family G member 5, and sterol regulatory element–binding protein 2 genes in non-FH-GH individuals and investigated whether the association of these SNVs with non-FH-GH could be explained by changes in the affinity of regulatory microRNAs (miRNA) targeting the sequences modified by the SNVs. Methods The study includes probands with non-FH-GH attending 2 lipid clinics in Spain. We performed functional analyses of selected variants using a luciferase reporter system. Through in silico target-prediction tools, we identified miRNAs, which binding to the 3′UTR could be affected by the presence of specific SNVs. We used analogs and inhibitors of these miRNAs to test this possibility. Results We identified 11 new SNVs showing significant association with non-FH-GH. We show that the presence of 4 of these SNVs leads to significant changes in the transcriptional levels of the reporter gene. Through mechanistic analysis, we identified 2 miRNAs (miR-27a and miR-133-3p) targeting the 3′UTR of sterol regulatory element–binding protein 2 and an additional miRNA (miR-92a) targeting the 3′UTR of low-density lipoprotein receptor–related protein-associated protein 1. Conclusion Our findings reveal novel regulatory links between certain miRNAs and key genes regulating cholesterol homeostasis. They also highlight the potential of miRNAs as therapeutic targets for the treatment of FH.

Background Armolipid Plus (AP) is a nutraceutical that contains policosanol, fermented rice with red yeast, berberine, coenzyme Q10, folic acid, and astaxanthin. It has been shown to be effective in reducing plasma LDL cholesterol (LDLc) levels. In the multicenter randomized trial NCT01562080, there was large interindividual variability in the plasma LDLc response to AP supplementation. We hypothesized that the variability in LDLc response to AP supplementation may be linked to LDLR and PCSK9 polymorphisms. Material and Methods We sequenced the LDLR 3′ and 5′ untranslated regions (UTR) and the PCSK9 5′ UTR of 102 participants with moderate hypercholesterolemia in trial NCT01562080. In this trial, 50 individuals were treated with AP supplementation and the rest with placebo. Results Multiple linear regression analysis, using the response of LDLc levels to AP as the dependent variable, revealed that polymorphisms rs2149041 (c.-3383C>G) in the PCSK9 5′ UTR and rs14158 (c.*52G>A) in the LDLR 3′ UTR explained 14.1% and 6.4%, respectively, of the variability after adjusting for gender, age, and BMI of individuals. Combining polymorphisms rs2149041 and rs14158 explained 20.5% of this variability (p < 0.004). Conclusions Three polymorphisms in the 3′ UTR region of LDLR, c.*52G>A, c.*504G>A, and c.*773A>G, and two at the 5′ UTR region of PCSK9, c.−3383C>G and c.−2063A>G, were associated with response to AP. These results could explain the variability observed in the response to berberine among people with moderate hypercholesterolemia, and they may be useful in identifying patients who could potentially benefit from supplementation with AP.

Gaucher disease (GD) is a disorder of glycosphingolipid metabolism caused by deficiency of lysosomal glucocerebrosidase (GlcCerase) activity, due to conformationally or functionally defective variants, resulting in progressive deposition of glycosylceramide in macrophages. The glucose analogue, N-butyldeoxynojirimycin (NB-DNJ, miglustat), is an inhibitor of the ceramide-specific glycosyltransferase, which catalyzes the first step of glycosphingolipid biosynthesis and is currently approved for the oral treatment of type 1 GD. In a previous work, we found a GlcCerase activity increase in cell cultures in the presence of NB-DNJ, which could imply that this compound is not only a substrate reducer but also a pharmacological chaperone or inhibitor for GlcCerase degradation. In this work we compare imiglucerase (the enzyme currently used for replacement therapy) and velaglucerase alfa (a novel therapeutic enzyme form) in terms of conformational stability and enzymatic activity, as well as the effect of NB-DNJ on them. The interaction between these enzymes and NB-DNJ was studied by isothermal titration calorimetry. Our results reveal that, although velaglucerase alfa and imiglucerase exhibit very similar activity profiles, velaglucerase alfa shows higher in vitro thermal stability and is less prone to aggregation/precipitation, which could be advantageous for storage and clinical administration. In addition, we show that at neutral pH NB-DNJ binds to and enhances the stability of both enzymes, while at mildly acidic lysosomal conditions it does not bind to them. These results support the potential role of NB-DNJ as a pharmacological chaperone, susceptible of being part of pharmaceutical formulation or combination therapy for GD in the future.

