Diego Tejedor

The BloodGen project: toward mass-scale comprehensive genotyping of blood donors in the European Union and beyond.

Neil D. Avent, Antonio Martinez, Willy A. Flegel, Martin L. Olsson, Marion L. Scott, Núria Nogués, Martin Písǎcka, Geoff Daniels, Ellen van der Schoot, Eduardo Muñiz-Diaz, Tracey E. Madgett, Jill R. Storry, Sigrid H. Beiboer, Petra A. Maaskant-van Wijk, Inge von Zabern, Elisa Jiménez, Diego Tejedor, Mónica López, Emma Camacho, Goedele Cheroutre, Anita Hacker, Pavel Jinoch, Irena Svobodova, and Masja de Haas

Molecular characterization of familial hypercholesterolemia in Spain.

Familial hypercholesterolemia (FH), characterized by isolated elevation of plasmatic low-density lipoprotein (LDL) cholesterol and premature coronary heart disease (CHD), is associated with mutations in three major genes: LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin 9 (PCSK9). We have analyzed 5430 Spanish index cases and 2223 relatives since 2004 with LIPOchip(®) genetic diagnostic platform, a microarray for the detection of Spanish common mutations in these three genes, including copy number variation (CNV) in LDLR, followed by sequencing analysis of the coding regions of LDLR and exon 26 of APOB, when the result is negative. Samples were received from hospitals of all around Spain. The preferred clinical criterion to diagnose FH was Dutch Lipid Clinic Network (DLCN) score. Our results show that there is a broad spectrum of mutations in the LDLR gene in Spain since about 400 different mutations were detected, distributed along almost the whole LDLR gene. Mutations in APOB (mainly p.Arg3527Gln) covered 6.5% of positive cases and only one PCSK9 mutation was detected. We found correlation between more severe mutations and the clinical diagnosis but also that 28% of FH patients harboring mutations do not have a definite clinical diagnosis. This study analyzes the mutation spectrum in Spain, remarks the importance of genetic diagnosis of FH patients, as well as the cascade screening, and shows how it is being carried out in Spain.

Frequency of Low-Density Lipoprotein Receptor Gene Mutations in Patients With a Clinical Diagnosis of Familial Combined Hyperlipidemia in a Clinical Setting

OBJECTIVES The purpose of this study was to determine the frequency of mutations in the low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) genes in consecutive patients with a clinical diagnosis of familial combined hyperlipidemia (FCH) in a nonresearch setting. BACKGROUND The lipid phenotype frequently overlaps in familial hypercholesterolemia (FH) and FCH. Detection of causative mutations in LDLR or APOB provides an unequivocal diagnosis of FH, but such genetic testing has not been systematically performed in FCH. METHODS We used Lipochip (Progenika, Derio, Spain), a microarray that includes 203 causative mutations in LDLR and 4 APOB defects, to investigate 143 unrelated FCH patients. RESULTS Mutations of LDLR were found in 28 patients (overall prevalence, 19.6%). No APOB defects were found. Compared with patients who had a normal LDLR gene, patients with mutations had lower waist circumference (p = 0.02); significantly (p < 0.005) higher total cholesterol, non-high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and apoB; nonsignificantly (p = 0.063) lower triglycerides; and a lower frequency of diabetes mellitus (22% vs. 0%, respectively; p = 0.002). Total cholesterol and apoB levels showed the best receiver-operator characteristics curves in the prediction of LDLR mutations, with areas under the curve (95% CI: of 0.750 (95% confidence interval [CI]: 0.647 to 0.853) and 0.744 (95% CI: 0.636 to 0.851), respectively. Total cholesterol of 335 mg/dl and apoB of 185 mg/dl were the best thresholds for diagnosis of LDLR mutations. CONCLUSIONS Screening for LDLR defects is advisable for patients with a clinical diagnosis of FCH showing high total cholesterol or apoB levels. Diagnostic criteria for FH should not exclude patients whose personal and familial lipid values appear to fit the clinical criteria of FCH.

Genetic diagnosis of familial hypercholesterolemia using a DNA-array based platform

OBJECTIVES The aim of this study was to validate the Lipochip genetic diagnostic platform by assessing effectiveness, sensitivity, specificity and costs for the identification of patients with familial hypercholesterolemia (FH) in Spain. This platform includes the use of a DNA micro array, the detection of large gene rearrangements and the complete resequencing of the low-density lipoprotein receptor gene. DESIGN AND METHODS DNA samples of patients with clinically diagnosed FH were analyzed for mutations by application of the Lipochip platform. Results obtained were confirmed by DNA sequencing and MLPA analysis by two other, independent laboratories. RESULTS Of 808 patients tested, Lipochip detected a mutation in 66% of the cases and of these 78% were detected by the micro array. A specificity of 99.5% at a sensitivity of 99.8% was reached. A positive test result could be reported within 22 days after start of analysis. The total average screening costs of

The Bloodgen Project of the European Union, 2003-2009

350 per case were significantly lower compared to other existing screening programs. CONCLUSION Lipochip provides a reliable, fast and cheap alternative for the genetic testing of patients with clinically diagnosed FH.

The C677T polymorphism in the MTHFR gene is associated with the toxicity of methotrexate in a Spanish rheumatoid arthritis population.

The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.