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.

Variations in the gene encoding the low-density lipoprotein receptor (LDLR) can cause familial hypercholesterolemia (FH), one of the most common inherited metabolic disorders in humans. The functional effects of the p.Gln92Glu and p.Asn564His alterations are predicted as benign, but the c.313 + 1G>C and p.Lys799_Phe801del changes are believed to cause disease. Although p.Gln92Glu and c.313 + 1G>C have been observed only in Spain, p.Asn564His and p.Lys799_Phe801del are widespread in Western Europe. In order to estimate the ages (t generations) of these four variants of the gene, to determine their possible origin and to consider the influence of age and selective pressure on their spread, we analyzed 86 healthy individuals and 126 FH patients in Spain. Most of the FH patients investigated carried two of these four LDLR variants simultaneously, while only one patient carried three of them simultaneously. Haplotype analyses were based on five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C and c.2232G>A. The results suggest that p.Gln92Glu and c.313 + 1G>C arose at about the same time (99 and 103 generations ago, respectively) in the CACTG haplotype and that p.Asn564His and p.Lys799_Phe801del appeared in the CGCCG haplotype and might be slightly more recent variations (92 and 95 generations ago, respectively). Low selective pressures could explain the maintenance of these variants in spite of their ages. The origin of p.Gln92Glu and c.313 + 1G>C appears to be in Spain whereas p.Asn564His and p.Lys799_Phe801del could have been introduced in Spain by Celtic migrations in the seventh to fifth centuries BC.

Resumen En la enfermedad de Gaucher (EG) se produce un desequilibrio en el sistema monocito-macrofago entre la formacion y la degradacion de la glucosilceramida, consecuencia de una baja actividad de la enzima lisosomal beta-glucorebrosidasa. Los enfoques terapeuticos en la EG estan dirigidos a disminuir la sintesis de glucosilceramida inhibiendo la enzima ceramida-glucosiltransferasa; aumentar su degradacion infundiendo una beta-glucorebrosidasa recombinante, o recuperar la actividad residual de las enzimas mutadas mediante chaperonas farmacologicas. Las moleculas inhibidoras de la encima ceramida-glucosiltransferasa utilizadas son analogos de glucosa o de ceramida. Las enzimas recombinantes presentan parametros cineticos similares, pero difieren en la secuencia de aminoacidos, asi como en sus cadenas de oligosacaridos. Las manosas de las cadenas de oligosacaridos de las enzimas recombinantes son fundamentales para su reconocimiento por las celulas. Destaca la velaglucerasa alfa por su alto contenido en manosas, con un promedio de 9 manosas por cadena.

Resumen Introduccion La hipercolesterolemia familiar (HF) es una enfermedad autosomica dominante, causada principalmente por mutaciones en la region 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 busqueda de mutaciones en sujetos clinicamente diagnosticados como HF revelo 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 Espana mediante el uso de la plataforma LIPOchip ® en pacientes con sospecha clinica de HF. Metodos Se realizo el analisis funcional de las mutaciones mediante ensayos de retardo de la movilidad electroforetica (EMSA) y transfeccion 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 patron de afinidad por las proteinas nucleares en los EMSA. Ademas, estas mutaciones produjeron una reduccion 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 analisis de funcionalidad de las variantes encontradas en la region promotora de LDLR con objeto de conocer su implicacion con el fenotipo HF.

Summary 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.

Gaucher disease (GD) is an autosomal recessive disorder characterized by defective function of glucocerebrosidase. GD presents a wide spectrum of manifestations, and patients and their relatives may develop neurological abnormalities more frequently than the general population. This study aims to determine the presence of neurological symptoms (NS) and Parkinson's disease (PD) in Spanish GD patients and their relatives. We surveyed 87 GD Spanish families and validated the information obtained on the neurological involvement through their physicians, as well as the historical data included in the Spanish Gaucher Disease Registry. Neurological abnormalities were correlated with the genetic characteristics. Statistical analyses included descriptive parameters, ANOVA, t-test, correlation study and Pearson coefficient. Information was obtained from 118 patients and 324 relatives. Out of 110 patients with type 1 GD, 32 (29.1%) reported NS and 7 (6.4%) had PD. In relatives, a total of 39 (13.1%) subjects had NS, including 16 with PD (5.3%). The prevalence of NS in genetic carriers (15.9%) was greater than that in non-carriers (5.9%; p 0.01). Patients with PD carried the following GBA mutations: S364R, D409H, L444P, R257Q, IVS4-2A G, c.500insT, and L336P. Relatives with PD exhibited a wide spectrum of mutations: L444P, N370S, V398I, R257Q, G202R, c.1439-1445del7, [E326K; N188S], and c.953delT. We observed a high incidence of PD in type 1 GD and relative's carriers. PD was more frequent in carriers of L444P and other rare GBA mutations. Therefore, it is important to perform a systematic neurological exam in patients with type 1 GD and carriers with high risk mutations.

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.

Recently, an acute restriction of imiglucerase has occurred as a result of viral contamination and manufacturing problems. A position statement from the European Working Group for Gaucher Disease and European Gaucher Alliance established a set of key recommendations for identifying and monitoring at-risk patients. In Spain, a profile of the shortage situation was obtained through follow-up of patients with Gaucher disease (GD) and compliance with the therapy recommendations. Here we describe a group of patients, with modified doses of imiglucerase, during the shortage. Fifty adult GD1 patients (25 males/25 females), previously on ERT, were analysed before and after the 6-month shortage. The mean age was 45.3 ± 15.3 years (range: 18-84). The mean Severity Score Index at diagnosis was 8.7 ± 3.8 (range: 3-19); 20% of patients were splenectomized; and 78% had bone disease. During the shortage, 23 patients (46%) discontinued therapy; as complications in this group only one patient suffered a bone crisis and another anaemia (Hb

Resumen Introduccion La apolipoproteina A-I Zaragoza (apo A-I Z, mutacion L144R) es una variante de la apolipoproteina A-I cuyos portadores tienen muy baja concentracion de colesterol-HDL (c-HDL) pero, aunque presenten factores de riesgo aterogenicos adicionales, paradojicamente no muestran signos de aterosclerosis a nivel subclinico. Estudios metabolicos in vivo realizados en los portadores heterocigotos de esta variante de apo A-I revelaron que tiene una tasa de catabolismo que duplica la de la proteina nativa sugiriendo la posibilidad de que tuviera un efecto favorable sobre el proceso global de transporte reverso de colesterol. Objetivo Establecer un sistema de expresion y purificacion de apo A-I nativa y apo A-I Z recombinantes de alto rendimiento y elevada pureza con el fin de comparar sus propiedades en distintos aspectos relativos al transporte reverso de colesterol, asi como otras caracteristicas antiaterogenicas de la apo A-I. Metodologia Partiendo del cDNA de apo A-I nativa se amplifico mediante PCR de la secuencia de la apo A-I madura y se disenaron los oligonucleotidos cebadores que permitieron el clonaje en un plasmido de expresion inducible pET-45 que incorpora una cola de Histidinas en la posicion N-terminal del peptido expresado. Mediante mutagenesis dirigida se introdujo la mutacion L144R en la secuencia madura de apo A-I para expresarla con el mismo sistema. Bacterias E. coli de la cepa BL21(DE3) fueron transformadas con los plasmidos preparados, la expresion de las correspondientes proteinas fue inducida y su purificacion realizada en condiciones no desnaturalizantes mediante cromatografia de afinidad en columnas de niquel. Resultados La expresion y la purificacion de las dos proteinas se realizo con exito y fue comprobada mediante SDS-PAGE y tecnicas inmunoquimicas. Se obtuvieron rendimientos medios superiores a 30 mg de proteina purificada por litro de cultivo con un grado de pureza del 94% para la proteina nativa y 93% para la mutante. Conclusiones Se ha conseguido un sistema de expresion y purificacion para apo A-I nativa y apo A-I Z de alto rendimiento y pureza adecuados que constituye la base para su posterior caracterizacion estructural y funcional de las propiedades antiaterogenicas de esta variante de la apo A-I.