Impact of low-density lipoprotein receptor mutational class on carotid atherosclerosis in patients with familial hypercholesterolemia

Objective: Methotrexate (MTX) is the first-choice drug for the treatment of rheumatoid arthritis (RA) patients. However, 30% of RA patients discontinue therapy within 1 year, usually because of adverse effects. Previous studies have reported conflicting results on the association of polymorphisms in the MTHFR gene with the toxicity of MTX in RA. The aim of this study was to assess the involvement of the C677T and A1298C polymorphisms in the MTHFR gene in the toxicity of MTX in a Spanish RA population. Methods: The study included retrospectively 468 Spanish RA patients treated with MTX. Single nucleotide polymorphism (SNP) genotyping was performed using the oligonucleotide microarray technique. Allele and genotype association analyses with regard to MTX toxicity and a haplotype association test were also performed. Results: Eighty-four out of the 468 patients (18%) had to discontinue therapy due to adverse effects or MTX toxicity. The C677T polymorphism (rs1801133) was associated with increased MTX toxicity [odds ratio (OR) 1.42, 95% confidence interval (CI) 1.01–1.98, p = 0.0428], and the strongest association was shown in the recessive model (OR 1.95, 95% CI 1.08–3.53, p = 0.0246). The A1298C polymorphism (rs1801131) was not associated with increased MTX toxicity (OR 0.94, 95% CI 0.65–1.38, p = 0.761). A borderline significant risk haplotype was found: 677T-1298A (OR 1.40, 95% CI 1.00–1.96, p = 0.0518). Conclusion: These results demonstrate that the C677T polymorphism in the MTHFR gene is associated with MTX toxicity in a Spanish RA population.

HLA-DRB1*1501 and Spinal Cord Magnetic Resonance Imaging Lesions in Multiple Sclerosis

BACKGROUND AND OBJECTIVES Defects in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), a highly atherogenic condition. The effect of different LDLR mutations on coronary heart disease (CHD) risk is insufficiently defined. We assessed carotid intima-media thickness (IMT), a surrogate marker of CHD, in relation to LDLR mutational class in FH. METHODS In 436 Spanish FH patients (223 men and 213 women, age 44+/-14 years) with known LDLR mutations, alleles were classified by standard criteria as null (n=269), defective (n=162), or undetermined (n=5). LDLR defects were detected using a microarray (Lipochip) designed to uncover prevalent mutations in Spain and gene sequencing when no mutations were detected. Carotid IMT and plaque were assessed in FH patients and 268 healthy subjects. RESULTS All carotid measurements were increased in FH patients versus controls (p<0.05), irrespective of genotype. After adjustment for gender and age, patients with null alleles compared with defective alleles had similar mean and maximum common carotid artery (CCA) IMT, but higher maximum IMT at any carotid segment, with median values (95% confidence interval) of 1.25 mm (1.19-1.31) and 1.11 mm (1.05-1.18), respectively. Multivariate analysis showed that null alleles were independently associated with maximum CCA-IMT (beta=0.09, p=0.033) with an impact similar to that of gender (beta=0.10, p=0.035). CONCLUSIONS FH patients show advanced carotid atherosclerosis in relation to LDLR mutational class. The findings support the utility of genetic testing in FH beyond providing a secure diagnosis.

Can we predict top‐level sports performance in power vs endurance events? A genetic approach

BACKGROUND Multiple sclerosis (MS) is a heterogeneous neurologic disease with extensive variation with respect to the most affected central nervous system region (brain vs spinal cord). OBJECTIVE To test the hypothesis that this variation in lesion location (brain vs spinal cord) might be (partially) genetically determined. DESIGN Candidate gene study. SETTING Academic research. PATIENTS Patients were selected for the availability of DNA material, clinical variables, and brain and spinal cord magnetic resonance images (evaluating T2-weighted lesion load in the brain and the number of spinal cord lesions). MAIN OUTCOME MEASURES For genotyping, we used a DNA chip containing a set of genes mentioned in previous publications noting their relation to different phenotypes of MS. We assessed the association between brain and spinal cord abnormalities and the genotypes of the patients. RESULTS One hundred fifty patients were included in the analysis. Five single-nucleotide polymorphisms within the major histocompatibility complex region were associated with the number of focal abnormalities in the spinal cord. The most significant was rs3135388 (surrogate marker for the HLA-DRB1*1501 allele). Carriers of HLA-DRB1*1501 had a median of 4 spinal cord lesions compared with 2 lesions for noncarriers (P < .001). No significant association was noted between the single-nucleotide polymorphisms and T2-weighted lesion load in the brain. CONCLUSIONS Carriership of HLA-DRB1*1501 (via rs3135388) was associated with the extent of focal abnormalities in the spinal cord. Spinal cord lesions might be an explanation for increased MS disease severity in patients carrying HLA-DRB1*1501.

Femoral Atherosclerosis In Heterozygous Familial Hypercholesterolemia Influence Of The Genetic Defect

The goal of our study was to discriminate potential genetic differences between humans who are in both endpoints of the sports performance continuum (i.e. world‐class endurance vs power athletes). We used DNA‐microarray technology that included 36 genetic variants (within 20 different genes) to compare the genetic profile obtained in two cohorts of world‐class endurance (N=100) and power male athletes (N=53) of the same ethnic origin. Stepwise multivariate logistic regression showed that the rs1800795 (IL6−174 G/C), rs1208 (NAT2 K268R) and rs2070744 (NOS3−786 T/C) polymorphisms significantly predicted sport performance (model χ2=25.3, df=3, P‐value <0.001). Receiver–operating characteristic (ROC) curve analysis showed a significant discriminating accuracy of the model, with an area under the ROC curve of 0.72 (95% confidence interval: 0.66–0.81). The contribution of the studied genetic factors to sports performance was 21.4%. In summary, although an individuals potential for excelling in endurance or power sports can be partly predicted based on specific genetic variants (many of which remain to be identified), the contribution of complex gene–gene interactions, environmental factors and epigenetic mechanisms are also important contributors to the “complex trait” of being an athletic champion. Such trait is likely not reducible to defined genetic polymorphisms.