Familial hypercholesterolemia (FH), an autosomal dominant inherited disorder resulting in increased levels of circulating plasma low-density lipoprotein (LDL), tendon xanthomas and premature coronary artery disease (CAD), is caused by defects in the LDL receptor gene (LDLR). Three widespread LDLR alterations not causing FH (c.1061-8T>C, c.2177C>T and c.829G>A) and one mutation (c.12G>A) with narrow geographical distribution and thought to cause disease were investigated. In an attempt to improve knowledge on their origin, spread and possible selective effects, estimations of the ages of these variants (t generations) and haplotype analysis were performed by genotyping 86 healthy individuals and 98 FH patients in Spain for five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C, and c.2232G>A; most patients carried two of these LDLR variants simultaneously. It was found that both the c.1061-8T>C (t = 54) and c.2177C>T alterations (t = 62) arose at about the same time (54 and 62 generations ago, respectively) in the CGCTG haplotype, while the c.12G>A mutation (t = 70) appeared in a CGCCG haplotype carrying an earlier c.829G>A alteration (t = 83). The estimated ages of selectively neutral alterations could explain their distribution by migrations. The origin of the c.12G>A mutation could be in the Iberian Peninsula; despite its estimated age, a low selective pressure could explain its conservation in Spain from where it could have spread to China and Mexico, since the sixteenth century through the Spanish/Portuguese colonial expeditions.

Niemann-Pick disease type C (NP-C) is an inherited neurovisceral lysosomal lipid storage disease characterized by progressive neurological deterioration. Different clinical forms have been defined based on patient age at onset: perinatal, early-infantile (EI), late-infantile (Li), juvenile and adult. We evaluated the efficacy and tolerability of miglustat in 16 symptomatic NP-C patients, with comparative reference to one neurologically asymptomatic, untreated patient. All patients were categorized according to age at neurological disease onset, and were assessed using a standardized clinical assessment protocol: disability and cognitive function scales, positron emission tomography (PET), and biochemical markers. PET and disability scale evaluations indicated that cerebral hypometabolism and neurological symptoms were stabilized during treatment in juvenile-onset NP-C patients. EI and Li NP-C patients, who had higher disease severity at baseline (treatment start), showed increased disability scores and progressive cerebral hypometabolism during follow up. Similarly, while cognitive scale scores remained relatively stable in patients with juvenile NP-C, cognition deteriorated in EI and Li patients. Plasma chitotriosidase (ChT) activity was lower in the juvenile NP-C subgroup than in EI and Li patients, and generally increased in patients who discontinued treatment. Plasma CCL18/PARC and ChT activities indicated greater macrophagic activity in EI and Li patients versus juveniles. Miglustat was generally well tolerated; frequent adverse events included diarrhea and flatulence, which were managed effectively by dietary modification and loperamide. Overall, miglustat appeared to stabilize neurological status in juvenile-onset NP-C patients, but therapeutic benefits appeared smaller among younger patients who were at a more advanced stage of disease at baseline.

Objective Hypercholesterolemia is an early risk factor for Alzheimer's disease. Low-density lipoprotein (LDL) receptors might be involved in this disorder. Our objective was to determine the risk of mild cognitive impairment in a population of patients with heterozygous familial hypercholesterolemia, a condition involving LDL receptor dysfunction and lifelong hypercholesterolemia. Methods By using a cohort study design, patients with familial hypercholesterolemia (N=47) meeting inclusion criteria and comparison patients without familial hypercholesterolemia (N=70) were consecutively selected from academic specialty and primary care clinics, respectively. All patients were older than 50 years. Those with disorders that could affect cognition, including history of stroke or transient ischemic attacks, were excluded from both groups. Thirteen standardized neuropsychologic tests were performed in all subjects. Mutational analysis was performed in patients with familial hypercholesterolemia, and brain imaging was obtained in those with familial hypercholesterolemia and mild cognitive impairment. Results Patients with familial hypercholesterolemia showed a high incidence of mild cognitive impairment compared with those without familial hypercholesterolemia (21.3% vs 2.9%; P =.00). This diagnosis was unrelated to structural pathology or white matter disease. There were significant differences, independent of apolipoprotein E4 or E2 status, between those with familial hypercholesterolemia and those with no familial hypercholesterolemia in several cognitive measures, all in the direction of worse performance for those with familial hypercholesterolemia. Conclusion Because prior studies have shown that older patients with sporadic hypercholesterolemia do not show a higher incidence of mild cognitive impairment, the findings presented suggest that early exposure to elevated cholesterol or LDL receptor dysfunction may be risk factors for mild cognitive impairment.

Gaucher disease is the first lysosomal disorder for which clinically effective enzyme replacement therapy has been introduced. Lifelong treatment with imiglucerase, the recombinant glucocerebrosidase manufactured by the Genzyme Corporation (MA, USA), is administered intravenously - usually at biweekly intervals. An acute shortage of imiglucerase (to 20% of prior global supply) has occurred as a result of viral contamination of the production facility; production was hafted, and a full supply of imiglucerase is not anticipated until January 2010. An urgent meeting of physicians, researchers, and patients was convened through the agency of the European Working Group for Gaucher Disease; this was instigated by patients internationally represented by the European Gaucher Alliance. Here we present a position statement based on the findings of the group, with key recommendations about identification and monitoring of at-risk patients threatened by the abrupt withdrawal of treatment, the equitable distribution of residual imiglucerase - and access to alternative treatments including those that have completed phase III clinical trials but have not yet been licensed. (C) 2009 Elsevier Inc. All rights reserved

There are few published data from real-world clinical experience with miglustat (Zavesca®), an oral inhibitor of glucosylceramide synthase, in type 1 Gaucher disease. We report data from a prospective, open-label investigational study that evaluated substrate reduction therapy with miglustat 100 mg t.i.d. as a maintenance therapy in patients with Type 1 Gaucher disease who had been switched from previous enzyme replacement therapy. Long-term data on changes in organ size, blood counts, disease severity bio-markers, bone marrow infiltration, overall clinical status and safety/tolerability were analyzed from 28 patients with Type 1 Gaucher disease who were attending routine clinic visits. Assessments were performed at six, 12, 24, 36 and 48 months of therapy. Disease severity biomarkers improved up to 48 months after initiation of miglustat, while other disease parameters remained stable. Miglustat showed an acceptable profile of safety and tolerability throughout treatment. In conclusion, miglustat is an effective therapy for the long-term maintenance of patients with Type 1 Gaucher disease previously stabilized with enzyme replacement therapy.

Nuestro objetivo es proporcionar al clinico los conocimientos basicos para la interpretacion de datos referidos a enfermedades geneticas que le sirvan de ayuda a la hora de tomar decisiones clinicas. Se hace un simil del genoma humano con una enciclopedia, la «enciclopedia de la vida», analizando como se transmite la informacion genetica, las causas, las consecuencias y las principales reglas de nomenclatura de las mutaciones. Se discute a continuacion que mutaciones y como pueden contribuir a la aparicion de la enfermedad cardiovascular. Por ultimo, se describe la contribucion de la genetica a la mejora del diagnostico y el tratamiento de la hipercolesterolemia familiar (HF), una de las entidades en que el tratamiento hipolipemiante es eficaz y coste-efectivo. Se justifica el empleo de tecnicas de deteccion genetica a gran escala como son los biochips que analizan las principales mutaciones de los genes que originan el HF.

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.

Uptake of low density lipoproteins (LDL) by their receptor, LDLR, is the primary mechanism by which cells incorporate cholesterol from plasma. Mutations in LDLR lead to familial hypercholesterolemia, a common disease affecting 1 in 500 of the human population. LDLR is a modular protein that uses several small repeats to bind LDL. The repeats contain around 40 residues, including three disulfide bonds and a calcium ion. Repeat 5 (LR5) is critical for LDL and β-migrating very low density lipoprotein binding. Based on the crystal structure of LDLR at endosomal pH (but close to extracellular calcium concentration), LR5 has been proposed to bind to the epidermal growth factor (EGF) precursor domain of LDLR in the endosome, thus releasing the LDL particles previously bound in extracellular conditions. We report here the conformational stability of LR5 as a function of temperature and calcium concentration under both extracellular and endosomal pH conditions. The repeat was very stable when it bore a bound calcium ion but was severely destabilized in the absence of calcium and even further destabilized at acidic versus neutral pH. The temperature and calcium concentration dependence of LR5 stability clearly indicate that under endosomal conditions the unfolded conformation of the repeat is largely dominant. We thus propose a new mechanism for LDL release in the endosome in which calcium depletion and decreased stability at acidic pH drives LR5 unfolding, which triggers LDL release from the receptor. Subsequent binding of LR5 to the EGF precursor domain, if it takes place at low calcium concentrations, would contribute to a further shifting of the equilibrium toward dissociation.

Fundamento y objetivo La enfermedad de Gaucher (EG) se caracteriza por su heterogeneidad clinica. Estudios previos indican que los familiares pueden presentar anomalias neurologicas con mayor frecuencia que la poblacion general. El objetivo de este trabajo ha sido conocer la presencia de manifestaciones neurologicas (MN) en pacientes con EG y sus familiares. Pacientes y metodo Entre enero y diciembre de 2006 se remitio una encuesta a 42 medicos y 92 familias con afectados de EG para obtener informacion sobre MN y correlacionarla con las caracteristicas geneticas. Se realizo un analisis estadistico descriptivo, con analisis de ANOVA, prueba de la t de Student y estudio de correlacion (coeficiente de Pearson). Resultados Se obtuvo informacion de 72 familias (un 78,3% de respuestas): 99 pacientes y 266 familiares. De los pacientes con EG tipo 1, 30 (32,6%) presentaban MN: temblor en 8 casos (8,7%), movimientos incoordinados en 9 (9,8%), falta de concentracion en 11 (11,9%), estrabismo en 7 (7,6%), hipoacusia en 8 (8,7%), enfermedad de Parkinson (EP) en 7 (7,6%) y neuropatia periferica en 10 (10,9%). En cuanto a los familiares (primer/segundo grado), 36 (13,5%) presentaron alguna MN: EP en 14 casos (4,9%), epilepsia en 8 (3,0%), temblor en 7 (2,6%), hipoacusia en 2 (0,7%) y otros en 5 (1,9%). La frecuencia de MN en portadores de mutacion en el gen de la enzima betaglucosidasa acida (GBA) fue del 17,3%, frente al 5,7% en los no portadores (p = 0,0096). Los pacientes con EG que presentaban EP tenian las mutaciones S364R, D409H, L444P [IVS4-2a → g; c.(–203)A → G], c.500insT y L336P. Los familiares con EP presentaban diversas mutaciones: L444P, N370S, V398I, G202R, c.1439-1445del7 [E326K; N188S] y c.953delT. En otras MN, las predominantes fueron D409H, G195W, R120W, R147X, L336P y G377S. Conclusiones La EP y otras MN tuvieron en pacientes con EG tipo 1 y sus familiares una incidencia superior a la esperada y aparecieron mas frecuentemente en portadores de L444P o mutaciones raras. Se recomienda realizar un examen neurologico sistematico a los pacientes con EG tipo 1 y a portadores de mutaciones de riesgo.

Civeira F, Pocovi M, Cenarro A, Garces C, Ordovas JM. Adenine for guanine substitution 78 base pairs 5′ to the apolipoprotein (APO) A-I gene: relation with high density lipoprotein cholesterol and APO A-I concentrations. Clin Genet 1993: 44: 307–312. © Munksgaard, 1993 A common mutation, adenine (A) for guanidine (G) substitution (G/A) has been located78 bp 5′ to the apo A-I gene. This region has been shown to be involved in the transcriptional regulation of the apo A-I gene. Previous studies have shown that this mutation is associated with altered high density lipoprotein cholesterol (HDL-C) levels, although these findings have not been consistent. We have studied the frequency of this mutation in 125 subjects (60 males and 65 females) selected because they had HDL-C levels below the 25th (low HDL) or above the 75th (high HDL) percentile of the population distribution. The presence of the mutation was detected by Msp I digestion of a 259 bp fragment of PCR amplified DNA. The allele frequency was similar in both groups (0.20 for the lowest HDL group and 0.28 for the highest HDL group, p>0.05), although a non-significant trend was observed in a higher frequency of the A/A genotype in the highest HDL females (17.5%) vs only 6.7% in the lowest HDL female group. In conclusion, in this population the G/ A mutation was not significantly associated with HDL-C or apo A-I plasma levels.

Introduccion La via de senalizacion del acido retinoico (AR) media la transcripcion inducida por ligando de genes del metabolismo lipidico a traves de los receptores nucleares, receptor X de retinoico y receptor de AR, y del coactivador proteina de union al AR tipo II. Se ha establecido una asociacion entre esta via de senalizacion y las alteraciones lipidicas en trastornos como la hiperlipemia familar combinada y la hiperlipemia inducida por el tratamiento con inhibidores de la proteasa (IP) en pacientes con infeccion por el virus de la inmunodeficiencia humana (VIH). Nuestro objetivo ha sido comprobar la hipotesis segun la cual el gen CRABP2 estaria involucrado en la modulacion de la dislipemia. Sujetos y metodos Se ha realizado un estudio de asociacion del polimorfismo -394T>C del promotor de CRABP2 en 3 poblaciones independientes (299 individuos sanos, 182 pacientes con infeccion por el VIH y 151 pacientes con hipercolesterolemia familiar [HF]). Todas las analiticas se determinaron en ausencia de tratamiento hipolipemiante. Se realizo ANOVA ajustando por edad, indice de masa corporal, sexo, y uso de IP cuando fue necesario. Resultados La frecuencia del alelo C fue del 0,03 en los 3 grupos estudiados. Entre los individuos sanos, los portadores del alelo C presentaban un aumento en la concentracion de colesterol total del 9% (p = 0,027) y de colesterol unido a lipoproteinas de baja densidad (cLDL) del 13% (p = 0,020). En los pacientes infectados por el VIH, se realizo un analisis multivariante de 4 medidas tomadas durante 1 ano, y se observo que los valores de cLDL eran significativamente mas elevados en los portadores del alelo C (p = 0,001), y que estos oscilaban entre el 10 y el 31%. Asimismo, los pacientes HF portadores del alelo C presentaban un aumento significativo en la concentracion de cLDL del 16% (p = 0,038). El alelo C estaba sobrerrepresentado en individuos hipercolesterolemicos (p = 0,001). Conclusiones Nuestros resultados muestran que el gen CRABP2 , involucrado en la via de senalizacion del acido retinoico, esta asociado con elevadas concentraciones de cLDL en plasma.

Type 1 Gaucher disease (GD1) is characterised by lack of central nervous system involvement; however, there are several reports of associated neurological manifestations. The aim of this study was to systematically evaluate neurological manifestations in 31 patients with GD1 (12 males and 19 females; mean age 39.4 (range 5-77) years). Participants underwent a complete neurological examination and cognitive tests. Investigation of symptoms and medication intake, and motor and sensory electroneurograms were obtained. 30.7% of adult patients had neurological deficits, including psychomotor delay, parkinsonism, dementia, impaired saccadic ocular movements and peripheral nerve dysfunction. Three patients were redefined as type 3 GD. Electrodiagnosis was performed on 15 patients; 26.7% had reduced amplitude and/or abnormal waveforms in at least three nerves, 33.3% had a mild reduction in amplitude of two nerves and 40% had amplitude reduction in one nerve. Patients with three or more affected nerves had additional neurological symptoms. Our results demonstrate that neurological alterations occur in patients diagnosed with GD1, and subclinical peripheral neuropathy is a frequent finding.

Background: Apolipoprotein E (apo E) plays a major role in lipid metabolism, and its genetic variations have been associated with cardiovascular risk. The objective of this study was to investigate the influence of the APOE promoter (−491 A/T, −427 T/C and −219 G/T) and coding region (APOEɛ2/ɛ3/ɛ4) polymorphisms in atherosclerosis disease by association and linkage disequilibrium analyses. Materials and methods: We analyzed these polymorphisms in a sample of 286 subjects with atherosclerosis disease: 153 subjects with atherothrombotic stroke (ATS) and 133 subjects with ischemic heart disease (IHD); and in two control groups, 103 newborns and 114 elderly subjects.Results: The ɛ4 allele was associated with more severe carotid stenosis in the ATS group, being the percentages of ɛ4 carriers 26.7% and 11.4% for the higher and lower carotid stenosis groups, respectively (p=0,066). The −491 T/T IHD subjects presented higher vessel scores than subjects A/A and A/T genotypes at that position (p=0,041), and the frequencies of −2 (5.1% versus 14.1%,p=0,060) and −427C (10.3% versus 24.4%,p=0,019) alleles were lower in IHD subjects with higher extent score versus lower extent score. The ɛ2 allele was in linkage disequilibrium with the −427C allele in all studied groups, and the −219T allele was associated with the ɛ4 allele in the IHD group.Conclusion: In summary, the ɛ2 allele was in linkage disequilibrium with the −427C allele in all studied groups, and only slight associations between the analyzed APOE polymorphisms in the promoter and in the coding region and carotid and coronary vascular disease have been observed.

Autosomal dominant hypercholesterolaemia (ADH) are a heterogeneous group of monogenic lipid disorders. The plasma level of lipoprotein(a) (Lp(a)) is a heritable trait associated with increased coronary heart disease (CHD) risk.To evaluate the frequency of elevated Lp(a) as a cause of ADH and the characteristics of subjects with high Lp(a) (hyperLp(a)).200 healthy subjects and 933 unrelated Spanish subjects with a clinical diagnosis of ADH who were screened for low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) gene mutations. Standard cardiovascular risk factors and blood lipid levels, including Lp(a), were evaluated. HyperLp(a) was defined as Lp(a) levelsor=95th centile of control values.Lp(a) was higher in 263 subjects without LDLR or APOB mutations (nonLDLR/nonAPOB group) than in 670 subjects with mutations (FH group): 40.0 mg/dl (interquartile range (IR) 15.0-89.0) versus 31.0 mg/dl (IR 11.0-73.7) respectively, p = 0.002. HyperLp(a) was present in 23% of ADH subjects (odds ratio (OR) 5.6 (95% CI, 2.9 to 10.7) versus controls) and 29% of nonLDLR/nonAPOB subjects (OR 7.7; 3.9 to 15.4). After adjusting for Lp(a), LDL cholesterol levels were95th centile in 28 (10.6%) nonLDLR/nonAPOB subjects and in 9 (1.3%) FH subjects. Lp(a) levels were nonsignificantly higher in ADH subjects with early-onset CHD than in those without (43.5 mg/dl, (IR, 12.0-82.0) versus 31.7 mg/dl (11.8-76.5), respectively).HyperLp(a) is responsible for ADH in approximately 6% of nonLDLR/nonAPOB subjects. HyperLp(a) would not appear to be a risk factor for early-onset CHD in ADH, independently of whether genetic defects have or have not been demonstrated.

Autosomal dominant hypercholesterolemia (ADH) is frequently caused by functional mutations in the low-density lipoprotein receptor (LDLR) or apolipoprotein B-100 (APOB) genes, but approximately 40% of ADH subjects disclose no such molecular defects, possibly pointing to alternative genetic mechanisms.Our objective was to test the hypothesis that increased intestinal cholesterol absorption might play a role in the lipid abnormalities of subjects with ADH without identified genetic defects.This is a cross-sectional study of consecutive subjects with primary hyperlipidemia identified during an 18-month period in two lipid clinics.A total of 52 subjects with a clinical diagnosis of ADH were examined for molecular defects in LDLR and APOB. No APOB defects were found. Functional LDLR mutations occurred in 31 (60%) subjects, who received a diagnosis of familial hypercholesterolemia (FH). Those for whom no mutations could be identified were labeled as non-FH ADH. In addition, 38 subjects with familial combined hyperlipidemia (FCH) and 45 normolipidemic control subjects were studied.Interventions were diagnostic.Serum noncholesterol sterols were used as markers for the efficiency of intestinal cholesterol absorption.Adjusted campesterol to cholesterol ratios increased in the order non-FH ADH more than FH more than controls more than FCH, with mean values (95% confidence interval) in 10(2) mmol/mol cholesterol of 505 (424-600), 397 (345-458), 335 (294-382), and 284 (247-328), respectively. Thus, cholesterol absorption was lowest in FCH and highest in non-FH ADH.Increased intestinal cholesterol absorption may partially explain the high cholesterol levels of non-FH ADH subjects. Serum noncholesterol sterols are a useful tool for the differential diagnosis of genetic hypercholesterolemias, especially FCH and ADH unrelated to LDLR or APOB defects.

Semi quantitative MRI is a very useful procedure for evaluating the bone marrow burden (BMB) in Gaucher disease (GD). Score systems have been applied to obtain a parameter for evaluating the severity of bone disease. Our purpose was to test a simple, reproducible and accurate score to evaluate bone marrow involvement in GD patients. MRI was performed in spine, pelvis and femora at diagnosis in 54 adult GD1 patients, 61.1% of whom were female. Three MRI patterns and punctuation in each location were defined: normal, 0; non-homogeneous infiltration subtypes reticular, 1; mottled, 2; diffuse, 3; and homogeneous infiltration, 4. This score was called Spanish-MRI (S-MRI). Two independent observers applied the S-MRI and bone marrow burden score and compared the differences using the non-parametric Mann-Whitney test. Correlation rank test was calculated. In 46 patients (85.2%), bone involvement was observed. Thirty-nine (72.3%) had their spine affected, 35 (64.8%) pelvis and 33 (61.2%) femora. Fourteen patients had bone infarcts, 14 avascular necrosis, 2 vertebral fractures and 2 bone crises. Correlation analysis between S-MRI and BMB was (r(2)=.675; p=.0001). No evidence of correlation was observed between CT activity and S-MRI nor between CT activity and BMB. We have found a relationship between genotype and bone infiltration according to S-MRI site and complications. S-MRI is a simple method that provides useful information to evaluate bone infiltration and detect silent complications. Our results correlated with the BMB score but offer higher sensitivity, specificity and accuracy for classifying the extent of bone disease.